Francis Fleurat-Lessard

Jean-Michel Savoie

Florence Richard-Forget

Christian Barreau

Nadia Ponts

Vessela Atanasova-Penichon

Laetitia Pinson-Gadais

Véronique Pronier

Thierry Gibard

Marie-France Neveux

Corine Grimaldi

Christos Athanassiou

Maria Otilia Carvalho

Shlomo Navarro

George Opit

Catherine Regnault-Roger

Matthias Schöller

Vaclav Stejskal

Phyllis Weintraub

S Savoldelli

Ali A Isikber

H K Tofel

R Guruprasad

Cornel Adler

Matthias Schoeller

Subramanyam

A A Hameed

Canadas Pedraza

De Paredes

Weichel L Birner
email: erich.birner@basf.com

and by-products

Keywords: 20 Suppression of Plodia interpunctella moth population by mating disruption Cephalonomia tarsalis control Integrated pest management, food industry, monitoring, insecticides. Session 1: Recent advances in IPSP and current trends in post-harvest Metal bin, grain, insects, molds, remote monitoring, phosphine recycling, fumigation, liquid insecticides 3 -Carifend™: an alternative method remote monitoring, phosphine recycling, fumigation, liquid insecticides Stored tobacco, insect, IPM, Carifend™, tobacco quality, tobacco

and Food Safety" of the INRA Research Centre of Bordeaux-Aquitaine.

This scientific meeting, held every two years, under the auspices of the International Organization for Biological and integrated Control (IOBC), regularly brings together scientists, engineers, industry players, stakeholders and professionals in post-harvest agrofood chain sanitation quality and safety to report the state of progress in applied research in stored product integrated protection. The socio-economic reality of increasing safety and quality traceability concern by foodstuffs purchasers, processed food manufacturers, food supply chains, consumer demand, and the application of recent European Regulations of "Hygiene package" and "Pesticide package" in all food chains accelerate the need for greater production of applied research results. The real challenge of developing new approaches and the application of necessary changes in current programs of integrated pest management in food processing plants and in stored products is impeded by a global reduction of human and material resources available for research and development on pests and pathogens contamination of durable agricultural products and derived food products.

Regardless of this critical situation, the 9th IPSP 2013 Conference is meeting the challenge for the transition towards an efficient control of post-harvest pests and pathogens with both the limitation chemical use and the improvement of risk control of infestation or contamination of durable foods by insect pests, molds or their impurities and toxins. At IPSP 2013 Conference, the optimal solutions for tackling this difficult challenge will be presented through 90 communications comprising the scientific program. Their authors come from more than 25 countries, confirming the interest of the international scientific community for this IPSP Conference.

The Local Organizing Committee of IPSP 2013 wishes to thank all the contributors making this Conference intriguing and relevant. We are grateful to IOBC and INRA for their advice and support for the organisation. We really appreciate the financial support of our sponsors and organizations and wish to thank the Scientific Advisory Committee for their advice in building the scientific program of this Conference. Thank you also to all the authors of communications for making the work of the Organizing Committee possible.

Avant-propos

La 9 ème Conference OILB-SROP sur la Protection Intégrée des Produits stockés (PIPS) est organisée pour la 1 ère fois par l'U.R. INRA "Mycologie et Sécurité des Aliments" (MycSA) du Centre INRA de Bordeaux-Aquitaine.

