Honey bee primer pheromones and colony organization: gaps in our knowledge

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Honey bee primer pheromones and colony organization:

gaps in our knowledge

Mark L. Winston, Keith N. Slessor

To cite this version:

Review

Honey

bee

_{primer pheromones}

and

_colony

organization:

gaps

in

our

knowledge

Mark L. Winston

a

Keith N.

Slessor

b

a

Department

of

_Biological

Sciences, Simon Fraser

_{University, Burnaby,}

BC V5A 1S6, Canada

b

Department of _Chemistry, Simon Fraser _{University, Burnaby,} BC V5A 1S6, Canada

(Received 21 November 1996;

accepted

20

_May

1997)

Abstract-The purpose of this article is to _{discuss gaps in} our

_{knowledge concerning}

how

_honey

bee

_{primer pheromones}

mediate worker activities and

_colony

functions. We first review the chemical structure _{and functions of queen mandibular}

_pheromone

(QMP), but then focus

_primarily

on areas

_{of potential}

future research interest. We discuss the mode of action that _{QMP may} have

on _workers, address the issue of

_{why QMP}

is a

_complex

blend of five _{components, propose} an

evolutionary pathway

for

_{pheromone complexity}

_{in the genus}

_Apis,

discuss biochemical

differ-ences _{between worker and queen mandibular}

_glands,

_{review the evidence for additional queen}

pheromones

besides QMP, examine how queen and brood

pheromones

interact to mediate

_colony

biology,

and discuss the relative

_importance

of

_congestion

and

_population

size in

_pheromone

transmission.

_Finally,

we _suggest some research areas that involve commercial

_applications

of

honey

bee

_pheromones

for

beekeeping

and crop

pollination.

© Inra/DIB/AGIB/Elsevier, Paris

Apis

mellifera

/

_{primer pheromones}

/ queen

pheromones

/

_{colony integration}

1. INTRODUCTION

The _purpose of this article is to discuss gaps in our

_{knowledge concerning}

how

honey

bee

_{primer pheromones}

mediate worker activities and

_colony

functions. There

is,

of course, a _great deal known about

_pheromones

in social

insects,

and

especially

the

_honey

bee

_{(Free, 1987;}

Win-ston,

1987, 1992). However,

most of our

knowledge

about social insect

_pheromones

involves releaser

_compounds

that elicit

specific

behaviors such as

_alarm,

orienta-tion,

trail

_marking,

mate attraction and others. In contrast, we know _very little

about

_{primer pheromones}

that exercise a more fundamental level of control

_by

influencing

worker

_{physiology, thereby}

mediating

worker and

_colony

reproduc-tion and

_influencing

broad

_aspects

of

colony organization,

caste structure and division of labour.

*

Correspondence

and

_reprints

The

_honey

bee is the

_only

social insect for which any

primer pheromone

has been identified

_chemically,

and that is the

queen’s

mandibular

_{gland pheromone}

(QMP).

The identities of some

_pheromone

components from the

_queen’s

mandibu-lar

_glands

have been known for over 35 years, and were _among the first insect

pheromones

to be identified

_(Barbier

and

Lederer, 1960;

Callow and

Johnston,

1960;

Butler and

_Fairey,

_1964;

Callow et

al., 1964). However,

in 1988 additional

compounds

were

_reported

_that,

in

combi-nation with

previously

identified compo-nents,

_provide

a full

_duplication

of the

queen’s pheromonal

activity

from the mandibular

_gland

secretions

_(Slessor

et

al., 1988).

Additional

_{activity originating}

from

_pheromones

secreted

_by

other _queen

glands

has _yet to be

_chemically

identified

(Slessor et al., 1997).

The

_queen’s

mandibular

_gland

pheromone,

or

_QMP,

consists of five

com-ponents,

including

(E)-9-keto-2-decenoic

acid

_{(9ODA), (R,E)-(-)-}

and

(S,E)-(+)-9-hydroxy-2-decenoic

acid

_(9HDA),

_methyl

p-hydroxybenzoate

(HOB),

and

4-hydroxy-3-methoxyphenylethanol

(also

called

_homovanillyl

alcohol or

_HVA).

This

blend _{may vary in} the

_proportions

of indi-vidual

compounds

(Slessor

et

al., 1990;

Pankiw et

_{al., 1996),}

but all five

compo-nents are _necessary to elicit the full _range of worker _responses to

_{QMP (Winston}

et

al., 1989, 1990, 1991). Functions of QMP

identified to date include the inhibition of queen

rearing by

workers

(Winston

and

Taylor,

1980;

Winston et

al., 1989, 1990;

Naumann et

al., 1993;

Pettis et

al., 1995a;

Melathopolous

et

_{al., 1996),}

attraction of workers to form a retinue around the _queen within colonies as well as

_externally

to

form a swarm cluster

_(Winston

et

al.,

1982,

1989;

Slessor et al.,

1988;

Kaminski

et

_{al., 1990),}

stimulation

_{of pollen}

forag-ing

and brood

_rearing

in

small,

newly

founded colonies

_(Higo

et

_{al., 1992),}

and

delay

of worker behavioral transitions

from within-hive to

_foraging

tasks

_(Pankiw

et

_{al., 1998).}

_Early

studies also

_reported

that

_QMP

inhibits worker ovaries from

developing,

but

_subsequent

research has

suggested

that

_QMP

alone has no role in

the

_suppression

of _{worker ovary}

develop-ment _{and egg}

_laying

_(Willis

et

_{al., 1990;}

Kaatz et al., 1992a;

Plettner et

_{al., 1993).}

Rather,

brood-produced

primer

pheromones

appear to

_provide

the most

significant

inhibition

_preventing

worker ovary

development,

in combination with non-mandibular _queen

_{pheromones (Jay,}

1970, 1972).

