Photoperiod and chemical signals determine Photoperiod and chemical signals determine synchronization of biological rhythms synchronization of biological rhythms

in tritrophic systemin tritrophic system

Le KangLe Kang

Institute of ZoologyInstitute of Zoology

Chinese Academy of SciencesChinese Academy of Sciences

Beijing, 100101, ChinaBeijing, 100101, China

APCE 2009

direct

IndirectHerbivores

Natural enemies

Plants

Direct

Tri-trophic interactions:

As well-known, plants recruit natural enemies to protect them from herbivores through herbivore-induced plant volatiles when plants are damaged by the herbivores (De Moraes et al., 1998; Arimura et al., 2005).

Natural enemies have to utilize different inforchemicals to locate the appropriate host plants and host prey in their whole life span (Vinson, 1976).

Primary producer

Consumer II

Consumer I

ParasitoidOpius dissitus

LeafminerLiriomyza huidobrensis

Lima bean

Phaseolus lunatus

Leafminer-Induced Volatiles

Lima bean-leafminer-parasitoid tritrophic system

L. huidobrensis L. sativae

Adults

Pupae

Larvae

Herbivorous leafminer species

The Leafminers here are Dipteran insects of the genus Liriomzya, which contains more than 300 species. 5 species considered to be truly polyphagous.

Adult flies usually lay their eggs in the tissues of leaves, and larvae of the leafminers create serpentine mines. The larvae mining causes serious damage of plants.

The larvae pupate on the surfaces of leaves or soil.

Parrella 1987 Ann. Rev. Ent.

Leaminers have caused

seirous damages on

agricultural crops in China

since they invaded to

China in the early 1990‘s

(Kang, 1996)

L. huidobrensis L. sativae

The two leaminer species

feed in different sections of

the leaf mesophyll, e.g. L.

sativae in the palisae

mesophyll, L. huidobrensis in

the spongy mesophyll and

through within the leaf viens.

Past resultsPast results We identified more than 90 constitutive and inducible compounds from host and n

on-host plants of leafminers. Most of the inducible volatiles could be divided into three major categories: green leaf volatiles, terpenoids, and oximes (Wei et al, 2006, Planta).

The leafminers locate host plants by the perception of a combination of general and host-specific volatiles based on EAG and behavioral tests (Zhao and Kang, 2002, Physiol Ent.; 2003, J. Appl. Ent.).

No behavioral responses of parasitoids were detected to health leaves of host plants and non-host plants. However, distinct responses were obtained from wound plants whatever host or non-host plants of leafminers (Zhao and Kang, 2002, J. Eur. Ent.; Wei et al., 2006,Chem. Sen.)

The parasitoids responded preferably to (Z )-3-hexen-1-ol, although six chemicals are attractive in behavior to the parasitoids (Wei et al, 2007, PLoS ONE).

Diurnal rhythms interacting among

different organisms was poorly understood

Circadian Rhythm is the central clock of

organisms

Diurnal rhythms of feeding, sleeping,

and mating in an organism can even

interact with each other

However

How do diurnal rhythms of each trophic level match with each other in the tri-trophic system?

So

Tritrophic system – Herbivore-plant-parasitoid

Each trophic level was closely correlated with others and

Various activities of them exhibit distinct diu

rnal rhythms

Daily rotation of the earth creates an approximately 24-h environmental oscillation of day and night.

HypothesisHypothesis

Plants， herbivores, and natural enemies in tritrophic system have their own circadian rhythms, which could regulate their activity, feeding, reproduction, defense, and adaptation to one another.If the rhythms are not synchronic(including circadian and photo-induced), the interactions of tritrophic system will be disorder.

Research system and methods

oral hook

oral hook

Pea leafminer: expressed as the oral hook moving frequency

Lima bean: Emission rhythms of the plant volatiles induced by leafminer larval feeding

Parasitoid: Emergence, locomotion, and oviposition

Three photoperiodic cycle:L15:D9, LL, and DD

3h as one time interval, 8 time intervals a day

ResultsResults-under LD (light : dark=15:9)-under LD (light : dark=15:9)

Firstly, we tested the volatiles from healthy lima Firstly, we tested the volatiles from healthy lima beans and biological rhythm of the three levels ibeans and biological rhythm of the three levels in the tritrophic system under 15L:9D (similar to tn the tritrophic system under 15L:9D (similar to the natural light dark cycle in summer).he natural light dark cycle in summer).

