Bioassays in invertebrates

Electrophysiological methods

Classical electroantennography on insect antennae

general method responses not specific dose-dependent responses very useful for analytical

purposes

Connected gas chromatography – electroantennography (GC-EAG, GC-EAD)

insect antenna as selective detector

Bioassays in invertebrates

Olfactometer is used for testing volatile stimuli. In choice-test we observe responses to different stimuli (control, neutral, positive, negative). We observe movement of tested animal in the direction of tested compounds.

Klasické uspořádání testu antibakteriálních ůčinků (testovaná látka je nanesena na papír a přiložena na plotnu s bakteriální kulturou)

Y- olfactometr

Use of dummies of different shape

artificial leaves and other plant parts for studies of interactions of plants and insects

conspecific and heterospecific individuals for studies of social behaviour

artificial females for studies of sexual behaviour

Oviposition test on an artificial leave (polyurethane impregnated with plant leave extract)

Bioassays in invertebrates

Wind tunnelWind tunnel

Observation and evaluation of behaviour in response to volatiles, e.g. sexual behaviour (sexual pheromone): activation, oriented flight, finding of odour source, copulation attempts

Original equipment by Carde et al. :Tested compound is adsorbed on a filter paper (dispenser), placed on left, insect introduced on right.

Bioassays in invertebrates

Test of trail pheromones:

Traces 1 and 2 are “drawn” by tested compound and movements of ants on the drawing are followed

Bioassays in invertebrates

Biotesty u savcůVery complicated and demanding, problems with learning,

responses influenced by the researchers (primates), costly in humans

Bioassays in mammals

Design and evaluation of bioassaysDesign a planning of a bioassays is crucial

asking clear questions simple design, controlled environmental conditions sufficient number of experimental animals in the same

physiological state sufficient number of repetitions independent values! recording, documentation, digitalisation, later evaluation

negative and positive controls necessary avoid contamination elimination of animals in different physiological repetition, choice of suitable statistical method

In the literature we often find mistakes and misinterpretation.

BumblebeesBumblebees primitive social structureprimitive social structure one-year cycleone-year cycle fertilised queen overwintersfertilised queen overwinters starting colonies in springstarting colonies in spring workers take care of the broodworkers take care of the brood reproductive individuals emerge in reproductive individuals emerge in

summersummer mating in the open airmating in the open air old queen, workers, and males die in the old queen, workers, and males die in the

autumnautumn

Chemical signals in Chemical signals in bumblebees bumblebees

females' signals:females' signals: ·· sex pheromone sex pheromone of virgin queens of virgin queens (premating phase)(premating phase) · · queen's pheromonequeen's pheromone (queen is the only egg layer)(queen is the only egg layer)

males' signals:males' signals: ·· marking (sex) pheromones marking (sex) pheromones (premating behaviour)(premating behaviour)

workers' signals:workers' signals:· · dominant signalsdominant signals· · orientation in the nestorientation in the nest

male

queen

Bombus terrestrisBombus terrestris

Males‘ premating behaviourMales‘ premating behaviour

W aiting near the nest Perching species Patro lling species

SP E C IE S U S IN G PH E R O M O N E

SPE C IE S N O T U S IN G PH ER O M O N E

Male marking (sex) pheromones Male marking (sex) pheromones

producproduction in the tion in the cecephphalicalic part part of the labial glandof the labial gland

cephalic part consists of cephalic part consists of numerous acini and the ducts numerous acini and the ducts connecting particular acini connecting particular acini

multicomponent mixturesmulticomponent mixtures

Kullenberg et al. 1973; Ågren et al. 1979, Bergström et al. 1981

Usual Usual typtypes of compoundses of compounds alialiphphatic alatic alccoholoholss a andnd aldehyd aldehydeses ethylethyl esteresterss of fattyof fatty acidsacids terpenic alterpenic alccoholoholss a andnd theirtheir ester esterss (mono-, (mono-,

sessesququi-i-,, a andnd diterpen diterpeneses))

compositions are species - specificcompositions are species - specific compositions may be used as a toolcompositions may be used as a tool

for chemotaxonomyfor chemotaxonomy

OH

OAc

Bombus confususBombus confusus - Lo - Loccalitality of occurrencey of occurrence

Bombus confususBombus confusus

prematingpremating strateg strategyy - - perchingperching ( (elevated perchelevated perch))

mmororphphologicologicalal adaptaadaptationtion – – big eyesbig eyes

literaturliteraturee – – species oriented species oriented optically in search for mate optically in search for mate

male B. confusus on a perch

LLabiabial gland extractal gland extract

45 50 55 60 65 m in45 55 65 min45 55 65 min

Main components of the Main components of the secretionsecretion

OR

OCOCH3

1, R = H2, R = CO(CH2)4CH3

3, R = CO(CH2)6CH3

4, R = CO(CH2)8CH3

5

6OCOCH3

Double bond positionDouble bond position

m/z0

100

%

43

41

17361

55

8169

79

87171

12395122

231

174 232 404

m/z 173 m/z 231

(CH2)3

SS M+ 404

OAc(CH2)5

Z-Configuration of double bond (FT-IR)

