 

THFSIS

This is to certify that the

dissertation entitled

FACTORS ASSOCIATED WITH THE HONEY
BEE COLONY AND THE FORAGING BEE THAT
CONTRIBUTE TO THE SPREAD OF BLUEBERRY
LEAF MOTTLE VIRUS TO HIGHBUSH BLUEBERRIES

presented by

Walter Lee Pett

has been accepted towards fulﬁllment
of the requirements for

Ph . D . degree in Entomology

 

{g 71v 76’0WW

Major prrofesg

Date October 1, 1993

 

MS U is an Afﬁrmative Action/ Equal Opportunity Institution 0-12771

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LIBRARY
Mlchlgan State
University

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FACTORS ASSOCIATED WITH THE HONEY BEE COLONY
AND THE F ORAGING BEE THAT CONTRIBUTE TO THE
SPREAD OF BLUEBERRY LEAF MOTTLE VIRUS TO
HIGHBUSH BLUEBERRIES

by

Walter Lee Pett

A DISSERTATION

Submitted to
Michigan State University
impartial fulﬁllment of the requirements
for the degree of

Department of Entomolgr

1993

ABSTRACT

FACTORS ASSOCIATED WITH THE HONEY BEE COLONY
AND THE FORAGING BEE THAT CONTRIBUTE TO THE
SPREAD OF BLUEBERRY LEAF MOTTLE VIRUS TO
HIGHBUSH BLUEBERRIES

By
Walter Lee Pett

Factors associated with honey bees that facilitate the spread of
pollen-home blueberry leaf mottle nepovirus (BBLMV) to highbush
blueberries were examined. These factors included the longevity of
infectious BBLMV in the honey bee colony and in-hive virus transfer
within and between colonies. Blueberry leaf mottle virus remained
infectious for at least 10 days within colonies of the honey bee, which is
the primary pollinator. Flowering blueberry plants were caged for
various periods with colonies of honey bees derived from a blueberry
farm where a high percentage of bushes were infected by BBLMV. Twelve
of 84 plants later tested enzyme-linked immunosorbent assay (ELISA)-
positive for BBLMV. Counts of pollen grains washed from "house bees"
(bees that have never left the colony) resulted in a average of 5, 149 pollen
grains per bee, indicating that in-hive pollen-virus transfer occurs and
can be a source of spread of virus -contaminated pollen within a colony.
Evidence of pollen transfer between colonies and to a lesser extent

between apiaries was also documented by observing the drifting

(wandering far away from the bee's hive and/ or visiting other hives)
behavior of honey bees. No 6-colony apiaries were situated at similar
blueberry plantings. Three colonies at each apiary were ﬁtted with
pigment dispensers which marked the bees' thorax with colored pigment
(unique for each colony) as they entered or left the colony. The
dispensers proved to be 99.7% effective in marking bees. Counts at non-
marking colony entrances were made by recording the number and color
of foragers. The percentage of foragers originating from different colonies
within an apiary ranged from 32 to 63%. Bees originating from colonies
located 600 m away comprised 1.3 to 4.5 % of the foraging force of the
unmarked colonies. This drifting phenomenon could contribute to the
maximum spread of BBLMV by bee-to-bee contact and multiple colony

visits.

To my sons Will and Liam.
I love you.

iii

ACKNOWLEDGMENTS

I wish to thank my guidance committee; Roger Hoopingarner, Don
Ramsdell, Karen Klomparens, Jim Hancock, and George Ayers, for
sharing their wisdom, knowledge and friendship with me. I also thank
them for their compassion and understanding. A special thanks to Ed
Graﬁus for his ﬁnancial and emotional support during the last 3 years.

I would also like to thank the entire Department of Entomologi
(especially the fourth ﬂoor) for all of the friendships that have evolved -
what a great place to work.

A special thanks to Sue Morris - a true friend.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ vi
LIST OF FIGURES .............................................................................. vii
INTRODUCTION ................................................................................. 1
LITERATURE CITED ...................................................................... 5
LITERATURE REVIEW ........................................................................ 7
LITERATURE CITED ...................................................................... l 5

A SELF-MARKING SYSTEM TO DETERMINE FORAGING
POPULATIONS OF HONEY BEES (Apis mellifera L)

(HYMENOPTERA: APIDAE) ................................................................. 19
ABSTRACT .................................................................................... 20
INTRODUCTION ............................................................................. 2 1
MATERIALS AND METHODS .......................................................... 22
RESULTS AND DISCUSSION ......................................................... 25
LITERATURE CITED ...................................................................... 29

HONEYBEE FORAGING BEHAVIOR, IN-HIVE SURVIVAL OF
INFECTIOUS, POLLEN-BORNE BLUEBERRY LEAF MOTTLE
VIRUS AND TRANSMISSION OF THE VIRUS 1N HIGHBUSH

BLUEBERRRY .................................................................................... 30
ABSTRACT .................................................................................... 3 1
INTRODUCTION ............................................................................. 32
MATERIALS AND METHODS .......................................................... 35
RESULTS ....................................................................................... 40
DISCUSSION ................................................................................. 45
LITERATURE CITED ...................................................................... 49

V

DRIFI'ING OF HONEY BEE FORAGERS WITHIN AND BETWEEN
APIARIES POLLINATING BLUEBERRY, VACCINIUM

CORYMBOSUM. .................................................................................. 5 1
ABSTRACT .................................................................................... 52
INTRODUCTION ............................................................................. 53
MATERIALS AND METHODS .......................................................... 54
RESULTS ....................................................................................... 55
DISCUSSION ................................................................................. 58
LITERATURE CITED ...................................................................... 60

CONCLUSION ..................................................................................... 6 1
LITERATURE CITED ...................................................................... 67

LIST OF REFERENCES ....................................................................... 68

vi

LIST OF FIGURES

Figure 1. An isometric and orthographic perspective of the
pigment dispenser used to mark foraging bees as they enter
or exit the colony. The dispenser is constructed of stock
lumber ﬁnished to the stated dimensions. Nylon bridal
veiling (ca. 50 openings per square cm) is stretched over
the bottom and the dispenser is ﬁled with pigment. ........................ 23

Figure 2. The average percent of foragers per colony and the
weight of the scale colony on the three days of data collection.
Bars equal the standard error of the mean foraging population
for each date. ................................................................................. 26

Figure 3. The number of foragers per colony plotted against
the total colony population. The data points represent the
four colonies that were photographed on June 1 l, 25 and
July 2, 1987. ................................................................................. 27

Figure 4. Arrangement of north (top) and south (bottom) apiaries
used for examining the drift of honey bees within and between
apiaries. The north apiary colonies are numbered 1-6 from
left to right. The south apiary colonies are numbered 7-12
from left to right. Squares represent marker colonies
(odd-numbered colonies) and the circles represent
the unmarked colonies (even-numbered colonies).
Colonies are separated by 2 m within the apiary and
600 m between apiaries. ................................................................ 39

Figure 5. Percentage of the total number of caged plants (21
per time period) that tested ELISA-positive for blueberry
leaf mottle nepovirus (BBLMV). Flowering Jersey plants
were caged 1. 3, 6, and 10 days after cages containing
colonies and BBLMV-infected pollen were established. ................... 41

Vii

Figure 6. Percentage of marked foragers (from odd-numbered
colonies) within north and south apiaries found at entrances
of the even-numbered unmarked colonies (See Fig. 1 for
diagram of apiaries and colonies within them). Apiaries
were located 600 m apart. Results are the mean of six,
3-min counts made over 12 days of the blueberry ﬂowering
period. "Unmarked colony" refers to bees that either were
entering their own unmarked colony or bees from a nearby
unmarked colony. .......................................................................... 44

Figure 7. Percentage of foreign foragres (marked honey bees)
originating 600 m away that were found entering unmarked
colonies. Colonies 1-6 were located at the northern apiary
and colonies 7-12 were located at the southern apiary. .................. 45

Figure 8. Percent of marked foragers (within apiary) found at
the entrances of the un-marked colonies two days after
establishment. ............................................................................... 56

Figure 9. Percent of marked foraers (within piary) found at
entrances of un-marked colonies eleven days after
establishment. ............................................................................... 56

Figure 10. Percent of marked foragers (originating 600 m away)
that comprise the foraging force of the un-marked colonies.
Cols. 1-6 are located at the southern apiary, 7—12 at the
northern apiary. ............................................................................. 57

viii

LIST OF TABLES

TABLE 1. Infection results of healthy potted Jersey blueberry
bushes that were caged over time with bees and a bee
hive obtained from a blueberry leaf mottle nepovirus
(BBLMV)-infected blueberry field and presumed to contain
BBLMV-infected pollen ................................................................... 42

ix

INTRODUCTION

Michigan is a major producer of highbush blueberries (Vaccinium
corymbosum L) in the US. with over 7,300 hectares in blueberry
production. Michigan blueberry growers face similar problems with
weeds, insects, and plant pathogens as other fruit and vegetable
producers do. However, with a perenial crop such as blueberries, where
peak production is not reached for seven to twelve years after plant
establishment, certain plant pathogens can have a devastating eﬁ'ect
compared to weed or insect damage. Growers can obtain some control
against weeds, insects and certain plant pathogens with the use of a
varity of pesticides. However, for several plant pathogens there are no
chemical means of control. In 1979, Ramsdell and Stace-Smith
described the physical and chemical properties of one such plant
pathogen, a virus, that was detected in a Michigan blueberry ﬁeld and
named the virus blueberry leaf mottle virus (BBLMV). BBLMV causes a
devastating disease to at least six cultivars of highbush blueberry
('Jersey', 'Rubel', 'Bluecrop‘, 'Elliot', 'Spartan', and ‘Blueray') (Sandoval
Briones 1992). The symptoms are most severe in 'Rubel'. Stems are
killed to the ground. Sparse regrowth from the crown exhibits leaf
distortion and mottling. Yield is nil. In 'Jersey', stem-kill occurs, but leaf
symptoms are very mild. The regrowth from the crown exhibits

shortened internodes and rosetting of leaves which are small and yellow-

2
green instead of deep green. The symptoms in all other known

susceptible cultivars are similar to 'Jersey'. 'Jersey' is the most
important cultivar in Michigan because it makes up 45% of the total
acreage. The disease causes extreme reduction of the bearing surface of
bushes, yield is greatly reduced with up to a 75-100% crop loss, and
eventual death of the bush. The causal virus has been detected and
serologically diagnosed in over 27 ﬁelds in the state (D. C. Ramsdell,
unpublished data).

Blueberry leaf mottle virus is classiﬁed as a nepovirus (Ramsdell
and Stace-Smith 1981 and 1983). Infective virus was associated with
pollen collected from diseased blueberry, and BBLMV antigen was
detected in triturated pollen (Childress and Ramsdell 1986). It has been
shown that the honey bee (Apis mellrfera L.) can transmit BBLMV from
blueberry to blueberry during pollination (Childress and Ramsdell 1987).

The importance of the honey bee as a pollinator for highbush
blueberries has been well documented (Dorr and Martin 1966). Michigan
blueberry growers use 2.5 to 10 colonies per hectare to ensure adequate
pollination (McGregor 1976). Due to the necessity of the honey bee for
pollination and proper fruit development, it is impossible to remove the
virus vector from the blueberry system. Therefore, we must understand
the factors that promote the spread of BBLMV, so possible means of
decreasing the spread of the virus can be devised.