Releasing of induced plant volatiles still display diurnal rhythm, when the lima beans are continuously damaged in diuarnal cycle by artificial simulation (Arimura et al., 2008).

Headspace sample analysis with GC

Healthy lima bean nearly don’t emit volatiles, but after damaged by insect ,they emit great amount of volatiles.

control

healthy lima

bean

healthy lima

bean

infected

lima bean

infected

lima bean

Feeding rhythm of tFeeding rhythm of the leafminer larvaehe leafminer larvae

Feeding frequency varieFeeding frequency varied greatly during the diurd greatly during the diurnal cycle, and the larval nal cycle, and the larval feeding was more active feeding was more active during the photophase tduring the photophase than during the scotophhan during the scotophase. ase.

The bar under the x axis refers to the photoperiThe bar under the x axis refers to the photoperiodic cycle, the black part refers to the scotophaodic cycle, the black part refers to the scotophase.se.

The total volatiles colleThe total volatiles collected from the leafminected from the leafminer damaged lima bean r damaged lima bean exhibit obvious rhythmexhibit obvious rhythms, and peaked s, and peaked one time interval after the l after the leafminer larval feedineafminer larval feeding rhythm.g rhythm.

The emission of total volatiles

(Z)-3-hexen-ol increased in the night, and peaked between 05:00 and 08:00. The emission of terpenes corresponded well with the rhythm of total volatiles peaked in the afternoon.

(A) (Z)-3-hexen-ol; (B) Terpenoids; DMNT:(3E)-4,8-dimethyl-1,3,7-nonatriene; TMTT:(3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene.

The parasitoids began to emerge during 05:00-08:00 in the late scotophase and peaked between 08:00 to 11:00.

Approximately 80% of the parasitoids emerged before 11:00, regardless of the sex.

Parasitoid emergence rhythm

In the scotophase, almost all fIn the scotophase, almost all female parasitoids were inaemale parasitoids were inactive (resting). ctive (resting).

In contrast, in the photophase,In contrast, in the photophase, locomotion and oviposition locomotion and oviposition activity displayed similar rhactivity displayed similar rhythms, both get peak in the ythms, both get peak in the afternoon.afternoon.

Parasitoid activity rhythms

Different kind of volatiles

Different kind of volatiles

Terpenes and lar

val feedingTerpenes and lar

val feeding

Parasitoid and volatiles

Parasitoid and volatiles

Summary: Summary: LD (15:9)LD (15:9)

1. Volatiles could be divided in to two kinds:1. Volatiles could be divided in to two kinds:A: Terpenes increased after the light was on and peaked in the A: Terpenes increased after the light was on and peaked in the

afternoon,afternoon,B: Fatty acid derived volatiles increased slowly when the larvae B: Fatty acid derived volatiles increased slowly when the larvae

began feeding in the early morning, but accumulated quickly ibegan feeding in the early morning, but accumulated quickly in the night.n the night.

2. 2. Rhythm of the terpenes emission was closely related to that of Rhythm of the terpenes emission was closely related to that of leafminer larval feeding.leafminer larval feeding.

3. 3. Emission of terpenes matched well with the parasitoid locomotEmission of terpenes matched well with the parasitoid locomotion and oviposition. However, the (ion and oviposition. However, the (ZZ)-3-hexen-ol well matche)-3-hexen-ol well matched parasitoid emergence with one postponed time interval.d parasitoid emergence with one postponed time interval.

4. 4. Under LD, most of the activities in tritrophic interactions show Under LD, most of the activities in tritrophic interactions show obvious rhythms, and the rhythms were closely correlated to obvious rhythms, and the rhythms were closely correlated to each other.each other.

ResultsResults-under LL and DD-under LL and DD

To distinguish rhythms in the system are circadian or photoperiod induced, we designed the experiments under constant light (LL) and dark (DD).

