A study of rapeseed (Brassica napus L. var. oleifera Metzger) flower nectar secretions

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A study of rapeseed (Brassica napus L. var. oleifera

Metzger) flower nectar secretions

J. Mesquida, R. Marilleau, M.H. Pham-Delègue, M. Renard

To cite this version:

A

STUDY OF RAPESEED

(BRASSICA

NAPUS

L. VAR.

OLEIFERA

_METZGER)

FLOWER

NECTAR

SECRETIONS

Jacques

MESQUIDA René MARILLEAU * Minh-Hà PHAM-DELEGUE * Michel RENARD

*

1.N.R.

A

.,

Centre de Recherches de Rennes Laboratoire de

_Zoologie

Domaine de la Motte au Vicomte B.P.

29,

F 35650 LE RHEU

**

LN.R.A., C.N.R.S.

Laboratoire de

Neurobiologie Comparee

des lnvert!bri’s La

_Guyonnerie

F 91440 BURES-SUR-YVETTE

***

I.N.R.A., Centre de Recherches de Rennes Station d’Am!lioration des Plantes Domaine de la Motte au Vicomte B.P.

29,

F 35650 LE RHEU

SUMMARY

Two nectar extraction

techniques, micropipetting

and

_{centrifugation,}

were

_compared

to establish a

reliable basis for

_analysis

and

_comparison

of nectar

_production

of

_rapeseed

lines under selection. All tests

used Kid cultivar.

Apart

from

_quantitative

measurements for nectar

_production,

nectar

_{glucid composition}

was assessed

_by

_gas

_{chromatography. Centrifugation provided larger quantities}

of

_{liquid (x}

4 -

_6),

but total

_glucid

content was not

_greater

than for

_{micropipetting. Centrifugation}

thus

_artificially

diluted nectar

and

_produced

_samples

_{unrepresentative}

of those

actually

encountered

by

insects. Glucose and fructose fractions were _{very similar}

_using

either method.

_{Micropipetting}

is therefore recommended for future work. Such

_sampling

may

provide

the basis for

_selecting

_{oilseed rape lines for} nectar

_quantity

and

_quality

to

_optimize

bee visits and cross

_pollination.

INTRODUCTION

Oilseed rape

(Brassica

napus L.

var

oleifera

M

ETZGER

)

is

partially

auto-gamous

(70 % ,

S

YLVEN

,

1920 ;

R

I

vES,

1957 ; M

ORICE

,

1960 ;

R

UD

-LOFF

&

_S

_CHWEIGER

_,

_1984),

but its abundant

nectar

and

_pollen

attract

numerous

insect

_pollinators.

Various studies

of

_{rapeseed pollinating agents}

have

indicated

that the

_plant

benefits from insect visitation

_(FREE

_{& Nurr}

_ALL

_,

_{1968 ;}

W

ILLIAMS

M

ESQUIDA

&

_R

_ENARD

_,

1981,

1982).

Insect

_pollination

has

more

effect

on

_plant

phenology

than

on

seed

_production

(R

ENARD

&

_M

_ESQUIDA

_,

_{unpublished data).}

Better

_{understanding}

of the

role

of

_pollinating

insects and factors

_affecting

plant

insect relations is

of

_great

_importance

to

Fl

_hybrid

seed

_{production using}

male sterile lines.

Field

_experiments

have shown that

_{honeybees (Apis mellifera L.)}

are

the

main

_pollinators

_(T

_ASEI

_,

_{1977 ;}

M

ESQUIDA

&

_R

_ENARD

_,

_1978,

1979

_(a),

1979

_{(b) ;}

R

ENARD

&

_M

_ESQUIDA

_,

_1983).

_Moreover,

_foraging

_honeybees

_{may show}

a

pre-ference

for

male fertile lines

as

_opposed

to

male sterile

lines

_(cytoplasmic

«

O

GURA

»

_type

male

sterility).

The

difference may reflect lower

nectar

secre-tion in

these

male

sterile

lines.

_Quality

and

_quantity

of

nectar

_production

have

been shown

to

be

_significant

for

selective

_foraging

behavior in

_honeybees

(M

ASSON

,

1983).

Therefore,

reliable

_techniques

for

_assessing

nectar

_production

of selected

lines

are

needed

if

_{entomophilous pollination}

is

to

be

_fully

assessed and

exploited

in

_selecting

Fl

_hybrid

seed

_parental

lines. The

_objective

is

to

equalize

bee distribution between male sterile and male fertile lines in

field

crops.