Cette Conférence biennale, placée sous les auspices de l'Organisation Internationale de Lutte Biologique et Intégrée (OILB), regroupe des scientifiques, ingénieurs, acteurs économiques et professionnels du monde entier intéressés par la maîtrise de qualité et la sécurité sanitaire dans les chaînes alimentaires post-récolte et pour faire l'état d'avancement de la recherche appliquée en Protection Intégrée des Produits stockés. Dans le contexte socio-économique actuel, la sécurité sanitaire et la traçabilité sont prioritairement exigées par les traders de produits alimentaires, les entreprises de transformations, les distributeurs ou les consommateurs. L'application des récents règlements européens du "paquet hygiène" et du "paquet pesticide" dans toutes les chaînes alimentaires accélère les besoins en résultats de recherche. Le challenge à relever consiste à développer de nouvelles approches et applications pratiques de solutions innovantes en protection intégrée contre les ravageurs et les contaminations des chaînes alimentaires après récolte. Cet objectif est contrecarré par la diminution constante du nombre de chercheurs qui se consacrent à ce domaine de recherche dans le monde. Toutefois, la 9 ème Conférence PIPS 2013 contribue à relever ce challenge de la transition vers une maitrise des ravageurs, agents pathogènes ou contaminants des denrées aprèsrécolte en limitant l'utilisation des pesticides tout en améliorant la prévision et la maîtrise des risques d'infestation ou de contamination des produits alimentaires durables par leurs impuretés ou leurs toxines. Pour la Conférence PIPS 2013, 90 communications ont été présentées pour contribuer à relever ce challenge. Leurs auteurs sont issus de plus de 25 pays différents, ce qui confirme l'intérêt de la communauté scientifique internationale pour cette thématique de la protection intégrée.

Le Comité d'Organisation de la Conférence PIPS 2013 remercie tous ceux qui ont contribué au succès et à l'attractivité de cette Conférence et en particulier l'OILB et l'INRA. Le soutien des entreprises et institutions qui ont participé financièrement à cette entreprise a été très apprécié. Le travail du comité scientifique pour effectuer la sélection difficile des communications orales ainsi que les efforts de l'ensemble des auteurs des communications (orales ou affichées) sont également à remercier pour la qualité et la rigueur scientifique de leurs prestations.

Francis Fleurat-Lessard Abstract: Acoustic detection of stored grain insects is based on their ability to generate sound (by feeding, flying, moving, probing for oviposition etc). In recent decades, acoustic devices of various kinds have been marketed for commodity inspection. The efficacy of acoustic devices in detecting the insects hidden inside the grain mass depends on many factors such as the sensor type, substrate structure, the interface between sensor and substrate, the assessment duration, the size and behavior of the insect, the density of the infestation, and the distance between the insects and the sensors. During the present study, adults and larvae of the most common pests of stored wheat have been detected in varying population density (0.1, 0.5, 1, 2, 10 & 50 per kg of wheat). Acoustic emissions of adults of Sitophilus oryzae, Rhyzopertha dominica, Tribolium confusum, Oryzaephilus surinamensis, Trogoderma granarium, Cryptolestes ferruginneus and Lasioderma serricorne and larvae of T. confusum, T. granarium, Ephestia kuehniella and Plodia interpunctella were recorded. The acoustic detector system consisted of a piezoelectric sensor mounted on the end of a probe that was pushed into the grain and a portable acoustic emission amplifier (AED-2010L, Acoustic Emission Consulting, Inc.) connected with a computer. All sound recordings took place in the lab using a sound insulated box in order to dampen background sound. Each recording lasted 3 minutes and was replicated 10 times. The system was 100% precise in negative predictions (predicting the absence of a pest when none is present) and considerably successful in positive predictions (predicting the presence of the pest when at least one individual is present in the grain mass). More analytically, prediction for infestation was rated falsely negative in 50-70%, 30-60%, 0-20% and 0-10% when actual population density was 0.1, 0.5, 1 and 2 adults / kg, respectively, irrespective of pest species. Respective values for larvae were 60-70%, 30-60, 0-40% and 0-30%. The system was always successful in detecting infestation when population density was > 2 individuals / kg irrespective of pest species or developmental stage. Our results are discussed on the basis of enhancing the use of acoustic devices as detection tools in storage facilities.

Keywords: Acoustic detection, stored wheat, piezoelectric sensor, sound analysis.

-The use of bio-test in the disinfestation of food industries

Abstract type: Poster

Süss L. 1 , Cassani S. 2 & Gallizia F. 1
Abstract: The proper use of bio-test in food industries, in the case of treatments with use of sulphuryl fluoride or hot air, is essential for assessing the results obtained.