There are several factors associated with the honey bee colony and
the foraging bee that may contribute to the spread of BBLMV these
include, but are not limited to: Longevity of infectious BBLMV within a
colony; the in-hive transfer of virus and / or pollen from bee to bee; and
the transfer of virus between colonies via honey bee drifting (movement of

3
foragers from their own colony to adjacent colonies). This study was

initiated to examine the role of honey bee colonies and foraging bees in
the spread of BBLMV to highbush blueberries under ﬁeld conditions and
to develop strategies to minimize the spread of virus in spite of the
necessary widespread use of honey bees in commercial blueberry culture.

This dissertation is a compilation of three published papers. The
papers describe results of experiments that were designed to investigate
the role of honey bees and the behavior of honey bee colonies with
respect to pollination and the movement of BBLMV.

The ﬁrst paper is titled "A self marking system to determine
foraging populations of honey bees (Apis mellifera L) (Hymenoptera:
Apidae)" and was published in BeeScience (Boylan-Pett et al. 1991). This
paper describes the ﬂuorescent pigment dispenser used to mark foragers
so that they could be distinguished from non-foraging bees. The method
also allows for identifying bees from a particular colony when the colony
is ﬁtted with a dispenser containing a pigment that is unique for that
colony (six colors were distinguishable in the ﬁeld).

The second paper is titled "Honeybee foraging behavior, in-hive
survival of infectious, pollen-borne blueberry leaf mottle virus and
transmission of the virus in highbush blueberry" and was published in
Phytopathology (Boylan-Pett et al. 1991a). This paper reports on the
longevity of infectious virus in the honey bee colony, the behavior of the
foraging honey bee, and the behavior of the colony itself, as they relate to
the spread of the virus. It is shown that these factors all play a critical
role in the short range spread (within the foraging range of the colony) of
the virus. It is also shown how these factors can contribute to the long

range (well beyond the foraging range of the colony) spread of the virus.

4
The third paper was published in Acta Horticulturae from the

proceedings of the 6th Pollination Symposium (Boylan-Pett and
Hoopingarner 1991). This paper was the ﬁrst to report on the long range
drifting of honey bees from one apiary to another located 600 m apart.
This drifting behavior, together with the in-hive transfer of pollen/virus,
provides us with information that helps us explain the movement of the
virus from ﬁeld to ﬁeld.

The three papers noted above provide information on methods that
examine the BBLMV / honey bee interaction, the interaction itself, and
discusses methods that can help minimize the spread of the virus.

LITERATURE CITED

Boylan-Pett, W. and R. Hoopingarner. 1991. Drifting of honey bee
foragers within and between apiaries pollinating blueberry, Vaccinium
corymbosum Acta Hort. 288:1 1 1- l 15.

Boylan -Pett, W., R. A. Hoopingarner and D. C. Ramsdell. 1991. A self-
marking system to determine foraging populations of honey bees (Apis
mellifera L) (Hymenoptera: Apidae). BeeScience 1: 199-202.

Boylan-Pett. W., D. C. Ramsdell, R. A. Hoopingarner. and J. F. Hancock.
1991a. Honeybee foraging behavior, in-hive survival of infectious, pollen-
bome blueberry leaf mottle virus and transmission of the virus in
highbush blueberry. Pytopathology 81: 1407— 1412.

Childress, A. M. and D. C. Ramsdell. 1986. Detection of blueberry leaf
mottle virus in highbush blueberry pollen and seed. Phytopathologi 76:
1 333- 1 337.

Childress, A. M. and D. C. Ramsdell. 1987. Bee-mediated transmission
of blueberry leaf mottle virus via infected pollen in highbush blueberry.
Phytopathology 77: 167-172.

Dorr, J. and E. C. Martin. 1966. Pollination studies on the highbush
blueberry, Vaccinium corymbosum L. Quart. Bull. Mich. Agric. Expt. Sta.
48: 437-448.

McGregor, S. E. 1976. Insect pollination of cultivated crop plants.
USDA Agriculture Handbook No. 496. Washington DC. 41 1pp.

Ramsdell, D. C. and R. Stace-Smith. 1979. Blueberry leaf mottle, a new
disease of highbush blueberry. Acta Hort. 95: 37-48.

Ramsdell, D. C. and R. Stace—Smith. 1981. Physical and chemical
properties of the particles and ribonucleic acid of blueberry leaf mottle
virus. Phytopathologr 71: 468-472.

6

Ramsdell, D. C. and R. Stace-Smith. 1983. Blueberry leaf mottle virus.
Descriptions of Plant Viruses. Commonw. Mycol. Inst. /Assoc. Appl. Biol.
Kew, Surrey, England. Unpaged. No. 267.

Sandoval Briones, C. R. 1992. Movement of bluebeny leaf mottle virus
(BBLMV) within and between cultivated and wild Vaccinium spp. MS.
Thesis. Michigan State University, E. Lansing. 186pp.

LITERATURE REVIEW

Associations between plant viruses and pollen have been known for
over ﬁfty years (cited in: Cooper et aL 1988). Since that time, the virus-
pollen interaction has been established for at least 46 different viruses
(Cooper et al. 1988). The two virus groups most often utilizing this
biological means of transmission are those belonging to the ilarvirus
(Fulton 1983) and nepovirus groups (Harrison 1977). In early studies of
nepoviruses, Callahan (1957) reported that elm mosaic virus (a strain of
cherry leaf roll virus) was transmitted to elm seedlings (Ulmus spp.) via
pollen. Lister and Murant (1967) observed transmission of raspberry
ringspot and tomato black ring viruses to raspberry, Rubus idaeus L.,
mother plants and seedlings via the pollen from infected plants.
Raspberry bushy dwarf virus was shown to be transmitted by hand
pollination from raspberry to raspberry, and the virus was shown to
spread in the ﬁeld using "infector" plants (Murant et al. 1974). Gilmer
and Way (1960) provided conclusive evidence that prune dwarf ilarvirus
and prunus necrotic ringspot (PNRSV) ilarvirus were transmitted by
pollen to seeds produced on healthy sour cherry trees (Prunus cerasus
L.). The transmission of virus from tree to tree via pollen was
documented by George and Davidson (1964) and Davidson (1976) in
studies involving bee and hand pollination of ﬂowers. Cherry leaf roll
virus from walnut was shown to be transmitted to nuts and seedlings of
English walnut (Juglans regia L.) by hand pollination with diseased

8
pollen (Mircetich et al. 1982). Cherry leaf roll virus was also shown to

infect birch (Betula pendula) embryos as a result of hand pollination with
infected pollen (Cooper et al. 1984). Virus movement from plant to plant
via pollen may have a devastating effect in a perennial crop in which all
plants could become infected over time.

In studying the virus-pollen interaction, plant virologists have
focused mainly on associating a particular virus with pollen using
electron microscopy or pollen washes, followed by enzyme-linked
irmnunosorbent assay (ELISA). Little information is available on the
spread of virus via pollen from plant to plant. Two exceptions were
studies by Mink (1983) and Childress and Ramsdell (1986 and 1987).
The study by Mink investigated stored pollen in honey bee colonies and
the long-distance spread of PNRSV from California to Washington sweet
cherries as a result of interstate movement of hives. He found that 20 of
40 hives tested contained PNRSV in infectivity tests on Chenopodium.
quinoa Willd. with pollen that had been stored in hive cells for 3 days. In
the same study, a hive from a California almond and cherry orchard
containing infected trees was caged with a healthy cherry tree in
Washington within 24 hours after leaving the California orchard. No
apparent transmission of PNRSV occurred.

In the ﬁrst Childress and Ramsdell study (1986) that examined
virus spread from plant to plant via pollen, they detected BBLMV antigen
in the pollen from 13 of 15 infected mature 'Jersey' bushes. They tested
the infectivity of the pollen by rub-inoculating the pollen onto C. quinoa
host plants. Fourteen days after inoculation, 43% of the inoculated
plants showed leaf symptoms typical of BBLMV infection. All of the
pollen causing infection tested ELISA-positive for the virus.

9
The second study by Childress and Ramsdell (1987) demonstrated

that honey bees were one causal agent in the transfer of BBLMV-infected
pollen from diseased to healthy bushes . They placed virus-free 2-year-
old potted bushes next to commercially planted healthy and infected
bushes during bloom. They also placed potted bushes within cages
containing infected or healthy commercial bushes with or without a
colony of honey bees. Highest rates of infection occurred among the
healthy potted bushes when they were caged with an infected bush and
bees. No infection occurred when the potted bushes were caged with
bees and a healthy bush. In the same study, 51.4% of the pollen loads
collected from foragers working a heavily BBLMV-infected ﬁeld tested
ELISA-positive for the virus.

Blueberry leaf mottle virus (BBLMV) was classiﬁed as a nepovirus
(Ramsdell and Stace-Smith 1981 and 1983). Unlike most nepoviruses,
whose primary vectors are nematodes, BBLMV has no known nematode
vector (Ramsdell and Stace-Smith 1983 , Childress and Ramsdell 1986).
Virus transmission through pollen and seed is usually considered of
secondary importance for nepoviruses. However, it does appear to be the
primary means of transmission for some viruses (Mink 1983).

BBLMV causes a devastating disease to at least six cultivars of
highbush blueberry ('Jersey’, 'Rubel', 'Elliot', 'Spartan', 'Bluecrop', and
'Blueray') (Sandoval Briones 1992). The symptoms are most severe in
'Rubel'. Stems are killed to the ground. Sparse regrowth from the crown
exhibits leaf distortion and mottling. Yield is nil. In 'Jersey', stem-kill
occurs, but leaf symptoms are very mild. The regrowth from the crown
exhibits shortened intemodes and rosetting of leaves which are small

and yellow-green instead of deep green. The symptoms in the other

10
susceptible cultivars are similar to 'Jersey'. 'Jersey' is the most

important cultivar in Michigan because it makes up 45% of the total
acreage. The disease causes extreme reduction of the bearing surface of
bushes, yield is greatly reduced with up to a 75-100% crop loss, and
eventual death of the bush results. The causal virus has been detected
and serologically diagnosed in over 27 ﬁelds in the state (D. C. Ramsdell,
unpublished data).

To understand certain behavioral aspects of honey bee colonies,
e.g.; number of foragers in a colony, the origins of bees found foraging in
ﬁelds, and drifting behavior (the movement of bees from their own colony
to adjacent colonies). it is useful to have techniques that allow for
distinguishing individual bees, or groups, of bees from one another. In
the apiculture literature, many studies have been reported that address
these behavioral characteristics of honey bee colonies. These studies
used a variety of methods to ascertain the origins of foraging bees and
the number of foragers from a particular colony. Gary et al. (1972 and
1978) used a magnetic retrieval system to study the distribution of
foraging bees used to pollinate alfalfa and almond. With this system, a
row of magnets is placed at the hive entrance in a manner that all
incoming bees must come in contact with it. Individual foraging honey
bees are anesthetized in the ﬁeld and a small numbered metal disc is
glued to the dorsal surface of their abdomen. The disc is located in a
manner that does not interfere with their wings and is light enough so as
not to disrupt ﬂight. Under ideal conditions a recovery rate of ca. 90% is
expected with this system (Gary 1971).

l 1
Mass marking of honey bees has been accomplished in a variety of

fashions including the use of radioisotopes, genetic markers, and
ﬂuorescent powders. Levin (1961) fed radioactive P32 labeled sugar
syrup to genetically marked Caucasian (black) bees. All of the Caucasian
bees that were captured in an alfalfa ﬁeld were found to be labeled with
P32. He concluded that there were no differences in using either
geneticaly marked or isotope labelled bees. Robinson (1966) used
genetically marked bees to examine the foraging range of honey bees in
citrus. Peer (1956) used bees with the cordovan color to study the
multiple mating of honey bee queens.