This

_paper

_compares

nectar

collection from

_{rape flowers}

_using

centrifuga-tion

versus

_glass

_{micropipettes.}

In

addition

to

_quantitative

_analysis

of

nectar

production,

nectar

_glucidic

_component

was

also

determined.

The method used

for

_{glucidic composition analysis}

was

derived

from

one

_{previously applied}

to

sunflower and oilseed rape

(F

ONTA

et

_{al., 1985 ;}

L

E

M

ETAYER

_pers.

_comm.).

MATERIALS AND METHODS

Plant material

All

_experiments

were done

_using

a winter cultivar

_Kid,

at the LN.R.A.

_experimental

station

(Rennes, France).

Samples

were collected in the

_{morning (8}

h

_GMT)

on

_{freshly opened}

_flowers,

on two

dates in

_April

1983. Plants in full flower were

_bagged

24 hrs

_{prior sampling}

to

_prevent

nectar collection

by foraging

insects.

Nectar extraction

_{by centrifugation}

Nectar was _{extracted from five flowers per}

_plant

on 15

_{April (first}

collection,

20

_plants)

and on 20

April (second

collection,

10

_plants).

_Sampling

and

_{centrifugation}

at 3 000 r.m. followed methods described

_by

Bosi

_(1973).

Nectar extract

_weight

was calculated from the difference in

_weight

before and after

_{centrifugation.}

Nectar extraction

using micropipettes

Glass

_{micropipettes (internal}

diameter 1 mm,

volume

5

_wl.),

were used to obtain

_samples

from up

Volumes of nectar collected were read

_directly

from

_micropipette

scales,

and

_samples

were then

weighed

before freeze storage

(—

25°

C)

for later

_analyses.

After

_micropipette

collection,

up to 5 flowers from 6

_plants

were

_subjected

to

_{centrifugation}

in the second collection.

Analysis of

nectar

_{glucid composition}

a)

Sample

preparation

Nectar was

_{partially dehydrated}

under vaccum

_overnight

_{in the presence of}

_{di-phosphorous}

pentox-ide,

after

_determining

flower nectar

_{weight. Trimethylsilylesters}

were

_{prepared following}

BROHST & Lorr

(1966)

and Bosi

_(1973),

the

_{sample being dehydrated}

in

_pyridine

_{(approximately}

1

_ml)

with addition of 0.9 ml of

_{hexamethyldisilazane}

and 0.1 ml of trifluoroacetic acid in that order. The solution was shaken for a few

_minutes,

then left for 12 hours to ensure

_complete

derivation of trisaccharides.

b)

Preparation of

standard

_reference

solutions

Two mg of reference sugars

(glucose,

fructose or

_sucrose)

were dissolved in 1 ml of

_pyridine,

and

dehydrated

as for

_samples,

so _{that sugars} were then at 1

I1g/1

concentrations.

c)

Chemical

_analysis

A GIRDEL 75 gas

chromatograph

equipped

with a pyrex

glass

column

₍₃

m x 2

_mm)

was

used,

filled with a

_{stationary phase,}

_type

OV 17 at 3 % on Chromosorb WHP. Column

_temperature

was

program-med from 185° C to 280°

C,

at 2° C/min. Detector

_temperature

was _{300° C. Carrier gas} was

_nitrogen

at 2

kg/cm

2

and a 24 ml/min flow.

Detector

_signals

were treated

_by

a HOUSTON

integrator

recorder,

recording

speed

5 mm/min.

Injected

volumes were 2

1

11.

Standard solution was

_injected

after each series of 4 or 5

_samples.

d)

Calculation

_of

_sugar

_proportions

_(following

_{Bosi, 1973)}

i.

_{Proportion of dry weight}

attributable to _{each sugar}

Ai :

_peak

area of a

_given

_{sugar in standard}

_{solution ;}

fi : response coefficient calculated from a standard

_solution,

relative to one _{of the reference sugars.}

Ar : area _{of reference sugar}

_peak

in standard solution ;

A’i: area of

_sample

_{sugar in standard solution.}

ii. Total sugar concentration

Considered to be

_equal

to nectar

_{dry weight}

since amino acid and other nectar _{components represent}

less than 0.03 % of nectar

_{dry weight}

for most flower nectars

_(BAKER,

cited

by

H

EINRICH

,

1975).