Under LL, the leafminer larval feeding retained a distinct rhythm, and was most active during 11:00-14:00, similar under LD, but did not significantly decrease during the dark period.

Under DD, leafminer larval feeding activity varied only slightly, and did not show any distinct rhythm .

The leafminer larval feedingunder LL and DD

Absolute amount of total volatiles

Under LL, total volatile emission increased gradually in the morning and peaked at noon or in the afternoon, then gradually decreased, similar under LD

Under DD, total volatiles increased gradually during the test period, but the rhythm disappeared under DD.

Under LL, all volatiles emitted after leafminer larval feeding presented similar rhythms to LD.The rhythms were disappeared in next day if keep LL or DD.

Under DD, the terpene volatiles were in low concentrations and did not show any obvious rhythm, In contrast, the fatty acid-derived volatiles, especially (Z)-3-hexen-ol, continuously increased.

Under LL, amount of terpenes was high. Under DD, volatile emission from damaged plants was low.

Thus, fatty acid-derived volatiles and terpenes had different responses to light, and their emission pattern was opposite..

Parasitoid emergenceParasitoid emergence

Parasitoid emergence rhythm under both LL and DD was similar to that under LD, indicating that parasitoid emergence was a genetic circadian rhythm.

During the LL, the parasitoids spenDuring the LL, the parasitoids spent most of their time in locomotion,t most of their time in locomotion, with a small portion of time for o with a small portion of time for oviposition.viposition.

During the DD, the parasitoids speDuring the DD, the parasitoids spent almost all of their times in restint almost all of their times in resting, a small portion in locomotion,ng, a small portion in locomotion, and nearly no oviposition activit and nearly no oviposition activity.y.

Therefore, parasitoid activities aTherefore, parasitoid activities are photo-inducible.re photo-inducible.

LLLL

DDDD

Parasitoid activitiesParasitoid activities

Summary: LL and DDSummary: LL and DD

1. Almost all rhythms of in the tritrophic system was photo-inducible, except that parasitoid emergence was a genetic circadian rhythm.

2. The leafminer larval feeding had an obvious rhythm, but were triggered by light.

3. Fatty acid-derived volatiles and terpenes had different responses to light, and their emission pattern was opposite. Rhythm of leafminer feeding was closely correlated to these volatiles under light.

4. Parasitoid activities are photo-inducible.

5. The rhythms of plant volatile emission, insect larval feeding, and parasitoid activities were perfectly correlated under LD, but the correlation was disrupted under LL, and even more so under DD.

Results-Results-artificially add the volatile artificially add the volatile concentrationsconcentrations

Volatile was an important signal for parasitoid activity. To prove whether volatile concentration can affect the activity rhythm of parasitoids, we artificially added volatiles to a concentration same as the peak concentration under the 15L:9D at two time intervals: 14:00-17:00 and 23:00-02:00.

Change of parasitoid locomotiChange of parasitoid locomotion and oviposition after artificion and oviposition after artificially adding the plant volatile cally adding the plant volatile concentrations in two time pointoncentrations in two time points.s.

Adding plant volatiles can not Adding plant volatiles can not promote the activities of paraspromote the activities of parasitoid locomotion and ovipositioitoid locomotion and oviposition under DD.n under DD.

(a) Parasitoid locomotion under LL(a) Parasitoid locomotion under LL(b) Parasitoid oviposition under LL (b) Parasitoid oviposition under LL (c) Parasitoid locomotion under DD (c) Parasitoid locomotion under DD (d) oviposition under DD(d) oviposition under DD

Artificially change the volatile concentrations

Results--naive and oviposition-experienceResults--naive and oviposition-experienced parasitoid response to the volatile rhythd parasitoid response to the volatile rhythm informationm information

As the larval feeding-induced volatiles could be dividAs the larval feeding-induced volatiles could be divided into two categories: terpenes peaked during 14:0ed into two categories: terpenes peaked during 14:00-17:00, and (0-17:00, and (ZZ)-3-hexen-ol peaked during 05:00-08:)-3-hexen-ol peaked during 05:00-08:00.00.