Brood

_pheromones

also are

involved in

_stimulating

cell

_{capping by}

adult workers

_(LeConte

et

_{al., 1990),}

and

promoting

larval

_feeding

and

_development

of adult

_glands

that

_produce

brood food

(LeConte,

1995; Mohammedi et

_{al., 1996),}

although

the substances involved are

prob-ably

different from those that inhibit worker ovary

development.

Although

there has been extensive basic research

_concerning

_queen

pheromones

for almost 4

decades,

and

some research

_{investigating potential}

func-tions of brood

_primer

_pheromones,

there still are

_significant

discontinuities in our

knowledge concerning

these semiochem-icals. Yet, we seem to have become

com-placent concerning

our

_{understanding}

of

honey

bee

_{sociochemistry.}

_Today,

_very little research is

_being

conducted world-wide on

_honey

bee or other social insect

pheromones. Perhaps

we assume that

_only

minor,

fine-tuning

research remains to be undertaken in this area.

On the

_contrary,

some of the most

exciting challenges

in insect

semiochem-istry

remain unanswered _{for any} social

insect,

and our

_knowledge

base

_concerning

honey

bees

_provides

an excellent

_starting

point

to address these

_questions.

For

exam-ple,

we know that

_QMP

inhibits _queen

rearing,

but to date we can

_{only speculate}

as to the neurochemical and/or hormonal mechanisms that mediate between

dis-covered that

_QMP

is a

_complex

_blend,

but

have _yet to understand

_why

there are so

many components, which worker activities

are influenced

by

which

_components,

why

such a

_complex

blend evolved in the first

place,

and whether workers

_produce

other

primer pheromones

in their mandibular

glands.

We have determined that the queen

produces

additional,

non-mandibular

pheromones,

but have little idea as to their

function,

origin

or

_identity.

We have been

aware for some time that brood

_produces

pheromones

involved in the inhibition of worker ovary

development,

but the inter-actions between brood and queen

pheromones

for this and other

functions,

as

well as the identities of the relevant brood

pheromones,

have _yet to be determined. We know that

_pheromone

_dispersal

is slowed in

_crowded,

_congested

colonies,

but have not yet discovered how the

queen’s

message is diluted so _{that queen}

rearing

can

_{begin prior}

to

_swarming.

Finally,

we have

_{only just begun}

to

_explore

the

_potential

_applications

of

_pheromones

for

_management

_purposes.

In the

_following

discussion,

we will

address these and related

_points,

suggest-ing

areas for future research and

problems

with our current

_{understanding}

of

_honey

bee

_pheromone

_biology.

2. HOW DOES

_QMP

ACT

ON WORKERS AND

_QUEENS?

There is a wealth of

_knowledge

avail-able

_{today concerning}

behaviours elicited

by

insect

_pheromones,

but _very little is known about how

_pheromones

act on

insects to induce

_particular

behaviours. This is

_especially

true for social

_insects,

where

_pheromones

such as

_QMP

are

involved in a

_complex

_array of individual

behaviours and

_colony-level

functions. One

_important

issue _{for queen} mandibular

_pheromone

is to determine how it acts on worker bees to

_delay

for-aging

and inhibit queen

rearing.

An advance in this area was made

_{recently by}

Pankiw et al.

_(1998),

who demonstrated that the presence of the queen and her

QMP

message slows the

ontogeny

from hive tasks to

_foraging

that is

_typical

of worker bee

_temporal

caste _ontogeny,

_by

inhibiting

the shift from within-nest work

to

_foraging

tasks. In these

_experiments,

the addition of

_{synthetic QMP}

to colonies caused workers to

_forage

at _{later ages} than workers from untreated colonies. In

addi-tion,

the Pankiw

_study

found

_{that juvenile}

hormone

(JH)

titres were lower in workers

from

_QMP-treated

colonies,

consistent with results _{from cage}

_{experiments by}

Kaatz et al.

_(1992a)

in which JH levels

were

_suppressed

in

_QMP-treated

workers.

Since an increase in JH titre

_normally

is associated with the

_change

from hive to

field tasks

_{(Robinson, 1992),}

these results

suggest that

_QMP

_may act

_{by inhibiting}

juvenile

hormone

_production

to

_regulate

the rate at _{which workers progress from}

within-nest to outside tasks.

This result is

_exciting

because it is the first demonstration of a

pheromone-hor-monal interaction for

_honey

bees.

How-ever, the

implications

of this

finding

for

the

_integration

of

_colony

functions are not

clear. A modulator such as

_{QMP might}

promote

a labor schedule that increases

efficiency

in task

_{performance by}

physio-logically delaying

a shift to the next _age caste. The _{presence of the}

_queen-based

inhibitor _may be

_adaptive

in

_preventing

all of a

_colony’s

_young workers from

developing

too

_rapidly

towards

_foraging

tasks in the event of a sudden loss of older

workers due to

_{predation, rapid}

weather

changes

or other

factors,

_detaining

some

for within-hive tasks.

_Conversely,

sudden queen loss could result in increased JH titres and a

_{corresponding}

decrease in

for-aging

ages. These

possibilities

need to be

rigorously

tested

_{through colony}

manipu-lations that

_{adjust temporal}

caste

the _{presence and absence of both} the _queen and

_synthetic

_QMP.

The

_juvenile

hormone _system also

might

be involved in

_solving

a

_paradox

in

QMP

biology:

what effect does

_QMP

have

on _queens themselves? This issue is

inter-esting

because queens re-absorb 36 % of their own

_daily

_{QMP secretion,}

_likely

across their

cuticles,

but also are

perma-nently

covered

_externally

with

_QMP

at

fairly high

concentrations,

about 1 ×

10

-3

of their

_gland

contents at _{any one} time

(Naumann et al., 1991).

Queens

could

_simply

catabolize or

ignore

their own

_QMP

_message, but the

experiments

demonstrating

QMP

inhibi-tion of JH in workers

(Kaatz

et al.,

1992a;

Pankiw et

_{al., 1998) suggest}

that

_QMP

also could act on adult queens to _suppress

their own JH

_production.