Nectar

_{dry weight}

was obtained from

V = dilution

volume ;

iii. Concentration

_of

_{each sugar in} nectar :

Data treatment

Effects of

_sampling

method and

_sampling

date,

respectively,

were

_{analyzed using}

Student’s t test

after

_checking

for

_{applicability (D}

_AGNELIE

_,

_1970).

RESULTS

Nectar

_production

Nectar

_weight

_per

flower

_(mg)

is shown in Table 1.

_{Centrifugate sample}

volumes

were

_always

4

to

6 times

_greater

than

for

_{micropipettes. Sample}

volumes

obtained

_{using micropipettes}

were

similar for

the

two

collection

dates,

but differed

_{significantly}

for

_{centrifugated samples.}

Glucid

content

The

two

_principle

_{sugars identified}

_{by chromatographic analyses}

were

glucose

and fructose.

o

Nectar sugar concentration

Sugar

concentration in

nectar

_{sampled by micropipette}

was

between

40

%

centrifu-gated

samples,

and

this

was

also

so

_{for each sugar}

considered

_separately

(tables

2b &

_2c)..There

was no

_significant

effect

of

_sample

date

for either

o

_{Sugar proportions-dry weight}

Glucoce

_{(Table 3)}

content was

_higher

than

that of fructose

_by

about

3

% -

4

%.

There

were

_slight

differences in estimates

for different

_{sugars with}

date and method

of

_collection,

but

no

_significant,

_except

for

centrifugated

samples

in the

second collection.

o

_Sugar

content in

nectar

_produced

_per

_flower

Table 4 shows

no

_significant

difference

between either

dates

or

_sampling

methods for

_{dry weights,}

or

fructose

or

_glucose

contents.

_{Total sugar}

_weight

in

nectar

_produced

_{per flower}

was

approximately

0.4 mg

(Table 4a).

o

_{Centrifugation after}

_micropipette

collection

Although

an

appreciable

amount

of

_liquid

was

extracted

by centrifugation

DISCUSSION

The whole sugar fraction of

nectar

was

obtained

by micropipetting,

although

four

to

six times

more

_liquid

was

extracted

by centrifugation.

Conse-quently, centrifugation

diluted sugar fractions in

samples

relative

to

real

content.

An

effect

on

collection method

on

estimation of

nectar

_production

was

_already

noted

for sunflower

_by

M

ADEUF

(pers. comm.)

and G

IRNIK

(1976)

who

_{preferred micropipette}

collection

to

both

_{centrifugation}

and

_washing.

Other methods for

_measuring

nectar

secretion

have

been described. S

WANSON

and S

HUEL

₍₁₉₄₉₎

considered

a

volumetric

_{centrifugation}

method

to

be reliable

for

_yield

data with

_large

scale

_sampling.

LivTZEVA

_{(1954) compared}

several

nectar

collection

methods,

and concluded

that

_{filter paper}

was

least reliable

(due

to

_evaporation)

as

_compared

with

pipette

or

_washing

methods.

Water

1893 ;

K

ENOYER

,

1917),

since

extract

_sugar

content

_{may include sugars}

lixivi-ated from

_plant

tissue cells. Most authors consider

_micropipette

collection

to

be

_preferable,

since

it

_{requires only}

the

_{simplest equipment}

and

is reliable

(V

ANSELL

et

_{al., 1942 ; V}

_ANSELL

_,

_{1943, 1944 ;}

S

WANSON

and

S

HUEL

,

1949).

Proportions

of fructose and

_glucose

in Kid

_variety

nectar were

_very

similar,

but

no

trace

of

sucrose was

found,

_althought

the latter has been

identified

at

low titres in other oilseed

_rape

varieties

_(P

_ERCIVAL

_,

_1961).

Such differences in estimates of

nectar

_{glucid composition}

_may

be

related

to

differences

in

_sensitivity

_{of gas}

_{chromatography techniques applied,}

but

a

varietal effect

cannot

be

excluded. It

_might

then be

_possible

to

detect and

define

nectar

_{glucidic compositions specific}

to

oilseed

_rape

_genotypes

as

sug-gested

by

F

ONTA

et

al.

₍₁₉₈₅₎

in

reference

to

sunflower.