3012 cm-1

Function of the labial gland Function of the labial gland secretionsecretion

Comparison of componentsComparison of components:: of the labial gland extractof the labial gland extract ofof h head-space ead-space sample of a marked perchsample of a marked perch of washing of a marked perchof washing of a marked perch ofof head-space head-space samplesample a andnd washing washing

of unmarked plants in the localityof unmarked plants in the locality

Labial gland extract

54

3222

1

Head-space of a marked perch (dry flower)

54

3

2

1

Head-space of and unmarked flower (blank)

2 0 3 0 4 0 5 0 6 0

Males’ marking behaviourMales’ marking behaviour 42 males marked individually42 males marked individually MarkingMarking – perches and – perches and

vegetation in the vicinity vegetation in the vicinity (straws) (straws)

Perches:Perches: dry flowers dry flowers Marking time:Marking time: morning, morning,

duration duration 18 min18 min

Number of marksNumber of marks of one male: of one male: 32-95 32-95

marking by B. confusus male

TerritoriTerritorieses of twoof two B. confusus B. confusus malesmales

ConclusionConclusion

B. confusus B. confusus is not a species oriented optically onlyis not a species oriented optically only Males produce a secretion in their labial glandMales produce a secretion in their labial gland SeSeccretretion is used for marking perches and surroundingsion is used for marking perches and surroundings B. confusus B. confusus is not exceptional among other perching speciesis not exceptional among other perching species

Methods used for the researchMethods used for the research: extra: extractionction, head-space, GC-MS, , head-space, GC-MS, derivatiderivatissaation withtion with DMDS, GC-FTIR DMDS, GC-FTIR, biological, biological observationsobservations..

Use of bioassays for localisation Use of bioassays for localisation of active components in mixturesof active components in mixtures

Identification of sex pheromone of horse chestnut leafminer (Cameraria ohridella) and its possible use for protection of chestnut trees

Cameraria ohridella Deschka et Dimić 1986 (Lepidoptera: Gracillariidae) originates from Macedonia; it is a dangerous pest feeding on horse chestnut, Aesculus hippocastanum (L).

First record in the Czech Republic: 1994 (South Moravia). Now it is spread in the whole country.

Biology (life cycle):4 generations / yearoverwintering in pupae first generation in March / April

Typical damage of leaves

Time of females’ calling activity

0

1

2

3

4

5

6

7

8

2 3 4 5 6 7 8 9 10 11 12

double bond position

EA

G r

esp

on

ses

(rel

)

EZ

A) Using EAG: Abdomens of calling females were dissected and extracted with hexane. EAG responses to saturated compounds (standards):

R-OH < R-Ac < 12:Ald < 14:Ald

Identification of sex pheromoneIdentification of sex pheromone

EAG map of 14:Ald monoenes:

B) Chemical approach:

Extract from 150 females didn’t give any good mass spectrum.

Strong EAG and GC-EAD signal on male antenna

Identification of sex pheromoneIdentification of sex pheromone

0

20

40

60

80

100

120

no reaction activation non-oriented flight oriented flight localization ofsource

3 FE

3 FE + PFBHA

Prove of aldehydic groupProve of aldehydic group

No response to standards of alcohols or acetates

PFBHA in MeOH was added to extract of females’ abdomens

EAG activity disappeared after derivatisation

Větrný tunel:

PFBHA

R-CH2-CH3

R-CH2-OCOCH3

R-CH2-OH

R-CH=O

+H2N

O

F

F

F

F

F

R-CH=NO

F

F

F

F

F

+

R-CH2-OH

R-CH2-OCOCH3

R-CH2-CH3

• 12:Ac, 14:Ald, and 14:OH have KI similar to the EAD-active peak

• (9E)-14:Ald (EAD-active) has a different KI from the pheromone

• KI of the EAD-active peak indicates two conjugated double bonds

C) Kováts index (KI):

Identification of sex pheromoneIdentification of sex pheromone

Conclusions from experiments A and B

• (9E)-14:Ald has a different KI and elicits lower EAG- and behavioural activity than the pheromone

• the pheromone may be tetradecadienal (TDDA) with double bonds located around C - 9

=> to prepare all isomers: 7,9-, 8,10- and 9,11-TDDA and test responses on GC-EAD

Identification of sex pheromoneIdentification of sex pheromone

D) GC-EAD of synthetic isomers:

8,10-TDDA

EAG of positional isomers TDDA:

9,11- < 7,9- << 8,10

GC-EAD recordings:

(E,Z)-8,10-TDDA elicits highest response of all isomers

KI and EAD responses to 8E,10Z-TDDA are identical with natural extract

Identification of sex pheromoneIdentification of sex pheromone

O

0

20

40

60

80

100

120

no reaction activation non-orientedflight

oriented flight localization ofsource

0,1 pg

1 pg

10 pg

100 pg

1000 pg

3 FE

Bioassays - Synthetic pheromone in the wind tunnel, dose response

(n = 25 males, 3-4 days old, day time 9-11 AM, air flow 0,4 m/s)

Field tests of the synthetic pheromone

0

100

200

300

400

500

6001st generation 2nd generation

Delta traps, checked daily

glue trap(420 males)

(n = 3)

Conclusions:• using combinatorial approach (EAG mapping and

Kováts retention index, (8E,10Z)-tetradeca-8,10-dienal was determined as sex pheromone of horse chestnut leafminer and synthesised in the laboratory

• chromatographic, EAG, and behavioural properties of synthetic and natural pheromone are identical

• synthetic substance is a specific attractant for malesin traps