Photoperiod and chemical signals determine synchronization of biological rhythms in tritrophic system Le Kang Institute of Zoology Chinese Academy of Sciences Beijing, , China Beijing, , China APCE 2009

direct Indirect Herbivores 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

Parasitoid Opius dissitus Leafminer Liriomyza huidobrensis Lima bean Phaseolus lunatus Leafminer-Induced Volatiles Lima bean-leafminer-parasitoid tritrophic system

L. huidobrensisL. 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 results   We identified more than 90 constitutive and inducible compounds from host and non-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 diurnal rhythms Daily rotation of the earth creates an approximately 24-h environmental oscillation of day and night.

Hypothesis 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 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

Results -under LD (light : dark=15:9)  Firstly, we tested the volatiles from healthy lima beans and biological rhythm of the three levels in the tritrophic system under 15L:9D (similar to the 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 limabean infected lima bean infected lima bean

Feeding rhythm of the leafminer larvae Feeding rhythm of the leafminer larvae Feeding frequency varied greatly during the diurnal cycle, and the larval feeding was more active during the photophase than during the scotophase. Feeding frequency varied greatly during the diurnal cycle, and the larval feeding was more active during the photophase than during the scotophase. The bar under the x axis refers to the photoperiodic cycle, the black part refers to the scotophase.

The total volatiles collected from the leafminer damaged lima bean exhibit obvious rhythms, and peaked after the leafminer larval feeding rhythm. The total volatiles collected from the leafminer damaged lima bean exhibit obvious rhythms, and peaked one time interval after the leafminer larval feeding 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 female parasitoids were inactive (resting). In contrast, in the photophase, locomotion and oviposition activity displayed similar rhythms, both get peak in the afternoon. Parasitoid activity rhythms

Different kind of volatiles Terpenes and larval feeding Parasitoid and volatile s

Summary: LD (15:9) 1. Volatiles could be divided in to two kinds: A: Terpenes increased after the light was on and peaked in the afternoon, B: Fatty acid derived volatiles increased slowly when the larvae began feeding in the early morning, but accumulated quickly in the night. 2. Rhythm of the terpenes emission was closely related to that of leafminer larval feeding. 3. Emission of terpenes matched well with the parasitoid locomotion and oviposition. However, the (Z)-3-hexen-ol well matched parasitoid emergence with one postponed time interval. 4. Under LD, most of the activities in tritrophic interactions show obvious rhythms, and the rhythms were closely correlated to each other.

Results -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 feeding under 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 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 spent most of their time in locomotion, with a small portion of time for oviposition. During the DD, the parasitoids spent almost all of their times in resting, a small portion in locomotion, and nearly no oviposition activity. Therefore, parasitoid activities are photo-inducible. LL DD Parasitoid activities

Summary: 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- artificially add the volatile concentrations 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 locomotion and oviposition after artificially adding the plant volatile concentrations in two time points.   Adding plant volatiles can not promote the activities of parasitoid locomotion and oviposition under DD. (a) Parasitoid locomotion under LL (b) Parasitoid oviposition under LL (c) Parasitoid locomotion under DD (d) oviposition under DD Artificially change the volatile concentrations

Results--naive and oviposition- experienced parasitoid response to the volatile rhythm information  As the larval feeding-induced volatiles could be divided into two categories: terpenes peaked during 14:00-17:00, and (Z)-3-hexen-ol peaked during 05:00-08:00.  We tested the olfactory responses of the naive and oviposition-experienced parasitoids to these two category volatiles using a Y-tube olfactometer.

 (A) The responds of naive and oviposition- experienced parasitoid to single volatile extraction and  (B) the responds of naive and oviposition- experienced parasitoid to two volatile blends, blend from 5:00-8:00 and blend from 14:00-17:00. * P<0.05; ** P<0.01. C / NC: Choice / No Choice; Ovi- Exp: Oviposition experienced. Differential responses of naive and oviposition- experienced parasitoids to volatiles

 Behavior experiments proved that parasitoids were able to distinguish volatiles emitted at different times in a diurnal cycle.  Naive and oviposition-experienced parasitoids could selectively use the rhythm of volatiles emitted to locate their host. But, oviposition-experienced parasitoids prefer the volatiles released in afternoon. Summary

Conclusions and discussion 1. Rhythms in tritrophic interaction exhibit closely correlation, derived from leafminer feeding to volatile emissions. 2. Light can dramatically affect the diurnal rhythm and the tritrophic interactions; it was a switch signal for insect activities in tritrophic system. 3. Behavior experiments proved that parasitoids were able to distinguish volatiles emitted at different times, and naive and oviposition-experienced parasitoids could selectively use the rhythm of volatiles emitted in different times to locate their host. 4. Taken together, we can find that the biological rhythms of the tritrophic system was effectively synchronized by a combination of programmed circadian rhythms, light- affected diurnal rhythms, directly induced responses, and experience-guided learning.

In addition, we found that fatty-acid derived volatiles and terpenoids have different diurnal rhythms and different responses to light. In addition, we found that fatty-acid derived volatiles and terpenoids have different diurnal rhythms and different responses to light. Furthermore, the parasitoid emergence rhythm was correlated to that of (Z)-3- hexen-ol, and parasitoid locomotion and oviposition rhythm were corresponding to the emission of terpenoids. Furthermore, the parasitoid emergence rhythm was correlated to that of (Z)-3- hexen-ol, and parasitoid locomotion and oviposition rhythm were corresponding to the emission of terpenoids.

In summary, we believe that the information from this study provides a better understanding of infochemical communications in a tritrophic system, and will help to implement a push-pull strategy in biorational pest management 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.

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