Since

nectar

_sugar

content, and

notably

that of

sucrose can

be

critical

for bee

_foraging

pre-ferences

_(P

_HAM

_-D

_ELEGUE

et

al. ,

1985),

screening

of

nectars

of

male fertile

and

male sterile

lines

_{for both sugar}

content

and

_{glucid composition}

_{may contribute}

to

_optimization

of

cross

_pollination.

In

_conclusion,

even

_though

no

_single

method is

_satisfactory

for all

_plants,

nectar

collection

_{using micropipettes}

is

_preferred

to

_{centrifugation}

for

_study

of

rapeseed

flower

nectars.

The latter

_provides

_larger

_{sample quantities,}

but these

are

_{unrepresentative}

of

nectar

_actually

accessible

to

_insects,

and modification

of chemical

_composition,

due

to

lesion of

_plant

tissues

cannot

be excluded.

Micropipette

nectar

collection offers the best available

_sampling

method,

for

assessing

quantitative

and

_qualitative

_aspects

of

nectar

_production

as

functions

of

_genotype

and

_phenological

state.

_{It may also}

be used

to

determine

pedo-climatic influences

on nectar

secretion.

Received

_{for publication}

in October 1987.

Accepted for

publication

in

_April

1988.

ACKNOWLEDGMENTS

The authors wish to thank Dr. C. MASSON for a critical review of the

_manuscript

and M. Le M

ETAYER

for

_helpful

_support

in data treatment.

RÉSUMÉ

ÉTUDE

DES

SÉCRÉTIONS NECTARIFÈRES

DES FLEURS DE COLZA

(BRASSICA

NAPUS L. VAR. OLEIFERA

_METZGER)

La

_{pollinisation}

du colza

(Brassica

napus L. var.

_{oleifera Mer}

_Z

_G

_ER

_),

assurée en

_partie

_{par le vent,}

dépend

toutefois de l’action des insectes

_{pollinisateurs,}

notamment dans le cadre de la

_production

de

domestiques ;

celles-ci manifestent des

préférences génotypiques

chez certaines

_lignées

mâles-stériles

_qui

pourraient

être liées à des différences de sécrétions nectarifères. Afin de

_disposer

d’une

_technique

fiable d’estimation et de

_comparaison

des

_productions

nectarifères de différentes

_lignées

en cours de

sélection,

deux

_techniques

_{d’extraction des nectars, par}

_{centrifugation}

et _par

_pipetage,

ont été

_comparées

_{pour les}

nectars de la variété Kid. Outre une mesure

_quantitative

des

_productions

nectarifères,

une méthode

d’analyse

par

chromatographie

en

_phase

gazeuse des constituants

glucidiques

a été

_appliquée.

La méthode d’extraction par

centrifugation

permet de recueillir des

_quantités

de solution

_plus

abondantes

(4

à 6

_fois)

_{que par}

_{pipetage (Tabl.}

_{1) ;}

toutefois la totalité de la fraction

_{glucidique représentative}

des

nectars est

_{intégralement présente}

dans des solutions

_prélevées

par

pipetage

(Tabl. 5) :

les sucres sont

donc dilués par

centrifugation.

La

_présence

de

_glucose

et de

fructose,

en

_proportions

_voisines,

a été mise

en évidence

(Tabl.

2,

3,

4). L’adoption

de la

technique

de

_{prélèvement}

par

pipetage,

qui

assure une

restitution

_plus

fidèle des sécrétions

_disponibles

pour les insectes

pollinisateurs,

est

_préconisée

et

_pourrait

permettre

la sélection de

_lignées

en vue d’une

_optimisation

de la

_{pollinisation}

croisée.

ZUSAMMENFASSUNG

UNTERSUCHUNGEN

ÜBER

DIE

BLÜTENNEKTARSEKRETION

VON RAPS

(BRASSICA

NAPUS L. VAR. OLEIFERA

_METZGER)

Raps (Brassica

napus L. var

_oleifera

_M

_ETZGER

_),

wird zum

_{größten}

Teil vom Wind bestäubt. Jedoch

hängt

vor allem die Produktion von Fl

_Hybrid-Samen

von der Aktivität bestäubender Insekten ab. Hierbei

_spielt

die

_Honigbiene

die

_{größte}

Rolle. Die Nektar sammelnden

_{Honigbienen zeigen}

eine Präferenz für männlich-fertile

Linien,

was dadurch zustande kommen

könnte,

daß Unterschiede in der Nektarsekretion bestehen. Um die im Laufe der Selektion entstandenen Unterschiede in der Nektarsekre-tion mit einer verläßlichen Methode zu

schätzen,

wurden zwei

Extraktionsmethoden,

die

Zentrifugation

und die

_{Pipettierung (anhand}

des Nektars der Varietät

_{Kid) verglichen.}

Über

die

_{quantitative Messung}

der

_{Nektarproduktion}

hinaus wurde eine

gaschromatographische Analyse

der

_{Kohlehydrat-Komponenten}

durchgeführt.