This

_possibility

is

_intriguing

because adult

_honey

bee queens are _{unusual among female}

egg-laying

insects in

_having

low levels

_{of JH,}

except for the first 1-2

_{days following}

adult _emergence, and JH

_apparently

is not

important

in

_maintaining

_egg-laying

_(Fluri

et

_{al., 1981;}

Robinson et

_{al., 1992;}

Fahrbach et

_{al., 1995).}

The _pattern of

_high

JH

_production

for the first 2

_days

of a

queen’s

life,

followed

_by

low levels

there-after,

is

_closely

and

_inversely

related to

the

_QMP

_{production profile}

_{of queens, in} which

_QMP

is low _{upon emergence but} rises

_{rapidly during}

the first 2

_days

post-emergence, and

usually

stays

high

for the

rest of the

_queen’s

life

_(Slessor

et

_{al., 1990;}

Pankiw et

_{al., 1996).}

Of course, this

rela-tionship

between

_QMP

and JH

_production

could be

_entirely

coincidental,

but appears

worthy

of

_{investigation}

to determine whether the queen mediates both her own

and workers’ JH

_{production through}

_QMP.

If so,

QMP

may transcend the

boundary

between

_pheromone

and

hormone,

which would be of considerable

_importance

in

understanding

the evolution and function of social insect semiochemicals.

Another

_significant

function of

_QMP

is the inhibition of _queen

_{rearing by}

adult worker

_honey

bees. Workers

_usually

will

not rear new _{queens in} the _presence of their

_colony’s

_{adult queen, but} removal of the queen and her

_pheromonal

_message deletes the inhibition

_{by QMP}

and leads to

the

_rearing

of new _queens within 12-24 h

(Winston

et

al., 1989, 1990;

Pettis et

_al.,

1995a;

Melathopolous

et

_{al., 1996).}

Work-ers do rear new _{queens in the presence} of the old queen

prior to reproductive

swarm-ing, probably

because

_pheromone

disper-sal is reduced in crowded colonies so that her inhibition is insufficient to _suppress

the queen

rearing

that

_precedes

_swarming

(Winston

and

_Taylor,

1980; Winston,

1987;

Winston et

al., 1991;

Naumann et

al., 1993).

The

_{physiological}

mechanism of

QMP’s

inhibition of _queen

_rearing

is

com-pletely

unknown,

but a useful

_approach

to address this issue

_{might exploit}

_genetic

differences in worker _responses to _queen

pheromone.

Some worker lines are

_highly

responsive

and others

_{respond poorly}

or

not at all to

_QMP

in

_{laboratory bioassays}

(Kaminski

et

al., 1990;

Pankiw et

al.,

1994, 1995, 1996, 1998).

Interestingly,

the

_high

_responding

workers are

consid-erably

more

_likely

to _{be involved in queen}

rearing

than the low

_responders,

for rea-sons we do not _yet understand

_(Pankiw,

1997). However, there

may be

physiolog-ical differences between

_high

and low

responding

workers in

_receiving

or

responding

to the

_{queen’s pheromonal}

message. If so, these differences could

provide

a

_{powerful tool to}

dissect the

neu-rochemical and/or hormonal

_{pathways by}

which

_QMP

_prevents workers from

per-forming

behaviours or

_secreting

the

spe-cialized food associated _{with queen}

rear-ing.

We have been

_conducting

some

sur-vey of brain and hormonal functions in workers

_rearing

and not

_rearing

_queens is needed to more

_{fully explore}

this

impor-tant

_topic.

3.

_QMP

COMPLEXITY AND BIOSYNTHESIS

Another

_key

issue for

_honey

bee

_primer

pheromone biology

is why

QMP

is such a

complex

blend.

_{Functionally,}

we know

that all five _components move

_through

the

colony

as a

unit,

at the same rate

(Nau-mann et

_{al., 1993),}

and all five are

neces-sary to elicit full retinue _response and to

suppress queen

rearing

(Slessor

et

al.,

1988;

Winston et

_{al., 1989). However,}

it is

not clear whether all five

_compounds

are

physiologically

active or whether some

components

are

_{important only}

as attrac-tants. We know that there are

_specific

antennal _receptors for 9ODA

_(Allan

et

al.,

1987),

and that workers whose antennae

have been removed do not

_respond

to

QMP

in

_{laboratory bioassays}

_(L-A

Kamin-ski,

unpublished

observations). Also,

9ODA alone is sufficient to

_inhibit

juve-nile hormone secretion in worker

bees,

and there were no differences in JH levels

between workers

_exposed

to 9ODA alone and those treated with the

_full,

five-com-ponent QMP

blend

(Kaatz

et

al., 1992a,

unpublished

observations).

This

_study

sug-gests that 9ODA _may be the

physiologi-cally

active

_ingredient

that inhibits

forag-ing

ontogeny while the other

_compounds

may be

important

in

enhancing

worker attraction to the _queen or in

_eliciting

worker-worker

_pheromone

transfers. Research

_concerning

_{neurophysiological}

responses to, and the

_{physiological}

effects

of,

separate and combined _components is necessary to confirm this

_hypothesis.

At an

_evolutionary

level,

it is not clear

why Apis mellifera

has five _components to

its

_QMP

blend. This condition seems to

be

_derived,

since other

_{species of Apis}

have fewer

_components

and show less dif-ferentiation between queen and worker mandibular blends. For

_example,

mandibu-lar

_glands

_{of queens from} the more

prim-itive

_{(Smith, 1991)}

_open-nesting

_species

A.

dorsata,

A.

_florea

and A.

_{andreniformis}

contain

_only

the three acid

_components

9ODA and +/-

9HDA,

and lack the

aro-matic HOB and HVA

_compounds.

A.

cer-ana, the other

cavity-nesting species

besides A.