We tested the olfactory responses of the naive and We tested the olfactory responses of the naive and oviposition-experienced parasitoids to these two catoviposition-experienced parasitoids to these two category volatiles using a Y-tube olfactometer. egory volatiles using a Y-tube olfactometer.

(A) The responds of naive (A) The responds of naive and oviposition-experienceand oviposition-experienced parasitoid to single volatild parasitoid to single volatile extraction ande extraction and

(B) the responds of naive a(B) the responds of naive and oviposition-experienced nd oviposition-experienced parasitoid to two volatile bleparasitoid to two volatile blends, blend from 5:00-8:00 ands, blend from 5:00-8:00 and blend from 14:00-17:00. nd blend from 14:00-17:00.

* P<0.05; ** P<0.01. * P<0.05; ** P<0.01.

C / NC: Choice / No Choice; Ovi-Exp: C / NC: Choice / No Choice; Ovi-Exp: Oviposition experienced.Oviposition experienced.

Differential responses of naive and oviposition-experienDifferential responses of naive and oviposition-experienced parasitoids to volatilesced parasitoids to volatiles

Behavior experiments proved that parasitoids were Behavior experiments proved that parasitoids were able to distinguish volatiles emitted at different timeable to distinguish volatiles emitted at different times in a diurnal cycle.s in a diurnal cycle.

Naive and oviposition-experienced parasitoids coulNaive and oviposition-experienced parasitoids could selectively use the rhythm of volatiles emitted to ld selectively use the rhythm of volatiles emitted to locate their host. But, oviposition-experienced parasocate their host. But, oviposition-experienced parasitoids prefer the volatiles released in afternoon.itoids prefer the volatiles released in afternoon.

Summary

Conclusions and discussionConclusions and discussion

1.1. Rhythms in tritrophic interaction exhibit closely correlatioRhythms in tritrophic interaction exhibit closely correlation, derived from leafminer feeding to volatile emissions. n, derived from leafminer feeding to volatile emissions.

2.2. Light can dramatically affect the diurnal rhythm and the trLight can dramatically affect the diurnal rhythm and the tritrophic interactions; it was a switch signal for insect activitrophic interactions; it was a switch signal for insect activities in tritrophic system. ities in tritrophic system.

3.3. Behavior experiments proved that parasitoids were able tBehavior experiments proved that parasitoids were able to distinguish volatiles emitted at different times, and naivo distinguish volatiles emitted at different times, and naive and oviposition-experienced parasitoids could selectivee and oviposition-experienced parasitoids could selectively use the rhythm of volatiles emitted in different times to ly use the rhythm of volatiles emitted in different times to locate their host. locate their host.

4.4. Taken together, we can find that the biological rhythms oTaken together, we can find that the biological rhythms of the tritrophic system was effectively synchronized by a f the tritrophic system was effectively synchronized by a combination ofcombination of programmed circadian rhythms, light-affeprogrammed circadian rhythms, light-affected diurnal rhythms, directly induced responses, and excted diurnal rhythms, directly induced responses, and experience-guided learning. perience-guided learning.

In addition, we found that fatty-acid deriveIn addition, we found that fatty-acid derived volatiles and terpenoids have different did volatiles and terpenoids have different diurnal rhythms and different responses to liurnal rhythms and different responses to light. ght.

Furthermore, the parasitoid emergence rhFurthermore, the parasitoid emergence rhythm was correlated to that of (ythm was correlated to that of (ZZ)-3-hexen-)-3-hexen-ol, and parasitoid locomotion and ovipositiol, and parasitoid locomotion and oviposition rhythm were corresponding to the emison rhythm were corresponding to the emission of terpenoids. sion of terpenoids.

In summary, we believe that the information fr

om this study provides a better understandi

ng of infochemical communications in a tritr

ophic system, and will help to implement a p

ush-pull strategy in biorational pest manage

ment programs in various agroecosystems (

Kang et al., 2009, Ann. Rev. Ent.).

Acknowledgment

Miss S.F. Zhang Dr. J.N. Wei

The research is supported by National Basic Research Program (973) and Innovative Team Program of NSFC.

Thank you for your attention!

E-mail:lkang@ioz.ac.cnE-mail:lkang@ioz.ac.cn