Fluorescent pigment has been used by a number of researchers to
mark honey bees for identiﬁcation purposes. Smith and Townsend
(1951) developed a hive entrance block, coated with ﬂuorescent pigment,
that bees were forced to walk over as they left the colony. They
determined that the entire foraging force of the colony was marked in
about 90 minutes. They determined the origins of foragers in the ﬁeld by
examining the captured bees with an ultraviolet light. Johansson (1959)
also used a "walk through" method, bees were marked not only at the
colony entrance, by walking over ﬂuorescent pigment, but also marked at
select cotton ﬂowers that were coated with ﬂuorescent pigment. Bees
and ﬂowers were examined with ultraviolet light to determine whether
they were visited by bees that were marked. Another method of marking
bees with ﬂuorescent pigment was developed by Frankie (1973). He
collected bees from ﬂowers and placed the bees in a nylon net bag. The
bag was placed in a container that had a thin layer of ﬂuorescent powder
on the bottom. A hand pump was ﬁtted to a small hole at the bottom of
the container, the container was sealed, and air was pumped into the

12
container to circulate the powder. He observed that the powder could be

detected on bees for up to 6 days after application. Dhaliwal and
Sharma (1972) developed a pigment dispenser that marked the thorax of
bees as they entered or left the colony. Duryea (1986) used a modiﬁed
version of the dispenser to mark foragers and concluded that the pigment
he used had no toxic effect on the marked bees.

Various techniques have been used to provide information about
the foraging force of a colony. Many of the techniques provide useful
information about the foraging force at a given time. Gary's (1967) ﬂight
cones provide data on the rate of foragers leaving the colony. An inverted
hardware cloth cone, with a small opening (at the small end) from which
bees can exit, is placed at the colonies' entrance with the cone opening
closed for a given period of time. Foraging bees accumulate in the cone
and the number of bees is recorded. This technique provides means of
determining when peak foraging occurs. Burrill and Dietz (1973)
developed a photoelectric counting device to record the number of
incoming and outgoing bees. They concluded that with the use of their
device a continuum of honey ﬂight behavior and activity can be
accurately assessed. Other methods for estimating foraging populations
were also based on the ﬂight activity at the colony entrance (Lundie
1925, Spangler 1969, Erickson et aL 1973, and Szabo 1980). These
methods all use entrance counts and are useful for determining the
number of foraging ﬂights at a speciﬁc time.

Various methods have been described for estimating the foraging
populations of individual colonies. These methods use trapping devices
that capture returning foragers. Danka and Gary'(l987) describe a
method that consists of 24 access tubes in the lid of a box—like trap

13
attached to the hive entrance. To trap bees the tubes are coated with

parafﬁn oil. The oil causes the return foragers to slip into a mesh bag
attached to the lid under the trap. The number of bees found in the trap
is estimated by weighing the trapped bees. The traps are left in place for
45 minutes. Szabo's (1989) device collects returning foragers in a trap
that is part of the hive. The trap is activated by closing a sliding
entrance gate and is left in this position for 45 minutes. After this time
the collected foragers are weighed to determine their number.

Drifting behavior of honey bees, the movement of bees from their
own colony to adjacent colonies, is a common phenomenon. The
majority of drifting bees are assumed to be young bees performing
orientation ﬂights where it is estimated that up to 50% of these bees may
drift. Drifting may also be extensive when colonies are established from
packages or colonies are moved to new locations (Corkins 1933).
Researchers have reported methods that may be used to help prevent
drifting within an apiary. These methods include painting the colonies
different colors (Free 1958) or arranging the colonies in a serpentine or in
a circular pattern (Jay 1971). Although methods to prevent drifting are
known, little information on the degree of the actual number of bees that
drift is available. One exception was study by Robinson (1979) who
used the magnetic retrieval system of Gary (1971) to determine the
number of tagged bees that drifted from their original colony. He tagged
150 bees per colony in 10 similar colonies arranged in a straight row.
Three days later he placed the magnets at the hive entrances to collect
the discs from the tagged bees. He recovered 73% of the discs and found
that little drift occurred from colonies at the ends of the row (1 and 2%,
respectivly, of the tagged bees from these colonies drifted) but that an

14
average of 21% of the tagged bees drifted from colonies that were within

the row. He concluded that end locations served as landmarks for the

foraging bees.

15

LITERATURE CITED

Burrill, R. M. and A. Dietz. 1973. An automatic honey bee counting and
recording device (Apicard) for possible systems analysis of a standard
colony. Amer. Bee J. 1 13:216-218.

Callahan, K. L. 1957. Pollen transmission of elm mosaic virus. (Abstr.)
Phytopathology 47: 5.

Chﬂdress, A. M. and D. C. Ramsdell. 1986. Lack of evidence for a
nematode vector of blueberry leaf mottle virus. Acta Hort. 186: 97-94.

Childress, A. M. and D. C. Ramsdell. 1986. Detection of blueberry leaf
mottle virus in highbush bluebeny pollen and seed. Phytopathology 76:
l 333- 1 337 .

Childress, A. M. and D. C. Ramsdell. 1987. Bee-mediated transmission
of blueberry leaf mottle virus via infected pollen in highbush bluebeny.
Phytopathologi 77: 167 -l72.

Cooper, J. I., P. R. Massalski and M. L. Edwards. 1984. Cherry
leaf roll virus in female gametophyte and seed of birch and its

relevance to vertical virus transmission. Ann. Appl. Biol. 105: 55-
64.

Corkins, C. L. 1933. The drifting of honeybees. Amer. Bee J. 73: 208-
209.

Cooper, J. 1., S. E. Kelley and P. R. Massalski. 1988. Virus-pollen
interactions. Pages 221-249. In. K. F. Haris (ed.): Advances in Disease
Vector Research. Vol. 5. Springer-Verlag, N.Y.

Danka, R. G. and N. E. Gary. 1987. Estimating foraging populations of
honey bees (Hymenoptera: Apidae) from individual colonies. J. Econ.
Entomol. 80:544-547.

Davidson, J. H. 1976. Field spread of prunis necrotic ringspot in sour
cherries in Ontario. Plant Dis. Rep. 60: 1080-1082.

Dhaliwal, H. S. and P. L. Sharrna. 1972. Mass marking honey bees for
behavior studies. Indian J. Entomol. 34: 85-86.

 

16

Duryea, D. K. 1986. A study of the use of diversionary plantings to
reduce pesticide-related honey bee (Apis mellifera) mortality. MS.
Thesis. Michigan State University. E. Lansing, MI. 101pp.

Erickson, E. H., L. O. Whitefoot, and W. A. Kissinger. 1973. Honey bees:
A method of delimiting the complete proﬁle of foraging from colonies.
Environ. Entomol. 2:531-535.

Frankie. G. W. 1973. A simple technique for marking bees with
ﬂuorescent powders. Ann. Ent. Soc. Amer. 66:690-691.

Free, J. B. 1958. The drifting of honey-bees. J. Agr. Sci. 51: 208-209.

Fulton, R. W. 1983. Ilarvirus Group. Description of plant Viruses. No.
275. Commonw. Mycol. Inst. /Assoc. Appl. Biol., Kew, Surrey, England.
Unpaged

Gary, N. E. 1967. A method for evaluating honey bee ﬂight activity at
the hive entrance. J. Econ. Ent. 60: 102-105.

Gary, N. E. 1971. Magnetic retrieval of ferrous labels in a capture-
recapture system for honey bees and other insects. Envir. Ent. 1: 71-7 8.

Gary, N. E., P. C. Witherell and J. M. Marston. 1972. Distribution of
foraging bees used to pollinate alfalfa. Envir. Ent. 2: 573-578.

Gary, N. E., P. C. Witherell and J. M. Marston. 1978. Distribution and
foraging activities of honeybees durring almond pollination. J. Apic. Res.
17: 188-194.

George, J. A. and T. R. Davidson. 1964. Further evidence of pollen
transmission of necrotic ring spot and sour cherry yellows viruses in
sour cherry. Can. J. Plant Sci. 44: 383-384.

Gilmer, R. M. and R. D. Way. 1960. Pollen transmission of necrotic
ringspot and prune dwarf viruses in sour cherry. Phytopathologr 50:
624-625.

Harrison, B. D. 1977. Nepovirus group. Description of plant Viruses.
No. 185. Commonw. Mycol. Inst. /Assoc. Appl. 8101., Kew, Surrey,
England. Unpaged.

Jay, S. C. 1971. How to prevent drifting. Bee World 52: 53-55.

Johansson, T. K. 1959. Tracking honey bees in cotton ﬁelds with
ﬂuorescent pigments. J. Econ. Ent. 52: 572-577.

Levin M. D. 1961. Distribution of foragers from honey bee colonies
placed in the middle of a large ﬁeld of alfalfa. J. Econ. Ent. 54: 482-484.

l7

Lister, R. M. and A. F. Murant. 1967. Seed transmission of nematode-
borne viruses. Ann. Appl. Biol. 59: 49-62.

Lundie, A. E. 1925. The ﬂight activites of the honeybee. US. Dept. Agr.
Bull. No. 1328. Washington DC.

Mink, G.I. 1983. The possible role of honeybees in long-distance spread
of prunis necrotic ringspot virus from California into Washington sweet
cherry orchards. Pages 85-91. In: Plumb, R. G. and M. J. Thresh (eds):
Plant Virus Disease Epidemiologi, Oxford, Blackwell.

Mircetich, S., A. Rowhani and J. Cucuzza. 1982. Seed and pollen
transmission of cherry leaf roll virus (CLRV-W), the causal agent of the
black-line (BL) disease of Walnut trees. (Abstr.) Phytopathology 72: 988.

Murant, A. F., J. Chambers and A. T. Jones. 1974. Spread of raspberry
bushy dwarf virus by pollination, its association with crumbly fruit. and
problems of control. Ann. Appl. Biol. 77 : 271-281.

Peer, D. F. 1956. Multiple mating of queen honey bees. J. Econ. Ent.
49: 741-743.

Ramsdell, D. C. and R. Stace-Smith. 1981. Physical and chemical
properties of the particles and ribonucleic acid of blueberry leaf mottle
virus. Phytopathology 71: 468-472.

Ramsdell, D. C. and R. Stace-Smith. 1983. Blueberry leaf mottle virus.
Descriptions of Plant Viruses. No. 267. Commonw. Mycol. Inst. /Assoc.
Appl. Biol. Kew, Surrey, England. Unpaged.

Robinson, F. A. 1966. Foraging range of honey bees in citrus groves.
Fla. Entomol. 49: 219-223.

Robinson, F. A. 1979. Foraging efﬁciency and drift among honey bee
colonies. Proc. IV Int. Symp. Poll., Md. Agr. Expt. Sta. Spec. Misc. Publ.
1:359-365.

Sandoval Briones, C. R. 1992. Movement of blueberry leaf mottle virus
(BBLMV) within and between cultivated and wild Vaccinium spp. MS.
Thesis. Michigan State University, E. Lansing. 186pp.