_mellifera,

has four of the five

components

but lacks HVA. In

addition,

mandibular

_gland

secretions from

open-nesting species

show less differentiation between worker and _queens

bees,

and

vir-gin

queens from A.

_mellifera

show the

most

_profound

differences from mated queens of all

Apis

species

(Plettner

et

al.,

1997).

These results

_{provide powerful}

evi-dence

_indicating

the evolution towards

more

_complex

blends and more

pro-nounced

_worker-queen

differences in

honey

bee

_pheromonal

_{systems. However,} the

_implications

of this

_finding

for

under-standing

the evolution of

_highly

social behaviour will not be

_{fully appreciated}

until we have more information

concern-ing pheromonal activity

of

_QMP

in other

Apis species.

Basic

_comparative

studies

concerning

how

_single

and

_multiple

pheromonal

components

influence queen

rearing,

foraging

and retinue behaviour in the _genus

_Apis

would

_{provide key}

infor-mation to

_interpret

how

_pheromonal

inte-gration

of worker and

_colony

functions may have evolved in

honey

bees,

and

per-haps

in all social insects.

Biosynthetic

studies of worker and queen mandibular

gland compounds

in A.

mellifera

and the other

_Apis

_species

also

might

enhance our

_{understanding}

of the evolution of _queen and worker castes.

Queen

and worker bees of A.

_mellifera

each

_produce

a

_{caste-specific}

blend of

functionalized 8 and 10 carbon

_fatty

acids in their mandibular

_glands,

and the

match the

_queen’s

_reproductive

and worker’s

_{non-reproductive}

roles in the

colony.

The

_{biosynthetic pathways}

of these substances result in

_{caste-specific}

mandibular

_gland

blends,

and are

particu-larly

important

in

_determining

the

pheromonal

nature of the

_queen’s

acidic

components

(Plettner

et

_al.,

_1996).

Com-parative

research on the

_biosynthesis

of

acidic

_compounds

_{in queens and workers} of other

_Apis

_{species might}

reveal

signif-icant information relevant to

understand-ing

the evolution and structure of caste

dimorphism

in social insects.

Similarly,

biosynthetic

studies of the aromatic

_compounds

HOB and HVA

might yield important findings

concern-ing

the evolution and function of these substances in the _genus

_Apis.

Studies

involving

HVA

_might

be

_especially

sig-nificant because of the chemical

_similarity

of HVA to the brain neurochemical

dopamine.

The

_biosynthetic

_relationship

between HVA and

_dopamine

is

_interesting

because one functions as a

pheromone

and

the other as a neuromodulator. It is

tempt-ing

to

_speculate

on how a

_biosynthetic

pathway

leading

to a neurochemical such as

dopamine might

have been modified in

A.

_mellifera

to

_produce

the

_pheromone

HVA,

and also whether HVA and

dopamine

have similar effects on brain

functions.

_Obviously,

this research area

is of

_potential

_significance

to

understand-ing pheromone

evolution and

pheromone-neurochemical interactions.

Another area of research interest con-cerns the control mechanisms that

deter-mine whether an adult female

_honey

bee

will

_produce

_queen or worker

_compounds

in her mandibular

_glands.

All

_Apis

_species

retain the

_ability

in workers and _queens

to

_synthesize

the acidic mandibular

com-pounds

of the other caste. Even A.

mellif-era workers will

_produce

_queen

com-pounds

under some

_{circumstances,}

particularly

if

_they

are

_queenless,

and queens

similarly

retain the

capability

to

synthesize

worker substances

_(Sasaki

et

al., 1989;

Plettner et

_{al., 1993, 1996, 1997).}

The mechanisms

_controlling

caste-spe-cific

_biosynthesis

are not

known,

nor do we understand how a worker or _{queen bee}

would switch to

_produce

the mandibular blends characteristic of the other caste.

In

addition,

the functions of the

worker-produced

acidic

_components

have never

been

_clearly

determined. The most abun-dant

_compound

in worker mandibular

glands

is

_10-hydroxy

decenoic acid

(10-HDA),

and it is

_commonly

believed that the functions of this substance are

nutri-tional,

as a

_component

of brood

food,

and

medicinal,

as an antibiotic

_(Winston,

1987). However,

10-HDA is most abun-dant in older workers of

_foraging

age

(Plet-tner et

_{al., 1997),}

_suggesting

a

foraging-related

_function,

_possibly

as a

primer

pheromone

involved in

_regulating

the _ages

at which workers

_begin

to

_{forage (Huang}

and

Robinson, 1992;

Pankiw et

_{al., 1998).}

If so, 10-HDA and/or other

worker-pro-duced mandibular

_compounds

could be the first

_{primer pheromones recognized}

from

_honey

bee workers.

_Clearly,

the

nature and function of worker mandibu-lar

_compounds

that are

_{chemically closely}

related to _{queen substances is} a research area

_deserving

considerable attention.

All of these

_{investigations}

would pro-vide information useful to resolve the issue of whether _{queens control} worker activi-ties or workers

_cooperate

in

_pheromonal

transfer and

_activity.

The control

hypoth-esis _{proposes that the} evolution of

multi-component

queen

pheromone

has been an

’arms race’ in which the _{queen has} evolved new

_components

to increase her control over worker activities

(Winston

and

_{Slessor, 1992).}

_{In contrast, it may be} in the workers’ interests to

_cooperate

with the queen in

pheromone

transfer even

though

that

_cooperation

results in

pre-venting

their own

_reproduction

(Keller

and Nonacs,

1993).

Data to date are not

but an increased

_{understanding}

of

pheromonal

evolution

_might

_prove

espe-cially

useful in this context.

4. OTHER

_QUEEN

PHEROMONES

It is evident that there are other

queen-produced pheromones

in addition to

_QMP,

but their

_glandular

sources, chemical

iden-tities and functions remain obscure. The existence of other

_pheromones

can be

deduced from the _gap between the effec-tiveness of

_synthetic

_QMP

and that of the queen in

effecting

worker behaviours

(Winston

et

al., 1989;

Winston and

Slessor, 1992; Slessor et al., 1997).