Die Methode der

_{Zentrifugation}

führt zu einer

_{größeren}

Ausbeute

₍₄

bis 6

mal)

als die

_Pipettierung

(Tab.

1).

Dennoch war die Gesamtheit der

_{repräsentativen Kohlenhydrat-Fraktion}

auch in den

pipettier-ten Proben

_vorhanden,

was darauf schließen

läßt,

daß in den

zentrifugierten

Proben die Zucker nur mehr verdünnt sind. Es konnte

_gezeigt

_werden,

daß Glukose und Fruktose in ähnlichen

_{Proportionen vorliegen}

(Tab.

2,

3 u.

_4).

Die Methode des

_{Pipettierens eignet}

sich

besser,

wenn man

_analysieren

_will,

welche

_{Sekretionsmenge}

dem bestäubenden Insekt effektiv zur

_Verfügung

steht. Außerdem könnte sie die Selektion von Linien im Hinblick auf eine

_Optimierung

der

_{Kreuzbestäubung begünstigen.}

REFERENCES

B

ONNIER G., 1893. - Influence du terrain

sur la

production

du nectar des

plantes.

Assoc. Fr. Av.

Sci.,

2, 567-569.

Bosi

G.,

1973. - Méthode

rapide

pour la determination par

chromatographie

en

_phase

gazeuse des

glucides

du nectar :

_technique

de

pr6l!vement

du nectar et de

_preparation

des ethers

_{trim6thylsilylds}

B

ROHST

_K.M.,

LOTI

_C.E.,

1966. - Determination of _some

components in corn _symp

_by

_gas

_liquid

chromatography

of the

_{trimethylsilyl}

derivatives. Cereal

Chem.,

43, 35-43. D

AGNELIEP., 1970. - Théorie _et methodes

statistiques.

Applications agronomiques.

2

vol., Duculot, S.A.,

Gembloux,

378 p. et 451 p.

F

ONTA

C.,

PHAM-DELEGUE

M.H.,

MARILLEAU

R.,

MASSON

C.,

1985. - R6le des _nectars de tournesol

dans le

_comportement

des insectes

_{pollinisateurs}

et

_analyse

_qualitative

et

_quantitative

des elements

glucidiques

de ces secretions. Acta

_{!’cologica,}

Œcol.

_Applic.,

6

_(2),

175-186. FREE

J.B.,

NurrALL P.M., 1968. - The

pollination

of oilseed rape

(Brassica

napus)

and the behaviour of bees on _{the crop. J.}

_Agric.

Sci.

_Camb.,

71, 91-94.

G

IRNIK

D.,

1976. -

[Productivité

en nectar du

_tournesoll.

_{[En russe].}

_{Pchelovodstvo,}

_8,

10-11. L

IVTZEVA

E.K.,

1954. -

[Methods

used in nectar secretion

_{investigations]. [In Russian].}

_{Pchelovodstvo,}

31

_(11),

33-39.

H

EINRICH B., 1975. -

Energetics

of

_pollination.

Annu. Rev. Ecol.

_Syst.,

6, 139-170. K

ENOYER L.A., 1917. - Environmental influences _{on nectar} secretion. Bot.

Gaz,

63, 249-365. M

ASSON

C.,

1983. - Role des médiateurs

chimiques

d’origine

animale et

_v6gdtale

dans la

_{pollinisation.}

C.R. 5‘

Symp.

Int.

Pollinisation,

Versailles, sept.

1983, 25-37.

M ESQU1DA

J., RENARD

_M.,

1978. - The

entomophilous pollination

of male sterile strains of winter

Rapeseed

(Brassica

napus L. var.

_{oleifera M}

_ETZGER

₎

and a

_{preliminary study}

of

_alternating

devices. Proc. 4th. Int.

_Symp.

Pollination,

Maryland,

49-57.

M

ESQUIDA

J.,

RENARD M.