Smith, M. V. and G. F. Townsend. 1951. A technique for mass-marking
honeybees. Can. Ent. 83: 346-348.

Spangler, H. G. 1969. Photoelectrical counting of outgoing and incoming
honey bees. J. Econ. Entomol. 62:1 183- 1 184

Szabo, T. I. 1980. Effect of weather factors on honeybee ﬂight activity
and colony weight gain. J. Apic. Res. 19: 164-171.

 

18

Szabo, T. I. 1989. Determining foraging populations of individual honey
bee colonies. Amer. Bee J. 129: 43-46.

19

A SELF-MARKING SYSTEM TO DETERMINE FORAGING POPULATIONS
OF HONEY BEES (Apis mellifera L) (HYMENOPTERA: APIDAE)

20

ABSTRACT

The number of foragers within a colony was evaluated using four
honey bee colonies. The colonies were ﬁtted with pigment dispensers
that marked the thorax of each bee as it entered or exited the colony. In
this study 99.7% of the bees were marked. Individual frames were
photographed and the number of marked (foragers) and unmarked bees
(house bees) were counted. The percentage of foragers ranged from 1 1.7

to 42.2% of the colonies' population.

21

INTRODUCTION

Understanding pollination and bee foraging behavior requires
knowing the number of foraging bees in a colony. Information about a
colony's foraging force is essential when recommending the number of
colonies to use for pollination services; too few colonies may result in
poor fruit set, too many adds an extra cost to the producer. In bee
foraging behavior studies, knowing the number of foragers in a colony
would provide the information required to determine the percentage of
foragers that were performing a speciﬁc task (e.g., observing marked bees
foraging different plants).

Various techniques have been used to provide information about
the foraging force of a colony. Gary's (1967) ﬂight cones provide data on
the rate of foragers leaving the colony, but the number of total foragers is
not addressed. Other methods for estimating foraging populations were
also based on the ﬂight activity at the colony entrance (Lundie 1925,
Spangler 1969, Burrill and Dietz 1973, Erickson et al. 1973, and Szabo
1980). These methods were useful for determining the number of
foraging ﬂights at a speciﬁc time, but they did not accurately estimate
the total number of foragers.

Danka and Gary (1987) and Szabo (1989) have described methods
for estimating the foraging populations of individual colonies. These
methods use trapping devices that capture returning foragers. During
the trapping period, the colony was closed off to prevent house bees (non-

22
foraging bees that have never left the colony) from entering the trap. This

closing also prevented foragers (those still in the colony) from leaving the
colony. During trapping (approximately 45 minutes) normal hive
activities were interrupted. The disruption of normal hive activities
should be considered when these methods are used repeatedly. Due to
the dynamic nature of foraging behavior (not all foragers leave the colony
at the same time) it is impossible to determine the total number of
foragers with either of these methods.

In this paper we describe a method for determining the total
number of foragers in individual honey bee colonies. Pigment dispensers
(similar to those of Dhaliwal and Sharma (1972) modiﬁed by Duryea
[1986]) were used to mark bees as they entered, or left, the colony. For
this study we deﬁne a forager as any bee that leaves the colony. The
number and proportion of foraging bees in a colony was determined by
counting the number of unmarked (house bees) and marked (foragers)

bees in photographs taken of the colony.

MATERIALS AND METHODS

Pigment Dispensers. The dispenser was constructed of wood (Fig. 1).
Nylon bridal veiling (ca. 50 openings per cmz) was stretched tightly over
the bottom of the compartment opening and stapled to the dispenser.
The dispenser was ﬁlled with Day-G10TM ﬂuorescent pigment (Day-G10
Color Corp. Cleveland, OH 44103) and the top of the dispenser was
covered with a piece of wood to prevent bees from entering the dispenser.

The dispenser was placed over the colony entrance, forcing foragers to

23
pass through the 19.3 cm long by 0.6 cm high opening. The 0.6 cm high

opening was an important dimension. This size opening ensured that
contact was made between the thorax of the bee and the dispenser. As a
forager entered or exited the colony its thorax rubbed against the bridal
veiling and was marked with pigment.

 

I

/ /

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

'7?

37.10111 >l' o 6 cm

 

 

 

 

 

 

 

 

I 3‘
'ﬁ—es cm * 19.3 cm * 8.9 sin—3| 7R
0.6 cm

Figure 1. An isometric and orthographic perspective of the
pigment dispenser used to mark foraging bees as they enter or exit
the colony. The dispenser is constructed of stock lumber ﬁnished
to the stated dimensions. Nylon bridal veiling (ca. 50 openings
per square cm) is stretched tightly over the bottom and the
dispenser is ﬁlled with pigment.

24
Eﬁ'ectiveness of Dispenser. Three colonies at the Michigan State

University Apiary, East Lansing, M1, were ﬁtted with pigment dispensers
5 days prior to data collection to allow bees to orientate to the reduced
entrance. Pigment was added to the dispenser and 3,000 individual bees
from three colonies (1,000 bees per colony) were observed as they left
their colony. The number of marked and unmarked bees was recorded.
Determining Forager Populations. On June 1, 1987, pigment
dispensers were placed at the entrances of four colonies at the Michigan
State University Apiary, East Lansing, MI. The dispensers contained no
pigment at this time. This allowed the bees to adjust to the reduced
entrance size of the colony. Pigment was added to the dispensers 24
hours before each data collection period. The following morning (prior to
ﬁrst ﬂights) each comb of the individual colonies was photographed
(Kodachrome 64, 35 mm slide ﬁlm) using a Minolta Maxxum 7000 auto
focus camera ﬁtted with a 28-mm wide-angle lens and cable release.
Each hive body was separated from the colony and covered to prevent
bee ﬂight before the individual combs were photographed. The combs
were photographed in a systematic fashion starting with the most
western facing comb. Both sides of each frame were photograpged. The
combs were then returned to an empty hive body in the same order in
which they were found prior to photographing. This procedure reduced
the posibility of photographing bees more than once. The bottom board,
irmer cover and the sides of the hive bodies were also photographed to
ensure accurate counts of all bees in the colony.

The processed slides were projected onto a grid and the number of
marked and unmarked bees on each comb was recorded. Colonies were

photographed on June 1 1, June 25 and July 2, 1987. One honey bee

25
colony (without dispenser) was placed on a scale and the weight of this

colony was recorded on the dates the experimental colonies were
photographed. The colony weight data served as an indicator of the

honey ﬂow in the foraging area.

RESULTS AND DISCUSSION

Eﬂ'ectiveness of Dispenser. Of the 3,000 bees that were observed
leaving the colony, 2,999 were marked (99.97%). It should be noted that
only those bees leaving the colony were recorded; the dispenser marks
bees as they enter or exit the colony, thus the lone unmarked bee would
probably have been marked when it returned to the colony.

For the ﬁrst several minutes after the pigment-containing
dispensers were in place, activity at the hive entrance was frantic. There
was an accumulation of pigment blocking the entrance and preventing
bees from entering or exiting the colony. The pigment dislodges from the
veiling quite easily when the dispensers are jarred. After the bees
removed the pigment from the hive entrance, the dispensers did not
appear to interfere with normal hive activity.

Forager Populations. The proportion of foragers ranged from 1 1.7 to
42.2% of the total colony population on the three dates the colonies were
photographed. The proportion of foragers was highest on June 10 (Fig.
2). A possible reason for the decline after June 10, was the availability of
forage on those particular dates. During the ﬁrst data collection period.
basswood (Tilia spp.) was in bloom. Our scale colony on June 1 1 , 1987

weighed 41.7 kg (during basswood bloom) and leveled off at 48.1 kg by

26
June 25, increasing to 48.5 kg on July 2. Apparently little forage was

available from June 1 1 to June 25. This information suggests that more
bees were recruited to forage when the rewards were abundant (June 11
in Fig. 2) and that fewer bees leave the colony when there is a nectar

dearth (July 2 and 7 in Fig. 2).

 

 

 

 

 

 

 

 

 

 

40 SO

"' 40
E” 30 ‘ 53
:60 +1
‘2 l - 30 3
>3 20 . +3
5 a
8 ’ 20 gr
I... 2
o i c
Q 10 " - 0

° “‘9— Foragers ' 10

- —°— Colony Wt.
0 1 l l 0
June 11 June 25 July 2
Date (1987)

Figure 2. The average percent of foragers per colony and the
weight of the scale colony on the three days of data collection.
Bars equal the standard error of the mean foraging population
ﬂnteach

No significant correlation between the number of foragers and
population was found (Fig. 3). This is in disagreement with Farrar (1937)
who suggested that large colonies with a high bee-to-brood ratio have a
greater number of foragers than smaller colonies with a low bee-to-brood
ratio. It should be noted that our colony populations were much lower

(15,000 to 26,000 bees) than Farrar's.

27
The pigment dispenser, along with photographs of marked and

unmarked bees, provide a means for determining the total number of
foragers in an individual colony. The dispensers do not seem to interfere
with normal foraging behavior. Although the colony is disrupted during
photographing, it is not disrupted when the colony is in its foraging

 

 

 

 

mode.
10
r=0.28 a
‘5‘ 8"
o
0
F1
3‘. El
22 6" '
v a
no a
r .. .. ..
h. 4" a
h-n
0
:5
Z 2‘ a
0 W I ' I ' I ' l ' I

 

 

 

14 16 18 20 22 24 26
Total Population (x 1000)

Figure 3. The number of foragers per colony plotted against the
total colony population. The data points represent the four
colonies that were photographed on June 1 1, 25 and July 2.
1987.

The pigment dispensers can serve as a means of identifying bees
from a particular colony (ﬁve colors, distinguishable in the ﬁeld. are
available). This technique should prove useful to researchers

28
investigating the many facets of honey bee behavior that require

knowledge of the origin and dispersal of individual honey bees from
individual colonies.

29
Literature Cited

Burrill, R. M. and A. Dietz. 1973. An automatic honey bee counting and
recording device (Apicard) for possible systems analysis of a standard
colony. Amer. Bee J. 1 13:216-218.

Danka, R. G. and N. E. Gary. 1987. Estimating foraging populations of
honey bees (Hymenoptera: Apidae) from individual colonies. J. Econ.
Entomol. 80:544-547.

Dhaliwal, H. S. and P. L. Sharma. 1972. Mass marking honey bees for
behavior studies. Indian J. Entomol. 34:85-86.

Duryea, D. K. 1986. A study of the use of diversionary plantings to
reduce pesticide-related honey bee (Apis mellifera) mortality. MS.
Thesis. Michigan State University. East Lansing, MI. 101pp.

Erickson, E. H., L. O. Whitefoot, and W. A. Kissinger. 1973. Honey bees:
A method of delimiting the complete proﬁle of foraging from colonies.
Environ. Entomol. 2:53 1-535.

Farrar, C. L. 1937. The inﬂuence of colony populations on honey
production. J. Agric. Res. 54:945-954.

Gary, N .E. 1967. A method for evaluating honey bee ﬂight activity at the
hive entrance. J. Econ. Entomol. 60:102-105.

Spangler, H. G. 1969. Photoelectrical counting of outgoing and incoming
honey bees. J. Econ. Entomol. 62:1183-1 184.

Szabo, T. I. 1980. Effect of weather factors on honeybee ﬂight activity and
colony weight gain. J. Apic. Res. 19:164-171.