How-ever,

synthetic

QMP

is identical in

activ-ity

to extracts from _{queen mandibular}

glands, indicating

that there are no addi-tional

_pheromonal

substances in these queen

glands

and that the sources of the

queen’s

additional

_{pheromonal activity}

lie elsewhere

_{(Slessor et al.,}

1988,

1997).

Var-ious sources for

pheromonal

queen

sub-stances have been

_{proposed, including}

ter-gite,

tarsal and Dufour’s

_glands.

The

_queen’s

_tergite

_glands

have

_long

been considered

_potential

_pheromonal

sources

_[reviewed

_by

Slessor et

_{al. ( 1997)].}

Proposed

functions for these

_glandular

secretions have included

_attracting

drones

or workers to the _{queen in} concert with

QMP

(Gary,

1961;

Vierling

and Renner, 1977; Smith

_{et al.,}

_{1993), recognizing}

kin

(Moritz and Crewe, 1988, 1991),

and other

primer pheromone

functions

_(Renner

and

Baumann, 1964; Velthuis, 1967, 1970).

Virgin

and young queen

tergite glands

contain considerable

_quantities

of decanoate esters,

especially decyl

decanoate,

that have been isolated and identified from the

_gland

_(Espelie

et

al.,

1990),

but their

_activity

has not been tested for

_specific

functions. In

_{addition, the}

ter-gite glands

do not _{appear to} be

_responsible

for

_attracting

workers to _{their queen}

(Slessor

et

_{al., 1997). Thus, the}

functions

of

_tergite

secretions remain

_speculative,

and the link between identified

_compounds

and functions is _as-yet unknown.

Queens

also

_deposit

substances on

comb that _may inhibit _queen cell

con-struction,

possibly

originating

from tarsal

glands

of mated _queens

_(Lensky

and

Slabezki, 1981). These

so-called

_footprint

pheromones

could be

_QMP,

since _queen mandibular

_pheromone

is

_{deposited by}

queens as

_they

walk on comb in

quanti-ties that could _{suppress queen}

_rearing

(Naumann

et

_{al., 1991). Nevertheless,}

research has not _yet ruled out the

_potential

existence of tarsal

_{gland footprint}

pheromone,

and if it does exist the func-tions of its active

_ingredients

remain

unex-plored.

A third queen

pheromone

appears to

be

_produced

in the Dufour’s

_gland,

and is

deposited

on _eggs as

_they

are laid

_by

the

queen

(Ratnieks,

1995).

The

proposed

function of this secretion is to

_identify

eggs as

_queen-laid

so

_{that they}

are not

con-sumed

_by

adult worker

bees,

as worker-laid eggs

frequently

are.

_Again,

_however,

the

_glandular

source, chemical

identity

and functional

_activity

of this

_pheromone

need to be confirmed and further

exam-ined.

Perhaps

the most

_interesting

of the unknown queen

pheromones originates

in the

head,

in a non-mandibular source.

Extracts from _{queen heads}

_provide

almost twice the attractiveness to workers than

QMP

alone,

and recent research has demonstrated that this additional

attrac-tion is not due to mandibular

_gland

com-ponents, either known or unidentified

(Slessor

et

_{al., 1997).}

We are

_currently

working

to

_identify

the

_compound(s)

and

glandular

source of this

secretion,

but as

for the other

_proposed

_queen substances its

_identity,

source and function remain

speculative. Clearly, there

are

_significant

A more fundamental issue concerns

why

the queen

might

need to

_produce

another

_pheromone.

One

_possibility

is that

an unidentified _queen

_pheromone,

espe-cially

from the

head,

might

be

_important

in

attracting

workers to attend the _queen.

QMP

is removed from queens

by

worker bees who attend her in a

_dynamic

retinue,

licking

and

_antennating

the _queen to obtain

pheromone

from her

_body.

The retinue bees attend the _queen for seconds to a few

minutes,

then

_{disperse through}

the

_colony

as _messengers that transmit her chemical

message to other workers

_through

antennal and

_mouthpart

contacts over a

_period

of

15 to 30 min

_(Seeley,

1979; Naumann,

1991;

Naumann et

al., 1991, 1992;

Wat-mough,

1996).

In

addition,

the queen and messenger bees

deposit

a

_relatively

minor

amount of

_pheromone

on the comb as

_they

walk

_through

_{the nest, which may then be}

picked

up

by

other workers

walking

on

the

_{pheromone-impregnated}

comb

(Nau-mann et

_{al., 1991).}

Our initial

_findings

_suggested

that

_QMP

was the retinue attractant, but

_subsequently

we discovered the existence of

low-responding

lines of worker bees that exhib-ited

_virtually

no _response to

_QMP

in

labo-ratory

bioassays (Kaminski

et

al., 1990;

Pankiw et

_{al., 1994, 1995).}

_Oddly,

workers that have no _response to

_QMP

will

never-theless form

_{normal-appearing}

retinues around the _queen, and their colonies show

no _apparent differences from colonies made up of

high-responding

workers

(Pankiw

et

al., 1995).

These observations _suggest

_that,

although QMP clearly

is attractive to

worker

bees,

it _{may be} of

_secondary

impor-tance in retinue formation

_compared

to an

unidentified _queen

_pheromone.

5.

_QUEEN

AND BROOD

PHEROMONE INTERACTIONS

Another

_possible

function of unidenti-fied queen

pheromones

is the

_suppression

of worker ovary

development.

This area

of

_pheromone

_biology

has been

_seriously

under-studied,

and is of unusual

signifi-cance because it appears to involve the interaction between queen

pheromones

and

_{brood-produced}

substances. There is

strong evidence

_indicating

that _brood pro-duces the

_primary

_signal

that inhibits worker _ovary

_development,

with a _queen

pheromone

providing

a

_{secondary signal,}

but this

_pheromonal

concert remains

unex-plored. In

addition,

brood _appears to

pro-duce a

_{secondary signal inhibiting}

_queen

rearing,

but the chemical nature and

bio-logical

function of this

_putative

_pheromone

is obscure.