_{1979 (a).}

-

Importance

de I’abeille

_domestique

_(Apis

_{mellifica L.)}

sur la

pollinisation

du colza d’hiver male-sterile

(Brassica

napus L. var.

_{oleifera M}

_ETZGER

₎

en

_production

de

semences

hybrides

Fl et

_consequences

sur les

_dispositifs

d’alternance.

_Symp.

Pollinisation

ITAPI-APIMONDIA,

Avignon,

49-60.

M

ESQUIDA

J.,

RENARD

_M.,

1979

_(b).

- Résultats

pr6liminaires

sur la

_{pollinisation}

du colza d’hiver mate-sterile et les modalites de

_production

de semences

_{hybrides. Inform.}

Tech.

_{CETIOM, ,}

65, 4-14.

M ESQUIDA

J., RENARD M., 1981. - Pollinisation du colza d’hiver male-fertile et male-sterile

(Brassica

napus L. var.

_{oleifera M}

_ETZGER

₎

_{par l’abeille}

_{domestique (Apis}

m.

_{mellifica L.) :}

effets sur la

phénologie

et le rendement.

_Apidologie,

12, 345-362.

M

ESQUIDA

J.,

RENARD

M.,

1982. - Etude de la

dispersion

du

_pollen

_{par le} vent et de

_{l’importance}

de la

pollinisation

andmophile

chez le colza

_(Brassica

napus L. var.

_{oleifera M}

_ETZGER

_).

_Apidologie,

13

_(4),

353-366.

M

ORICEJ., 1960. - La selection du colza d’hiver basée

sur t’etude des composantes du rendement. Ann. Amelior.

Plantes,

1, 85-116.

O

LSSON

G.,

1955. -

Vindpdlinering

hos

_{korsblomstriga oljeväxter. (in}

Swedish,

English Summary),

Sver.

Utsadesfoi.,

65, 418-422. P

ERCIVAL

M.S.,

1961. -

Types

of nectar in

_Angiosperms.

New

_Phytol.,

60,

235-281. P

HAM -D

ELEGUE

_M.H.,

MASSON

C.,

BONJEAN

_A.,

ETIEVANT

_P.,

MARILLEAU

_R.,

1985. - Etude des

param6tres

chimiques

impliquds

dans la

_{pollinisation}

_{du tournesol par les abeilles} en

_production

de

semences

_hybrides.

C.R. xr

_Conf

Int.

Tournesol,

Mar del Plata

_(Argentine),

mars

1985,

559-564. P

LANTA

A. von, 1886. -

Über

die

Zusammensetzung einiger

Nektar-Arten. Z.

_Physiol.

Chem., 10,

227-247.

R

ENARD M., MESQUIDA

J.,

1983. - Pollinisation

entomophile

du colza male-sterile en

_production

de semences

_hybrides

Fl dans différentes

_regions

de France. C.R. VI! congr. Int. sur le

Colza, Paris,

552-557.

RIVES

M.,

1957. Etudes sur la selection du colza d’hiver. Ann. Amelior.

Plantes,

1, 61-107. R

UDLOFF E., SCHWEIGER W., 1984. - Eine Methode _zur

Schätzung

der

_{Fremdbefruchtungsrate}

bei

Qualitatsraps (Brassica

napus

L.).

Arch.

Ziichtungsforsch.,

Berlin,

14, 327-334. S

WANSON C.A., SHUEL R.W., 1950. - The

centrifuge

method for

measuring

nectar

_yield,

Plant

_Physiol.,

25

_(3),

513-520.

S

YLVEN N., 1920. - Om

T

ASEI

_J.N.,

1977. - Observations

sur le colza _{d’hiver-floraison-sécrétion nectarif!re-visite par les abeilles.} Bull. tech.

Apic.,

4,

9-16.

V

ANSELL

_G.H.,

WATKINS

_W.Q.,

BISHOP

_R.K.,

1942. -

Orange

nectar and

_pollen

in relation to bee

activity.

J. econ.

Entomol., 35,

321-323. V

ANSELL

_G.H.,

1943. - Alfalfa _nectar and the

honey

bee. Am. Bee

_{J., 83,}

106-107. V

ANSELL

G.H.,

1944. - Cotton _nectar in relation _to bee

activity

and

_{honey production.}

J. econ.

Entomol. ,

37, 528-530. W

ILLIAMS

I.H.,

1978. - The