Szabo, T. l. 1989. Determining foraging populations of individual honey
bee colonies. Amer. Bee J. 129:43-46.

 

30

HONEYBEE FORAGING BEHAVIOR. IN-HIVE SURVIVAL OF
INFECTIOUS, POLLEN-BORNE BLUEBERRY LEAF MOTTLE VIRUS AND
TRANSMISSION OF THE VIRUS IN HIGHBUSH BLUEBERRRY

 

31

ABSTRACT

Factors associated with honey bees that facilitate the spread of
pollen-borne blueberry leaf mottle nepovirus (BBLMV) to highbush
blueberries were examined. These factors included the longevity of
infectious BBLMV in the honey bee colony and in-hive virus transfer
within and between colonies. Blueberry leaf mottle virus remained
infectious for at least 10 days within colonies of the honey bee, which is
the primary pollinator. Flowering blueberry plants were caged for
various periods with colonies of honey bees derived from a blueberry
farm where a high percentage of bushes were infected by BBLMV. Nelve
of 84 plants later tested positive for BBLMV by enzyme-linked
immunosorbent assay. Counts of pollen grains washed from "house
bees" (bees that have never left the colony) resulted in a average of 5,149
pollen grains per bee, indicating that in-hive pollen-virus transfer occurs
and can be a source of spread of virus-contarninated pollen within a
colony. Evidence of pollen transfer between colonies and to a lesser
extent between apiaries was also documented by observing the drifting
(wandering far away from the bee's hive and / or visiting other hives)
behavior of honey bees. Only 42.6% of the total foragers of a colony
originated from their own colony and 2.4% of the foragers were from
colonies located 600 m apart. This drifting phenomenon could
contribute to the maximum spread of BBLMV by bee-to-bee contact and

multiple colony visits.

32

INTRODUCTION

Associations between plant viruses and pollen have been known for
over ﬁfty years (Cooper et aL 1988). Since that time, the virus-pollen
interaction has been established for at least 46 different viruses (Cooper
et al. 1988). The two virus groups most often utilizing this biological
means of transmission are those belonging to the ilarvirus (Fulton 1983)
and nepovirus groups (Harrison 1977). In early studies of nepoviruses,
Callahan (1957) reported that elm mosaic virus (a strain of cherry leaf
roll virus) was transmitted to elm seedlings (Ulmus spp.) via pollen.
Lister and Murant (1967) observed transmission of raspberry ringspot
and tomato black ring viruses to raspberry, Rubus idaeus L., mother
plants and seedlings via the pollen from infected plants. Raspberry
bushy dwarf virus was shown to be transmitted by hand pollination from
raspberry to raspberry, and the virus was shown to spread in the ﬁeld
using "infector" plants (Murant et al. 1974). Gilmer and Way (1960)
provided conclusive evidence that prune dwarf ilarvirus and prunus
necrotic ringspot (PNRSV) ﬂarvirus were transmitted by pollen to seeds
produced on healthy sour cherry trees (Prunus cerasus L.). The
transmission of virus from tree to tree via pollen was documented by
George and Davidson (1964) and Davidson (1976) in studies involving
bee and hand pollination of ﬂowers. Cherry leaf roll virus from walnut
was shown to be transmitted to nuts and seedlings of English walnut
(Juglans regia L.) by hand pollination with diseased pollen (Mircetich et

33
al. 1982). Cherry leaf roll virus was also shown to infect birch (Betula

pendula) embryos as a result of hand pollination with infected pollen
(Cooper et al. 1984). Virus movement from plant to plant via pollen may
have a devastating effect in a perennial crop in which all plants could
become infected over time.

In studying the virus-pollen interaction, plant virologists have
focused mainly on associating a particular virus with pollen using
electron microscopy or pollen washes, follwed by enzyme-linked
irnmunosorbent assay (ELISA). Little information is available on the
spread of virus via pollen from plant to plant. One exception is a study
by Mink (1983) that investigated stored pollen in honey bee colonies and
the long-distAnce spread of PRNSV from California to Washington sweet
cherries as a result of interstate movement of hives.

In the apiculture literature, many studies pertaining to honey bee
foraging behavior and pollination have been reported. These studies
have utilized several techniques to ascertain the origins of bees foraging
on particular plants. Gary et aL (1978) used a magnetic retrieval system
to study the distribution of honey bees foraging almonds during
pollination. They concluded that the distance honey bees ﬂew to forage
was dependent on the foraging competition within the orchard. Bees
foraged at a greater distance from their colony when the number of
colonies at their apiary was high. Other researchers have used
radioisotopes (Levin 196 1), genetic markers (Robinson 1966) and
ﬂuorescent pigment (J ohansson 1959) to study the foraging behavior of
honey bees in a variety of pollinating systems. The drifting of bees has
been studied between adjacent colonies in an apiary (Free 1958).

 

34
However, according to an extensive literature search, no studies have

involved an infectious plant virus associated with pollen.

Blueberry leaf mottle virus (BBLMV) is classiﬁed as a nepovirus
(Ramsdell and Stace-Smith 1981, 1983). Infective virus was associated
with pollen collected from diseased blueberry, and BBLMV antigen was
detected in triturated pollen (Childress and Ramsdell 1986). It has been
shown that the honey bee (Apis mellifera L.) can transmit BBLMV from
blueberry to blueberry during pollination (Childress and Ramsdell 1987).
BBLMV causes a devastating disease to at least three cultivars of
highbush blueberry ('Jersey', 'Rubel', and 'Blueray'). The symptoms are
most severe in 'Rubel'. Stems are killed to the ground. Sparse regrowth
from the crown exhibitss leaf distortion and mottling. Yield is nil. In
'Jersey', stem-kill occurs, but leaf symptoms are very mild. The regrowth
from the crown exhibits shortened intemodes and rosetting of leaves
which are small and yellow-green instead of deep green. Yield is greatly
reduced. The symptoms in 'Blueray' are similar to 'Jersey'. 'Blueray' is
of minor importance in Michigan. 'Jersey' is the most important cultivar
in Michigan because it makes up 45% of the total acreage. This disease
causes extreme reduction of the bearing surface of bushes, 7 5-100% crop
loss, and eventual bush death. The causal virus has been detected and
serologically diagnosed in over 27 ﬁelds in the state (D. C. Ramsdell,
unpublished data).

The importance of the honey bee as a pollinator for highbush
blueberries has been well documented (Dorr and Martin 1966). Michigan
blueberry growers utilize from 2.5 to 10 colonies per hectare to ensure
adequate pollination (McGregor 1976). Due to the necessity of the honey
bee for pollination and proper fruit development, it is impossible to

35
remove the vector from the blueberry system. Therefore, we must

understand the factors that promote the spread of BBLMV, so possible
means of decreasing the spread of the virus can be devised.

The factors associated with the honey bee colony and the foraging
bee that contribute to the spread of BBLMV include, but are not limited
to: Longevity of infectious BBLMV within the colony: the in-hive transfer
of virus and/ or pollen from bee-to-bee: and the transfer of virus between

colonies via honey bee drifting (movement of foragers from their own

Iii... I

colony to adjacent colonies). This study was initiated to examine the role

1....—
I

of the honey bee colonies and foraging bees in the spread of BBLMV to
highbush blueberry (Vaccinium corymbosum L.) under ﬁeld conditions
and to develop strategies to minimize the spread of virus in spite of the

necessary widespread use of honey bees in commercial blueberry culture.

MATERIALS AND METHODS

BBLMV longevity and transmission. At the start of bloom, three
honey bee colonies were placed at an Ottawa 00., MI, blueberry farm
near West Olive known to have a large number of cv. Jersey blueberry
bushes infected with BBLMV. The colonies were kept at this location
until petal fall (for 2 weeks), at which time they were returned to East
Lansing, MI, and individually placed in cages with seven healthy
ﬂowering 2-yr-old potted Jersey bluebeny bushes. Seven additional
ﬂowering bushes were introduced to each cage on days 3, 6, and 10 of
the study. All plants were kept in the cage until petal fall, a period of 2
weeks. Additionally, a control cage containing a colony that had not

36
been used for blueberry pollination was established at the same time.

Four healthy ﬂowering plants were placed in this cage following the same
time schedule as noted above. A total of 100 potted, healthy, virus-
tested plants were used in the experiment. Eighty-four bushes were
caged with colonies from the infected ﬁeld, and 16 bushes were placed in
the control cage.

At the petal fall stage the plants were removed from the cages and
placed in a greenhouse until leaf drop in the autumn (a period of 4
months), at which time they were placed in cold storage for vemalization
for 8 weeks at 40 C. On their removal from cold storage, (approximately
10 months after exposure to bees), all plants were allowed to develop
leaves and were tested for BBLMV using an established ELISA protocol
(Childress and Ramsdell 1986) with the following alteration: The leaf
tissue was homogenized in extraction buffer, ﬁltered through cheese
cloth, and the ﬁltered extract was collected and kept at 5° C overnight
before plating. This procedure does not alter the ELISA absorbance
(A405nm) values of the healthy controls (compared to samples directly
plated after ﬁltering) but did produce higher absorbance values from the
infected samples. These higher absorbance values may be due to the
release of virus particles that may be sequestered or bound to plant
material. Leaf tissue from plants that tested ELISA-positive for BBLMV
were mechanically inoculated onto Chenopodium quinoa Willd. for
veriﬁcation of virus infection. The infected blueberry tissue was ground
with a mortar and pestle in a 0.01M phosphate buffer (pH 7.2, containing
2% nicotine [v/v)) and rubbed to Carborundum-dusted plants. Plants
that developed symptoms typical of BBLMV infection 7- 10 days after
inoculation were tested by ELISA.

37
In-hive pollen transfer. One drone bee and four house bees

(nonforaging bees) were collected from colonies used for blueberry
pollination in a commercial ﬁeld near Douglas, MI, as described later. To
distinguish house bees from foragers, colonies were ﬁtted with
ﬂuorescent pigment dispensers that marked bees as they enter or left the
colony. The dispensers are a modiﬁcation of those previously described
(Dhaliwal and Sharma 1972). Bees that were not marked with pigment
were considered to be house bees. The bees were individually stored in
vials and kept frozen until pollen counts were made.

The pollen was rinsed from the bees by adding 20 ml of 50%
ethanol to the vial containing the bee. The vial was then gently agitated
so that the bee was washed with ethanol. Then, the bee was removed
and the ethanol-pollen mix was ﬁltered through a 0.45-um ﬁlter.
Increasing concentrations of ethanol (75, 95 and 100%) were passed
through the ﬁlter to dehydrate the pollen and prepare the sample for
scanning electron microscopy (SEM). After the last 100% ethanol
treatment the ﬁlters were ﬁxed to 10 mm aluminum stub with a "Double
Stick" tab and placed in a desiccator to dry. The stubs were gold coated
for three minutes at 20 mA (about 21 nm of gold layer) and examined
using a JEOL 35CF SEM (15 kV, magniﬁcation of X400). The stubs were
scanned and all pollen was counted. Blueberry pollen was readily
identiﬁed by the fact that it is in tetrads, which have a mean diameter of
33.4 um and a diameter range of 24-41 um (Megalos and Ballington
1987). Pollen exine patterns can be used to determine a particular

cultivar if one has access to a scanning electron microscope.