The influence of brood in

_preventing

worker _ovary

_development

was first

described

_{by Jay}

_{(1970, 1972)}

in a series

of

_{elegant experiments}

that remain the benchmark in this field. His work involved

presenting

colonies with various combi-nations of extracts _{from queens and brood.}

The results demonstrated that both brood and the queen were _necessary to inhibit ovary

development in

adult worker

bees,

and that this effect had a

_pheromonal

basis.

In

addition,

Jay

found that the brood

sig-nal alone

_provided

a

_stronger

_inhibitory

effect than _{the queen}

_pheromone.

Subse-quent

research

(Willis

et

_{al., 1990;}

Kaatz

et

_{al., 1992b)}

added the

_surprising

find-ing

that

_QMP

was not the _queen

pheromone

that

_prevented

_{worker egg}

lay-ing. Virtually

no _{other progress} has been made in

_identifying

the brood

_pheromone

or in further

_exploring

the interaction

between queen and brood substances.

However, there

have been some

tanta-lizing

reports that

_suggest

further

explo-ration of the mechanisms involved in worker ovary inhibition

_might

reveal a

complex pheromonal

control _system with

profound implications

for our

under-standing

of social insect

_colony

organiza-tion.

_{Ovary development}

in insects involves two

_{processes, the production}

of

development

and _egg

_production

itself. It appears that these two _processes may be mediated

_by

different

_pheromonal

_systems in the

_honey

bee.

_Preliminary

work

sug-gests that _{queens may} have an

_important

function in

_suppressing

the

_synthesis

of

vitellogenin, possibly

due to

_QMP

_activity

(Kaatz

et

_{al., 1992b),}

while brood is

_likely

to be more

_significant

in

_suppressing

ovary

development

and egg

laying

in workers

(RW Currie,

unpublished

observations).

Further work in this area will

_provide

important

information

_addressing

two of the

_key

issues in social insect

_biology,

the

proximate

nature of the

_signals

_mediating

reproductive

differences between _queens and workers and the ultimate

_{pathways by}

which

_colony

structure evolved.

A second interaction between _queen and brood

_pheromones

involves the inhi-bition _{of queen}

_{rearing. Surprisingly,}

there is an inverse

_relationship

between the number of queens reared and the

quantity

of _young brood

_provided

to workers in

queenless

colonies

_(Pettis

et

_{al., 1997).}

This result was

_unexpected

because work-ers use the _younger larval _stages to initiate queens, yet the addition of _{young larvae} to

colonies _{suppresses queen}

_rearing.

One

explanation

for this

_phenomenon

is that workers receive two

_signals

to inhibit queen

rearing,

one from

_QMP

and a

sec-ond from young brood. Pettis and his

co-authors have

_proposed

that the _queen

sig-nal is more

_important

in

_mediating

_queen

rearing

associated _{with queen loss and}

swarming,

whereas a decline in the brood

signal

may initiate queen

supersedure.

Again,

however,

this

_brood-queen

inter-action remains

largely

unexamined.

6. PHEROMONE DISPERSAL AND WORKER CONGESTION

One of the most

_interesting

_aspects of

honey

bee _queen

_pheromone

is that it does

not

_prevent

_queen

_{rearing prior}

to

swarm-ing,

in

_spite

of the

_queen’s

_{presence in the}

colony.

Two

_hypotheses

have

_developed

to

explain

this

_paradox:

1)

queen

pheromone

production

declines

_prior

to

_swarming,

and

2)

colony crowding

interferes with

the movement _{of queen substances}

through

the nest. The _queen

_production

hypothesis

appears

unlikely,

at least as far as

_QMP

is

concerned,

since the level of

QMP

produced by

pre-swarming

queens is identical to the level found in

non-swarm-ing

queens

(Seeley

and

Fell, 1981).

Recently,

we

_provided

evidence that

queen

pheromone dispersal

does

change

in colonies that are

_beginning

to become crowded with adult workers

_(Naumann

et

al., 1993).

We tracked the movement of tritiated 9-ODA contained in

_QMP

in crowded versus uncrowded

colonies,

and found that about 90 % of uncrowded workers had detectable amounts of

_QMP

radiolabel,

whereas

_only

about 45 % of workers in crowded colonies showed detectable

_quantities.

However,

_population

size and conges-tion did not appear to have an effect on

the relative rates of movement from the

center to the

_periphery

of the nest. That

is,

congestion

did not

_change

the

_pattern

of

pheromone

distribution;

workers that had received

_pheromone

had similar amounts

on their bodies in both crowded and

uncrowded

_situations,

and showed simi-lar distribution

_patterns

within the nest.

Rather,

the effect of

_{crowding appeared}

to be in the

_proportion

of workers not

encountering pheromone

rather than in its distribution. This result suggests that pop-ulation size rather than

_congestion

is

important

in

_diluting

worker contacts with queen

pheromone,

leading

to diminished

efficacy

of the adult

_{queen’s inhibitory}

influence on the

_rearing

of new _queens.

This

_study

raised a number of

_important

issues

_concerning

_{queen influence} on

worker activities.

First,

the

_question

of

population

versus

_congestion

needs to be

populations

and

_degrees

of

_congestion.

We

_only

examined a level of

crowding

characteristic of colonies

_prior

to the

com-mencement of

_pre-swarming

_queen

rear-ing.

Also,

our

_study

tracked the movement

of radiolabel in

colonies;

breakdown of

pheromone

into non-active

_components

might

have influenced our

_conclusions,

and a more definitive resolution of this

issue awaits

_techniques

that can track

pheromone compounds directly.

In

addi-tion,

the eventual identification of other queen

pheromones might

reveal a different

distribution _pattern in nests, and

require

a new

_{interpretation}

of the relative

signif-icance of

_population

size versus

conges-tion.

_Finally,

_queen

_rearing

_in

pre-swarm-ing

colonies also could result from a

change

in worker _response to queen

pheromone brought

on

_by

other

factors,

such as season,

_congestion,

or others. All

of these issues remain

unresolved,

but are

important

to understand the

_biological

basis of

_swarming

and to

_develop

man-agement

techniques by

which

_swarming

might

be

_delayed

or

_prevented.

7. COMMERCIAL APPLICATIONS OF HONEY BEE PHEROMONES FOR BEEKEEPING AND CROP POLLINATION

A final research area that

_begs

atten-tion is the

_application

of

_honey

bee

pheromones

for

_beekeeping

_{and crop}

pol-lination. The broad

_body

of fundamental

knowledge concerning

queen

pheromone

has led to a number of recent

_management

applications. QMP

has been formulated under the trade names ’Bee Boost’ for

bee-keeping

use and ’Fruit Boost’ as an attrac-tant for _crop

_pollination,

and both

prod-ucts are

_being

used

_commercially

for these

applications.

For

_example,

’Bee Boost’ has proven effective at

_enhancing

_mating

success and queen survival in commercial

queen

rearing operations

and as a

substi-tute _{for queen bees in}

_shipping

bulk

pack-ages of worker bees

long

distances

(Nau-mann et

al., 1990;

Pettis et

al.,

1993;

Win-ston and

Slessor,

1993).

’Fruit Boost’ is

being

sold as a

_honey

bee attractant for

use on

_highbush

blueberries,

_pears, some

apple

varieties and

kiwifruit,

and can

enhance fruit size and

_yield

_{significantly}

enough

to increase _grower income

$ 1 000

to

$4

000 US _per acre

_(Currie

et

al.,

1992

a,

b;

Winston and

Slessor, 1993;

Naumann

et al., 1994).

It is

_only

within the last 10 _{years that}

honey

bee

_{synthetic pheromones}

have been sold

_{commercially, primarily}

as

worker attractants to catch swarms

(Schmidt et al.,

1993;

Winston

_{et al., 1993)}

and

_pollinate

_crops

_(Currie

et

al., 1992a,

b;

Naumann et

al., 1994;

Winston and

Slessor, 1993).

Pheromone sales for these functions have reached

economically

noticeable

_levels,

but there is consider-able

_potential

for

_{significant expansion}

in the use of

_pheromones

for these and other

purposes.

For

_example,

the addition of

_synthetic

QMP

to colonies can

_prevent

_swarming

for _up to 2 months

_(Winston

et

al., 1991),

providing

a

_potential

swarm control

tech-nique

that could cost as little as

_$3

US

annually

per

colony.

However,

beekeepers

need an

_appropriate

release device that

would

_dispense

_{the proper}

_daily

pheromone

dose over a 1-2 month

period

in order to take

_advantage

of

_QMP’s

swarm-preventing

properties.

This _type of research is

_challenging

_{because any} release device must not

_only

have the proper

biological

effect,

but its

purchase

price

must be

_inexpensive

_enough

to make it a viable

_product.

The

_impact

of a

suc-cessful swarm control

_technique

could have real

_utility

for

_honey

bee manage-ment, but success in this area will

_require

a

_strong

interaction between

_private

indus-try

with

_expertise

in

_pheromone

release

com-mercial

_{applications.}

We have

_barely

begun

to

_explore

the wide

_variety

_{of crops} that

_might

benefit from attractant _sprays

that enhance

_pollination,

as well as modes of

_application

or formulations that would

improve

the

_efficacy

of

_pheromone

attrac-tants. Both worker Nasanov

_pheromone

(formulated

and sold under the label ’Bee

Scent’)

and

_QMP

_(’Fruit

_Boost’)

have been used

_separately

in

_commercially

available attractant

_products,

but to date

no one has tested the two

_pheromones

used

_together

as an attractant for

pollina-tion.

_Queen

or other

_{pheromones applied}

within the

_colony

could benefit

beekeep-ers

_by

_{increasing pollen}

and/or

_honey

pro-duction,

but

_application

methods,

dosages,

blend

_composition

and the

_colony

condi-tions under which

_pheromones

would be beneficial remain as areas for future work.

Finally,

’Fruit Boost’ works within a

very narrow

_dosage

_range

_(Currie

et

al.,

1992a, b)

outside the

hive,

without a

demonstrable

_{dose-dependent}

_response. In contrast, within-hive functions such as

queen

rearing

exhibit the

expected

dose-dependent

response over a wide

_dosage

range

(Winston et al.,

1989, 1990, 1991),

yet

unusually high

concentrations of

_QMP

have no effect on unrelated worker behaviours

_(Pettis

et

_{al., 1995b).}

A

com-plete

assessment of

_{dose-response}

rela-tionships

and thresholds for various behaviours

_might

be a fruitful area of research for _sensory

_{physiologists}

inter-ested in behaviour to consider.

8. CONCLUSIONS

We have

_attempted

in this article to

describe some of the

_challenges

_remaining

in the field of

_{primer pheromone}

biology,

especially

with _queen

_pheromones

but also with

_{primer pheromones produced by}

brood and adult worker bees. To

date,

we

know the

_identity

of

_only

one _queen

pheromone,

while other _queen substances

as well as brood and worker

_pheromones

remain unknown. We know a consider-able amount about the functions in which these

_pheromones

are

_active,

but the

modes of

action,

_{physiological}

effects and

precise

chemical nature of

_specific

pheromonal

activities remain as

_subjects

for future research. There

_clearly

are

com-mercially

useful

_applications

for research in these areas, but our basic

_knowledge

concerning honey

bee

_pheromones

will need to

_expand

_dramatically

to take full

advantage

of these substances for

bee-keeping

and _crop

_pollination.