38
Honey bee drift within and between apiaries. Two apiaries (six

colonies at each location) were established in a single row facing south
(two meter spacing between colonies) at an Allegan Co., Michigan
blueberry farm (Fig. 4) near Douglas. One apiary (colonies labeled 1-6)
was located at the northern 2 hectare planting made up equally of cvs.
'Rubel' and 'Jersey'. The other apiary (colonies labeled 7-12) was located
600 m away at the southern 1.5 hectare planting and also included equal
amounts of cvs. 'Rubel' and 'Jersey'. The two plantings are separated by
a stream and wooded area. The odd numbered colonies were ﬁtted with
dispensers that marked the thorax of foraging bees with ﬂuorescent
colored pigment as the bees entered or left the colony. Each marker
colony's dispenser marked the bees with a colored pigment that was
unique for that particular colony, allowing for the determination of the
forager's origin. A forager is deﬁned as any bee that ﬂew from the colony.
The even numbered colonies were also ﬁtted with dispensers but
contained no pigment. Three l-minute counts recording the number and
color of marked foragers as well the number of unmarked foragers
entering or leaving each even-numbered colonies (unmarked colonies) at
the two sites were made on 6 days of the 12-day blueberry ﬂowering
period. A mean of 54.4 bees were counted and classiﬁed at each

observation.

39

 

Blueberries

r
l
I
I
I
I
I
I
I

Blueberries

 

Figure 4. Arrangement of north (top) and south (bottom) apiaries used
for examining the drift of honey bees within and between apiaries. The
north apiary colonies are numbered 1-6 from left to right. The south
apiary colonies are numbered 7-12 from left to right. Squares represent
marker colonies (odd-numbered colonies) and the circles represent the
unmarked colonies (even-numbered colonies). Colonies are separated by
2 m within the apiary and 600 In between apiaries.

40
RESULTS

BBLMV longevity and transmission. ELISA tests indicated that
twelve of the 84 healthy Jersey plants that were caged with colonies
previously exposed to BBLMV became infected with the virus. Figure 5
shows the percentage of the total number of infected plants
corresponding to the day the plant was introduced into the cage. There
is no apparent trend with respect to the day the plants were caged with
the infected colonies and the number of plants that became infected.
None of the blueberry plants showed symptoms of blueberry leaf mottle
disease. This is not unexpected, because plant may remain symptomless
for as long as 4 years (Ramsdell and Stace-Smith 1981). The results
demonstrate that BBLMV was infectious and accessible to bees for
transmission to healthy plants for at least 10 days in the colony.

The ELISA absorbance values of the 12 infected blueberry bushes.
the day the bushes were placed in the cage, and the results of the
mechanical inoculation of leaf tissue from BBLMV-infected blueberry to
C. quinoa are shown in Table 1. Only six of the 12 C. quinoa plants,
which were mechanically inocculated with plant tissue from blueberries
that tested ELISA-positive for BBLMV, were infected. High summertime
greenhouse temperatures (up to 34° C) may have interfered with the
maximum transmission of BBLMV to C. quinoa. None of the 16 plants
that were caged with the control colony were infected.

41

 

2O

 

 

 

 

 

 

 

 

 

 

 

A

3

 

T

Day Caged

Figure 5. Percentage of the total number of caged plants (21 per time
period) that tested ELISA-positive for blueberry leaf mottle nepovirus
(BBLMV). Flowering Jersey plants were caged 1, 3, 6. and 10 days after
cages containing colonies and BBLMV-infected pollen were established.

42

TABLE 1. Infection results of healthy potted Jersey blueberry bushes
that were caged over time with bees and a bee hive obtained from a
blueberry leaf mottle nepovirus (BBLMV)-infected blueberry field and
presumed to contain BBLMV-infected pollen

 

ELISA values (A405 nrnl

 

 

Daya Healthy Caged plants Infectivity on
controlsb exposed to infected Chenopodium
pollenC guinoad

1 0.108 0.169 —
1 0.108 0.200 +
1 0.253 0.538 +
1 0.253 0.613 +
3 0.330 0.378 —
6 0 253 0 683 +
6 0. 108 0.168 -
6 0. 1 13 0.432 —
6 0.330 0.372 -
10 0.253 0.536 +
10 0.330 0.394 —
10 0.253 0.570 +

 

a Healthy 2-yr-old potted Jersey plants (three, seven-bush
replicates/ time period) were placed in mesh cages with a hive
containing bees and BBLMV-infected pollen at day indicated after
the infected pollen source was placed in the cage. Plants shown here
are the 12 bushes that developed infections based on ELISA
(enzyme-linked irnmunosorbent assay).

b Mean A405 um value + 3 SD of four healthy control plants placed in
a cage with bees and a hive containing uninfected pollen.

C Mean A405 nm value (duplicate wells) of leaf extracts from each test
plant after exposure to bees carrying BBLMV-infected pollen.
followed by incubation in isolation for at least 6 mo.

9 Results of mechanical inoculation with triturated plant tissue from
ELISA positive test bushes to Chenopodium quinoa. Infectivity was
based on development of terminal leaf mottle/ collapse symptoms
characteristic of BBLMV in C. quinoa, followed by conﬁrmatory tests
by ELISA.

43
In-hive pollen transfer. The house bee pollen counts from four

individuals resulted in counts of 6,034, 5,872, 5,631, and 3,059, or a
mean count of 5, 149 (sd = 1,403) pollen grains per bee. A nonworker
drone bee also had a pollen count of 7,024 grains. These bees were not
marked with ﬂuorescent pigment and had not left the colony. Thus, any
pollen found on these bees was the result of in-hive pollen transfer.
More than 90% of the pollen found on these bees was blueberry pollen.

Honey bee drift within and between apiaries. A total of 3,1 15
drifting foragers were observed on the 6 days of data collection. Figure 6
shows the percentage of marked and unmarked foragers found at the
entrances of the unmarked colonies within each apiary location. The
data are means of all counts made during the ﬂowering period. The
colonies placed in the row (colonies 2 and 4 at the north apiary and
colonies 8 and 10 at the south apiary) had 62.6 and 57.4%, respectively,
of their foragers originating from colonies other than the unmarked
colonies. The colonies at the end of the row (colony 6 and 12) had
greatest number of unmarked foragers. However, 46% of the foragers
from colony 2 and 32.4% of those from colony 12 originated from
neighboring colonies.

The drifting of bees was not conﬁned to the apiary where the
colonies were located. Figure 7 shows the percentage of bees that
originated from one apiary and were part of the foraging force observed at
the unmarked colonies in the second apiary 600 m away. The results are
a mean of all counts made during the study. The number of bees that
drifted between apiaries declined over the duration of the experiment.
However, bees that originated 600 m away were found in all colonies,

except one, 12 days after the colonies were established.

 

 

 

 

 

 

 

 

 

 

 

 

80
I Colony] North
4 I Colony 3
I Colony 5
._. 60 - Unmarked Colony
‘3
2.. 7
a Z
i g
. a 7 x
[A 15;; g if; Z é
/ 32:25 / /
/ 2233: / /
/ .23, / /
é 3 g
Colony Number
80
. l Colony 7 South
«- Gorge. %
60 - Uggked Colony é)
a x
z - %
as - 7 a
a: 4" g é
a . a
- 7 a
. é %

 

 

   

8 10 12
Colony Number

Figure 6. Percentage of marked foragers (from odd-numbered colonies)
within north and south apiaries found at entrances of the even-
numbered unmarked colonies (See Fig. l for diagram of apiaries and
colonies within them). Apiaries were located 600 m apart. Results are
the mean of six, 3-min counts made over 12 days of the blueberry
ﬂowering period. "Unmarked colony" refers to bees that either were
entering their own unmarked colony or bees from a nearby unmarked
colony.

45

 

 

Colony 1
Colony 3
Colony 5
Colony 7
Colony 9
Colony 11

Ila-II

 

 

 

Foragers (%)
to
I

 

 

 

 

 

 

 

.11.

2 4 6 8 10 12
Unmarked Colony Number

 

Figure 7. Percentage of foreign foragres (marked honey bees) originating
600 m away that were found entering unmarked colonies. Colonies 1-6

were located at the northern apiary and colonies 7-12 were located at the
southern apiary.

DISCUSSION

Michigan is a major producer of highbush blueberries (7,300
hectares) with a high concentration of farms in the southwest region of
the state. Many of the farms border one another, and the spread of a
pollen-home virus via honey bee colonies used for pollination is probable. ,
The results presented in this paper provide information on the behavior

of the honey bee colony and foraging bees that could facilitate the spread
of BBLMV.

46
The phenomenon of bee-to-bee spread of virus-infected pollen,

coupled with visits by bees to several colonies, some as far as 600
meters, could lead to rapid spread of BBLMV from one or more infection
foci in a plantation.

The longevity of infectious BBLMV within a honey bee colony would
allow for the long distance spread of the virus. Infected colonies used for
pollination in one area of the state and then moved to a farm in a later
blooming area could transfer the virus over 100 miles. In a previously
reported study by Mink (1983), the long distance transfer of PNRSV from
California to Washington was examined. He found that 20 of 40 hives
tested contained PNRSV in infectivity tests on C. quinoa with pollen that
had been stored in hive cells for 3 days. In the same study, a hive from a
California almond and cherry orchard containing infected trees was
caged with a healthy cherry tree in Washington within 24 hours after
leaving the California orchard. No apparent transmission of PNRSV
occurred. In the present study, stored pollen was not tested for virus,
because pollen stored in the cells of the hive is inaccessible to bees for
possible virus spread. Stored pollen is used as a protein source for the
larvae of the colony (Gojmerac 1980). Instead, the BBLMV-infected
colonies were allowed to pollinate healthy plants in a cage. Plants placed
in the cage up to 10 days after the colnies were removed from the virus
ﬁeld source became infected with BBLMV. These results indicate that
the colony serves as a reservoir of virus; bees inadvertently move virus-
infected pollen form insect to insect in the hive. The result is the spread
of BBLMV from the hive to previously healthy bushes.

47
Our results are in agreement with those of Free and Williams

(1972), who conducted extensive studies determining the number of
pollen grains on nonforaging bees. The transfer of pollen within the hive
from bee-to-bee has also been documented in the apple orchard system
(DeGrandi et al., 1986). Because virus is associated with pollen and
pollen exchange does occur between bees, the spread of virus from one
plant to any plant in the foraging range of the colony is possible. The
foraging range of honey bees is dependent on many environmental
factors and conditions within the colony but may be as great as 4 km
(Free and Williams, 1972). Thus, virus transferred between bees foraging
in opposite directions could spread 8 km from the infected plant.

The large number of drifting foragers observed during this study
would suggest that virus transfer occurs not only within a single colony,
but between all colonies within an apiary. The drift of foragers between
apiaries would also promote the spread of BBLMV to adjacent blueberry
ﬁelds.

It is not known whether BBLMV was vectored by bees that injured
blossom tissues during pollination, thus allowmg direct inoculation by
deposition of Infected pollen in wounds, (pollen bears the virus both
externally and internally [Childress and Ramsdell 1986]), or by ovule
infection following fertilization by infected sperm.