Our

_experience

in

_pheromone

research has led us to one final conclusion that _may appear

obvious,

but is worth

_emphasizing

nonetheless. This _type of work is

_truly

interdisciplinary,

and

_requires

a _strong and

mutually supportive

interaction between

honey

bee

_biologists

and semiochemists.

Progress

in our

_{understanding}

of the role

pheromones play

in

_colony

_{organization,}

and the commercial

_applications

of this

knowledge,

are not

_possible

without a true

meeting

of the

_biological

and chemical fields. Science has

_{always progressed by}

taking

new

_approaches

to

_problems

based

on

_{interdisciplinary}

interactions,

and the

future of

_pheromone

research will be a

strong and

_fascinating

one

_only

if we

_bring

to bear the diverse

_approaches

that can come from

_crossing

disciplinary

bound-aries.

ACKNOWLEDGMENTS

We

_{gratefully acknowledge}

the contribu-tions of the numerous

_{undergraduate}

and

grad-uate students,

postdoctoral

fellows, technicians and

_colleagues

who have

_participated

in our

research program. Heather

Higo

read the

manuscript

and

_provided

useful comments. We also

_{deeply appreciate}

financial _support pro-vided

_by

_grants from the Natural Sciences and

Engineering

Research Council of Canada, the Science Council of British Columbia, the British Columbia

_Ministry

of

_Agriculture,

Producers Association, the Canadian

_Honey

Council, and Phero Tech Inc.

Résumé - Les

_phéromones

modifica-trices et

_{l’organisation}

de la colonie : lacunes dans nos connaissances. Le but de cet article est de discuter les lacunes dans nos connaissances sur la

_façon

dont

les

_phéromones

modificatrices influent

sur les activités des ouvrières et les fonc-tions de la colonie d’abeilles

_(Apis

melli-fera

L.).

Les

_phéromones

modificatrices,

en

_agissant

sur la

_physiologie

des

ouvrières,

exercent une influence sur la

reproduction

des ouvrières et de la

colonie,

sur la structure des castes et sur la divi-sion du travail. L’abeille est pourtant le seul insecte social chez

_lequel

une

phéro-mone modificatrice

_{ait jamais}

été identifiée

chimiquement ;

il

_s’agit

de la

_phéromone

de la

_glande

mandibulaire de la reine

(QMP).

Nous décrivons d’abord sa struc-ture

_chimique

et ses

_fonctions,

_puis

souli-gnons les domaines

qui

présentent

un

inté-rêt

_potentiel

_pour les recherches à venir. Nous discutons le mode d’action de la

QMP

sur les ouvrières et examinons le rôle de l’HJ comme médiateur de l’action

de la

_QMP.

Nous abordons ensuite la

question

de savoir

_pourquoi

la

_QMP

est

un

_mélange

_complexe

de

_{cinq composés.}

Pour

_{expliquer la complexité}

de cette

phé-romone chez le _genre

_Apis

nous propo-sons un chemin

_{évolutif qui suggère}

_que le nombre accru de

_composés

et les

diffé-rences entre les reines

_vierges

et les reines fécondes sont des

_{caractéristiques}

déri-vées. Nous discutons les différences

bio-chimiques

entre les

_glandes

mandibulaires des ouvrières et celles des reines. Nous

présentons

ensuite _{les preuves} de

l’exis-tence de

_{phéromones royales}

autres _que

la

_QMP

et démontrons l’existence d’un

autre

_{complexe phéromonal}

_{sécrété par}

des

_glandes

non encore identifiées dans

la tête des reines. Nous étudions aussi la

façon

dont

_{interagissent}

la

_phéromone

royale

et la

_phéromone

de couvain _pour

influer sur la

_biologie

de la colonie et

dis-cutons

_{l’importance}

relative _{de la} conges-tion de la colonie et de la taille de la popu-lation dans la transmission de la

phéromone.

Enfin,

nous

_suggérons

quelques pistes

de recherches

_susceptibles

de donner lieu à des

_applications

com-merciales des

_phéromones

_{d’abeilles pour}

l’apiculture

et la

_{pollinisation}

des cultures. ©

Inra/DIB/AGIB/Elsevier,

Paris

Apis

mellifera

/

_phéromone

modifica-trice /

_{pheromone royale}

/

_intégration

colonie

Zusammenfassung - Primerpheromone

und

_{Kolonieintegration}

bei

Honigbie-nen: Lücken in unserer Kenntnis. In diesem

_Beitrag

sollen Lücken unseres

Kenntnisstandes über die Funktion von

Primerpheromonen

in der

_Vermittlung

zwischen den Aktivitäten der

Arbeiterin-nen und den Funktionen des Bienenvolkes diskutiert werden.

_{Primerpheromone}

wir-ken auf die

_Physiologie

der Arbeiterinnen ein und beeinflussen hierdurch mittelbar die

_Reproduktion

der Arbeiterinnen und des

_Bienenvolks,

die Struktur der Kasten und die

_{Arbeitsteilung. Honigbienen}

sind die

_einzigen

sozialen

Insekten,

für die

jemals

ein

_{Primerpheromon}

chemisch identifiziert wurde. Dies ist das Pheromon der Mandibeldrüsen der

_Königin

_(QMP).

Wir beschreiben zunächst die chemische Struktur und die Funktionen des

_QMP

und stellen danach

heraus,

welche Gebiete von

möglicher Bedeutung

für

_zukünftige

Untersuchungen

sind. Wir

diskutieren,

auf welche Weise

_QMP

auf die Arbeiterinnen einwirken könnte untersuchen die

mögli-che Rolle von Juvenilhormon als

Akti-onsvermittler von

_QMP.

Danach

_gehen

wir auf die

_Frage

ein,

warum

_QMP

eine

komplizierte

Mischung

aus fünf

ver-schiedenen chemischen

_Komponenten

ist. Hierbei

_schlagen

wir einen

_möglichen

Evolutionspfad

zur

_Entwicklung

der