The current recommendations for the control of BBLMV is to rogue
out all infected plants as soon as the virus is detected visually or by
ELISA. This study indicates that additional steps can be taken to
minimize the spread of BBLMV to highbush blueberries. Honey bee drift
can be minimized by arranging colonies of an apiary in a serpentine
pattern, or in a circle, with all colony's entrances facing different

48
directions (Jay 1971). This arrangement may give bees a better reference

as to the locationof their own hive. However, no sure method of
controlling long range drifting is known. The prior location of the
colonies used for pollination must be known and colonies used
previously in blueberries or located near blueberry plantations should be
avoided. The most effective means of controlling the spread of BBLMV is
to identify all infected plants by ELISA tests, kill and remove them, and
replant with a cultivar resistant to BBLMV. Presently, the only cultivars
known to be susceptible to BBLMV are Jersey, Rubel, and Blueray.
Studies are underway to identify suitable virus-resistant replacement

cultivars.

49

LITERATURE CITED

Callahan, K. L. 1957. Pollen transmission of elm mosaic virus. (Abstr.)
Phytopathology 47: 5.

Childress, A. M. and D. C. Ramsdell. 1986. Detection of blueberry leaf
mottle virus in highbush blueberry pollen and seed. Phytopathology 76:
1333-1337.

Childress, A. M. and D. C. Ramsdell. 1987. Bee-mediated transmission
of blueberry leaf mottle virus via infected pollen in highbush blueberry.
Phytopathologl 77: 167-172.

Cooper, J. 1., S. E. Kelley and P. R. Massalski. 1988. Virus—pollen
interactions. Pages 221-249. In. K. F. Haris (ed.): Advances in disease
vector research. Vol. 5. Springer-Verlag, N.Y.

Cooper, J. I., P. R. Massalski and M. L. Edwards. 1984. Cherry leaf roll
virus in female gametophyte and seed of birch and its relevance to
verticle virus transmission. Ann. Appl. Biol. 105: 55-64.

Davidson, J. H. 1976. Field spread of prunis necrotic ringspot in sour
cherries in Ontario. Plant Dis. Rep. 60: 1080-1082.

DeGrandi-Hoffrnan, G., R. A. Hoopingamer and K. Klomparens. 1986.
The inﬂuence of honey bee (Hymenoptera: Apidae) in-hive pollen transfer
on cross-pollination and fruit set in apples. Enxiron. Ent. 15: 723-725

Dhaliwal, H. S. and P. L. Sharma. 1972. Mass marking honey bees for
behavior studies. Indian J. Entomol. 34: 85-86.

Dorr, J. and E. C. Martin. 1966. Pollination studies on the highbush
blueberry, Vaccinium corymbosum L. Quart. Bull. Mich. Agric. Expt. Sta.
48: 437-448.

Free, J. B. and I. H. Williams. 1972. The transport of pollen on the body
hairs of honeybees (Apis mellifera L.) and bumblebees (Bombus spp. L.).
J. Appl. Ecol. 9: 609-615.

Gary, N. E., P. C. Witherell and J. M. Marston. 1978. Distribution and
foraging activities of honeybees durring almond pollination. J. Apic. Res.
17: 188-194.

50

George, J. A. and T. R. Davidson. 1964. Further evidence of pollen
transmission of necrotic ring spot and sour cherry yellows viruses in
sour cherry. Can. J. Plant Sci. 44: 383-384.

Gilmer, R. M. and R. D. Way. 1960. Pollen transmission of necrotic
ringspot and prun dwarf viruses in sour cherry. Phytopathologl 50: 624-
625.

Jay, S. C. 1971. How to prevent drifting. Bee World 52: 53-55.

Johansson, T. S. K., 1959. Tracking honey bees in cotton ﬁelds with
ﬂuorescent pigments. J. Econ. Ent. 52: 572-577.

Levin M. D. 1961. Distribution of foragers from honey bee colonies
placed in the middle of a large ﬁeld of alfalfa. J. Econ. Ent. 54: 482-484.

Lister, R. M. and A. F. Murant. 1967. Seed transmission of nematode-
bome viruses. Ann. Appl. Biol. 59: 49-62.

McGregor, S. E. 1976. Insect pollination of cultivated crop plants.
USDA Agriculture Handbook No. 496. Washington DC. 41 1pp.

Mink, G.I. 1983. The possible role of honeybees in long-distance spread
of prunis necrotic ringspot virus from California into Washington sweet
cherry orchards. Pages 85-91. In. Plumb, R. G. and M. J. Thresh (eds):
Plant Virus Disease Epidemiologr, Oxford, Blackwell.

Mircetich, S., A. Rowhani and J. Cucuzza. 1982. Seed and pollen
transmission of cherry leaf roll virus (CLRV-W), the causal agent of the
black-line (BL) disease of Walnut trees. (Abstr.) Phytopathologl 72: 988.

Murant, A. F., J. Chambers and A. T. Jones. 1974. Spread of raspberry
bushy dwarf virus by pollination, its association with crumbly fruit, and
problems of control. Ann. Appl. Biol. 77: 271-281.

Ramsdell, D. C. and R. Stace-Smith. 1981. Physical and chemical
properties of the particles and ribonucleic acid of blueberry leaf mottle
virus. Phytopathology 71: 468-472.

Ramsdell, D. C. and R. Stace-Smith. 1983. Blueberry leaf mottle virus.
Descriptions of Plant Viruses. No. 267. Commonw. Mycol. Inst. /Assoc.
Appl. Biol. Kew, Surrey, England. Unpaged.

Robinson, F. A. 1966. Foraging range of honey bees in citrus groves.
Fla. Entomol. 49: 219-223.

51

DRIFTING OF HONEY BEE FORAGERS WITHIN AND BETWEEN
APIARIES POLLINATIN G BLUEBERRY. VACCHVIUM CORYNBOSUM.

52

ABSTRACT

Two 6-colony apiaries were situated 600 m apart at similar blueberry
plantings. Three colonies at each apiary were ﬁtted with ﬂorescent
pigment dispensers which marked the bees' thorax with colored pigment

as they entered or left the colony. Each dispenser-ﬁtted colony marked

 

bees with a colored pigment that was unique for that colony. Counts at
non-marking colony entrances were made by recording the number and
color of foragers. The percentage of foragers originating from different
colonies within the apiary ranged from 32 to 63 percent. Bees
originating from colonies located 600 m away comprised 1.3 to 4.5

percent of the foraging force of the un-marked colonies.

53

INTRODUCTION

Drifting behavior of honey bees, the movement of bees from their
own colony to adjacent colonies, is a common phenomenon. The
majority of drifting bees are assumed to be young bees performing
orientation ﬂights where it is estimated that up to 50% of these bees may
drift. Drifting may also be extensive when colonies are established from
packages or colonies are moved to new locations (Corkins 1933).
Researchers have reported methods that may be used to help prevent
drifting within an apiary. These methods include painting the colonies
different colors (Free 1958) or arranging the colonies in serpentine or in a
circular pattern (Jay 1971). Although methods to prevent drifting are
known little information on the degree of the actual number of bees that
drift is available.

Michigan is a major producer of highbush blueberry (Vaccinium
corymbosum L.). Most Michigan blueberry producers utilize from 2.5 to
10 colonies per hectare to ensure adequate pollination (Dorr and Martin
1966). In the blueberry system the drifting of honey bees has the
potential for both beneﬁcial and adverse effects on fruit production. A
beneﬁcial effect would be the mix of compatible pollen within colonies via
in-hive pollen exchange similar to that found in the apple system
(DeGrandi-Hoffrnan 1986). An adverse effect would include the
possibility of transferring a pollen-borne virus (Blueberry Leaf Mottle
Virus [Ramsdell and Stace-Smith 1980]) between colonies (Boylan-Pett et

54
al. 1991a). Honey bee drift also has the potential for negetive effects on

the honey bee colony itself. These would include the spread of European
foulbrood (VVardell 1982) or other bee diseases, and the rapid spread of
parasitic mites.

In this paper we report on the drifting behavior of honey bees
between colonies within an apiary established for blueberry pollination.
We also report on the drifting of foraging honey bees between apiaries
located 600 meters apart.

MATERIALS AND METHODS

Two apiaries (six colonies at each location) were established in a
single row facing south (two meter spacing between colonies) at a Allegan
Co., Michigan blueberry farm. One apiary (colonies labeled 1 through 6)
was located at the southern 1.5 hectare planting and the other (colonies
labeled 7 through 12) was located 600 m away at the northern 2 hectare
planting. The two 'Rubel'l'Jersey' plantings are separated by a stream
and wooded area.

The odd numbered colonies were ﬁtted with dispensers which mark
the thorax of a forager with ﬂuorescent colored pigment. The dispensers
are a modiﬁcation of those described by Dhaliwal and Shanna (1972).
Each marker colony‘s dispenser marked the bees with a colored pigment
that was unique for that particular colony allowmg for the determination
of the forager's origin. A forager is deﬁned as any bee that ﬂies from the
colony. The even numbered colonies were also ﬁtted with dispensers but

contained no pigment. Three one-minute counts recording the number

55
and color of marked foragers as well the number of unmarked foragers

entering or leaving the even numbered colonies (unmarked colonies) were

made on six days of the blueberry ﬂowering period.

RESULTS

Drifting within an apiary. A large number of drifting foragers
were observed on the six days of data collection. Figure 8 shows that two
days after the colonies were established 68.2% of the foragers of the
unmarked colonies originated from neighboring colonies. Those colonies
that were placed in the row (colonies 2, and 4 at the south apiary and
colonies 8 and 10 at the north apiary) had 78.2% and 70.0% respectively,
of their foragers originating from colonies other than the unmarked
colonies. The two colonies that were at the end of the rows (colony 6 and
12) had a greater number of unmarked foragers. Still 45.8% of the
foragers from colony 6 and 54.9% of those from colony 12 originated from
neighboring colonies.

Eleven days later the percentage of bees that originated from
neighboring colonies and comprised the foraging force of the un-marked
colonies had decreased to 58%. The colonies placed in the row had
slightly less than 50% of the foragers originating from colonies other than
the home colony (Fig. 9). The colonies located at the end of the row had
33.8% (colony 6) and 38.4% (colony 12) of the foragers originating from

different colonies.

56

 

 

 

Forager (%)
Forager (%)

 

 

 

Colony Number

Colony 1 South E3 Colony 7 North
Colony 3 Colony 9
Colony 5 60‘ I Colony 11
. Unmarked Col
I my I Unmarked Colony

 

8 IO 12

Colony Number

Figure 8. Percent of marked foragers (within apiary) found at the
entrances of the un-marked colonies two days after establishment.

 

 

80 80
Colony 1 South E]
D Colony 3 a
I Colony 5 I

60 — I Unmarked Colony 60 _ I

Foragers (%)
Foragers (%)

 

 

 

 

Colony Number

Colony 7

North
Colony 9

Colony 11
Unmarked Colony

 

Colony Number

Figure 9. Percent of marked foraers (within piary) found at entrances of

un-marked colonies eleven days after establishment.

57
Drifting between apiaries. The drifting of bees was not limited to

the apiary where the colonies were located. Figure 10 shows the percent
of bees originating 600 m away that comprise the foraging force of the
un-marked colonies the ﬁrst day after the colonies were established at
the planting. The number of bees that drifted between apiaries declined
over the duration of the experiment. However, bees that originated 600 m
away were found in all colonies, except one, 12 days after the colonies

were placed into blueberries.

 

   
 

IO
Colony 1
Colony 3
8 " I Colony 5
.3 I Colony 7
9:: 6 _ I Colony 9
5:0 I Colony 1 1
{2

 

 

 

 

Colony Number

Figure 10. Percent of marked foragers (originating 600 m away) that
comprise the foraging force of the un-marked colonies. Cols. 1-6 are
located at the southern apiary, 7- 12 at the northern apiary.

58
DISCUSSION

There was a large variation between colonies in the contribution to
the drifting population. Unfortunately, we did not make a population, or
age structure analysis of these similar-sized colonies to know if these
factors had a bearing on this phenomenon.

The differences in the amount of drifting between colonies placed
in the row and those at the end of the row are in partial agreement with a
previous report by Robinson (1979). In his experiments bees drifted to
end colonies but there was little drift of bees from colonies at the end of
the row. Our results shows that bees from colonies 1 and 7 (colonies at
the opposite end of the rows) made up a considerable portion of the
foraging force of the colonies placed next to them (colonies 2 and 8
respectively) (Fig. 8). However, it was also observed that a greater
movement of bees between colonies occurred from colonies placed in the
row (Fig. 8, bees originating from colonies 3, 5, 9, and l l). Colonies
placed at the end of the row are unique in that they have no adjacent
colony on one side. This distinguishing feature may serve as a landmark
for returning foragers.

While the drifting within an apiary could possibly show that bees
recognize placement, i.e., ends of rows versus the middle, this
recognition of placement did not seem to hold when drifting was between
apiaries. This was in spite of the fact that the apiaries had similar
shape, orientation, and number of colonies.

Michigan has about 30,000 colonies that are annually moved into
orchards and fields for pollination. These colonies are then returned to
their permanent apiary sites located throughout the state. With nearly
10 colonies/ hectare used in blueberry pollination, there are often

59
pollination apiaries that are located less than 500 meters apart. This

density, and drifting between apiaries, would help explain the very rapid
dissemination of diseases and mites within our honey bee population.
We do not feel that 600 meters is a remarkable distance for drifting
of foragers between apiaries. This experiment was initiated because we
had previously found marked foragers at colonies over 1.6 k from their
parent colony.
Robbing of honey between colonies should not be a factor in this

study since abundant nectar is found in the blueberry ﬂowers.

60

LITERATURE CITED

Boylan-Pett, W., D. C. Ramsdell, R. A. Hoopingarner. and J. F. Hancock.
1991. Honeybee foraging behavior, in-hive survival of infectious, pollen-
bome blueberry leaf mottle virus and transmission of the virus in
highbush bluebeny. Phytopathologl 81: 1407-1412.

Corkins, C. L. 1933. The drifting of honeybees. Amer. Bee J. 73:208-
209.

DeGrandi-Hoffrnan, G., R. Hoopingamer and K. Klomparens. 1986.
Inﬂuence of honey bee (Hymenoptera: Apidae) in-hive pollen transfer on
cross-pollination and friut set in apple. Environ. Entomol. 15:723-725.

Dhaliwal, H. S. and P. L. Sharma. 1972. Mass marking honey bees for
behavior studies. Indian J. Ent. 34: 85-86.

Dorr, J. and E. C. Martin. 1966. Pollinatin studies on the highbush
bluebeny, Vaccinium corymbosum L. Mich. Agr. Expt. Sta. Quart. Bul.
48:437-448.

Free, J. B. 1958. The drifting of honey bees. J. Agric. Sci. 51:294-306.
Jay, S. C. 1971. How to prevent drifting. Bee World 52:53-55.

Ramsdell, D. and R. Stace-Snrith. 1983. Blueberry leaf mottle virus.
Description of plant viruses. No. 267. Commonw. Mycol. Inst., Assoc.
Appl. Biol. Kew, Surrey, England. Unpaged.

Robinson, F. A. 1979. Foraging efﬁciency and drift among honey bee
colonies. Proc. IV Int. Symp. Poll., Md. Agr. Expt. Sta. Spec. Misc. Publ.
1:359-365.

Wardell, G. 1982. European foulbrood: Association with Michigan
bluebeny pollination, and control. Ph.D. Thesis. Michigan State
University, East Lansing. 84pp.

61

CONCLUSION

The association between bluebeny leaf mottle virus (BBLMV) and
highbush bluebeny (Vaccinium corymbosum L.) pollen was ﬁrst detected
by Childress and Ramsdell (1986). This pollen/ virus relationship led
these researchers to speculate that the honey bee (Apis mellifera L.)
played a vital role in the spread of the disease. Honey bees are the
primary pollinator of highbush blueberries in Michigan (McGregor 1976).
The role of the honey bee as a vector of BBLMV to highbush blueberries
was first documented by Childress and Ramsdell (1987) and again by
Boylan-Pett et al. (1991a).

Understanding the virus / honey bee interactions that are
associated with the transmission of BBLMV allows for the development of
recommendations to help prevent the spread of the disease in the ﬁeld.
Factors that are associated with the honey bee colony and the foraging
bee that contribute to the spread of BBLMV include, but are not limited
to : longevity of infectious BBLMV within a colony; the in-hive transfer of
virus and/ or pollen from bee to bee; and the transfer of virus between
colonies via honey bee drifting (movement of foragers from their own
colony to adjacent colonies) (Boylan-Pett et al. 1991a).

Longevity of infectious BBLMV within a colony can lead to the long
range spread of the virus. Infected colonies used for pollination in one
area of the state and then moved to a later blooming area could transfer

the disease over 100 miles. The longevity of infectious BBLMV within a

62
colony can also lead to the spread of the disease within the same area

between susceptible cultivars with different blooming periods. BBLMV
remained infectious and accessible to honey bees for 10 days when
colonies used for pollination in a known BBLMV-infected ﬁeld were caged
with healthy plants (Boylan-Pett et aL 1991a). These results indicate
that the colony serves as a reservoir of virus and that bees may come in
contact with the virus through in-hive virus/ pollen transfer.

ln-hive virus/ pollen transfer (the transfer of virus-infected pollen
from bee-to-bee within the hive) is a factor associated with the honey bee
colony that may play a vital role in the spread of BBLMV. When honey
bees foraging on BBLMV infected bushes return to their colonies' they
come in contact with other returning foragers. During this contact,
virus-infected pollen may be transferred from a virus vector bee to a non-
infectious bee. This new virus vector leaves the colony and may infect a
healthy bush in its foraging range. In-hive virus transfer is one
explanation for the random and spotty distribution of infected plants
found in diseased ﬁelds as described by Childress and Ramsdell (1987) .
In-hive pollen transfer has been documented in the apple orchard system
(DeGrandi-Hoffman et al. 1986).

Examination of non-foraging honey bees collected from colonies
used for bluebeny pollination resulted in an average count of 5, 149
pollen grains per bee (Boylan-Pett et aL 1991a). These results are in
agreement with Free and Williams (1972) who conducted extensive
studies determining the number of pollen grains on non-foraging bees.
Because the virus is associated with pollen and pollen transfer occurs
between bees, the spread of the virus from one plant to any plant in the

foraging range of the colony is possible. The foraging range of honey bees

63
is dependent on many environmental factors and conditions within the

colony but may be as great as 4 km (Free and Williams 1972). Thus,
virus transfer between bees foraging in opposite directions could spread
8 km from the infected bush.

The drifting behavior of honey bees is an important factor
associated with the spread of BBLMV. Results from our experiments
indicate that high levels of drifting occurred within an apiary. No days
after colonies were established 68.2% of the observed foragers had drifted
from their original colonies (Boylan-Pett and Hoopingamer 1991, Boylan-
Pett et al. 1991a). Drifting declined over the 12 days of the experiment;
still, 58% of the foragers observed on the last day of the experiment had
drifted from their original colony. Drifting between apiaries was also
observed at an unexpected rate. The ﬁrst day after the colonies were
established as many as 8% of the observed foragers had drifted from
colonies located 600 m away. This between apiary drifting also declined
over the duration of the experiment. However, bees that had drifted 600
m were found in all colonies, except one, 12 days after the colonies were
established. The observed drifting behavior together with in-hive
virus/ pollen transfer indicated that virus transfer could occur not only
within a single colony, but between all colonies of the apiary. Long range
drifting, between apiaries, promotes the spread of BBLMV to adjacent
bluebeny ﬁelds.

To investigate the drifting behavior of honey bees, and to
differentiate foragers from non-foragers for pollen counts, a method to
distinguish the origins of foragers with respect to their original colony
was employed (Boylan-Pett et aL 1991) . This method marks the thorax
of foragers with ﬂuorescent pigment as they enter or leave the hive. The

64
method was 99.97% effective since 2,999 of the 3.000 bees observed

leaving the colony were marked. It should be noted that only bees
leaving the colony were recorded; and that the bees are marked as they
enter or leave the colony. thus the lone unmarked bee would have
probably been marked when she returned to the colony. This system
was also used to determine the foraging populations of four honey bee
colonies on three days in 1987. The foraging populations ranged from

1 1.7 to 42.2% of the colony's population. The highest number of foragers
occurred on June 10 when basswood (Tilia spp.) was in bloom. No
signiﬁcant correlation between the number of foragers and colony
population was found.

The three published papers that comprise this dissertation provide
information on the factors associated with the honey bee colony and the
foraging bee that contribute to the spread of BBLMV to highbush
bluebeny. Recommendations based on this information to prevent the
spread of BBLMV include:

1. Due to the longevity of infectious virus within a colony the
prior location of honey bee colonies used for bluebeny pollination
must be known.

ii. Avoid colonies previously used for bluebeny pollination or
located near blueberry plantations. No action is required, and none
available, to prevent in-hive virus / pollen transfer.

iii. Due to the high occurrence of honey bee drift, within the
apiary, observed in the blueberry plantation, colonies should be
arranged in a serpentine or circular fashion to prevent drifting.

Many beekeepers paint their hive bodies different colors to
minimize bee drift. However, it should be noted that the hive

65
entrances of the colonies used in my experiments all had unique

colors, due to pigment dislodging from the dispensers, and drifting
was high.

iv. No method to prevent long range drifting is known.
However, providing some type of landmark for each colony may
help minimize bee drift.

v. The most effective method to control the spread of BBLMV
is to identify all infected plants by ELISA tests, kill and remove the
plants, and replant with healthy cultivars that are resistant to
BBLMV.

These studies not only investigate the interactions of honey bees
and the spread of BBLMV, but they also provide methods and
information that are useful for researchers studying various aspects of
pollination biology or honey bee behavior. Techniques from these studies
would be helpful in answering many of the concerns that have been
raised over the release of genetically engineered plant material and the
risks associated with their introduction to the environment. Of great
concern is the escape of genes from transgenic plants to wild populations
causing the development of new weedy plants. The most probable means
of gene escape is via pollen. The studies that have examined pollen
movement in the past have generally used small plots and have provided
useful information on only one type of honey bee pollen movement. The
research from this dissertation suggests that there are three distinct
types of pollen movement associated with bees. The ﬁrst type of pollen
movement, would be the movement of pollen from individual bees as they

move from ﬂower-to-ﬂower. The second type of pollen movement would

66
be the movement of pollen associated with in-hive pollen transfer. Pollen

from one plant, located at one end of the colony's foraging range, could
be distributed to a plant at the other end of the foraging range by the
bee-to-bee transfer of pollen in the hive. The third type of pollen
movement involves the long range movement of pollen that would be
associated with the long range drifting of honey bees. Studies using the
information and techniques described in this dissertation could provide
information that could be used to help make better informed decisions
regarding the risks associated with the release of engineered plant

material.

67

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