In 2012 were given to several Pest Control Companies n. 490 bio-tests. Two special containers were prepared: one sized cm 7 x 5 x 4 h for placement on floors or in the crevices and the other, of sized cm 6 x 6 x 7 h to be hanged at different heights in empty silos. The treatments were carried out in mills and food industries. The species employed in the bio-tests were Tribolium confusum (stage egg, larvae, pupa, adult or mix population), Ephestia kuehniella (eggs and larvae), Plodia interpunctella (eggs and larvae), Sitophilus oryzae (adults and a mix population of eggs, larvae and pupae in rice) and Lasioderma serricorne (adults and a mix population of eggs, larvae and pupae in biscuits). Detailed operational guidelines were provided. Three TNT bio-test for controlling the pre-treatment mortality were also prepared for each group of bio-test at any treatment; one of the three to be kept in the laboratory and two others to be transported together with the Bio-test submitted to treatments. All the insects of any stage placed in the TNT-Bio-test resulted fully alive. Overall complete mortality occurred in 368 of 387 total, while in 19 cases survival was recorded. Obviously, at some points of the work processes have not been reached the desired saturation of sulphuryl fluoride at the temperature of hot air required to achieve a complete mortality. In these few cases and few sites localized treatments with pesticides became indispensable. These mechanisms for survival under conditions of water stress have been studied in several species. Despite its importance in stored grains there has been no study on the water balance in S. zeamais. Thus, this work aims to assess the water balance and aggregation behavior in pyrethroid resistant (with and without associated fitness cost) and susceptible strains, seeking to understand their potential. Our objective was to compare female and male adults of susceptible and resistant strains observing: (1) the moisture necessary for the survival;

Keywords

(2) differences in longevity (this response may be due to a balance of water in particular) and if these results may be related to insecticide resistance (and associated fitness costs); and also (4) if the aggregation behavior of this species could regulate water loss through the formation of groups of insects. The results indicated that S. zeamais was extremely vulnerable to low relative humidities in the absence of food. The three strains showed loss of water of approximately 30% which is a relatively low value, but enough to significantly compromise their survival. It was still possible to observe differences between the individual and the group in the conservation of water, but there are no differences between group size. The group retains more water than the insect isolated.

Keywords

:

  Sitophilus zeamais, water balance, insecticide resistance

14:00 -15:50 Session 2: Biology and ecology of S-P pests and pathogens in IPSP Wednesday 03 July Room "Badiane" 08:40 -10:20 Session 5: Chemical control in stored product IPM programs 15:10 -15:40 Coffee break (Poster removal) 15:40 -16:50 Session 6: IPSP decision support tools and integrated approach Chairpersons: Bhadriraju Subramanyam & Francis Fleurat-Lessard Page

	15:40 -16:10	Aspects related to decision support tools and IPM in food chain	P. Trematerra	119
		Technical and economic analysis of
	16:10 -16:30	pest management practice in 14 grain	K. Crepon	120
		elevators
		Influence of storage practices and kind	M.-P. Leblanc &
	16:30 -16:50	of structure on insect presence in grain	F. Fleurat-
		bulks	Lessard

121 16:50 -17:15 Closing Ceremony

	C. Athanassiou & F.
	Fleurat-Lessard

List of posters Session 1: Recent advances in IPSP and Post-Harvest IPM current trends Chairpersons: Phyllis Weintraub & M. Otilia Carvalho No. Page

	Integration of Isaria fumosorosea with DE for R. dominica control	Waqas Wakil	7
	Combination of B. hebetor and γ-radiation on Ephestia control in dates	A.A. AL-Taweel	8
	Using the parasitoid T. evanescens with
	pheromone traps in date palm orchards and in	A.A. AL-Taweel	9
	date warehouses
	Session 2:

Biology and ecology of S-P pests and pathogens useful in IPSP Chairpersons: George Opit & Christos Athanassiou

	Occurrence and identification of insect pests in sunflower seeds and rape	S. Dauguet	18
	Population density estimation of stored grain pests based on their acoustic emissions	P.A. Eliopoulos	19
	Detection of insect infestation in stored wheat based on their acoustic emissions	P.A. Eliopoulos	19 2
	The influence of plant characteristics on the field infestation	M. A. Amro	20
	Susceptibility of different pulse grain to infestation of cowpea beetle	M.A. Amro	21
	Effect of different temperatures and host density of Ephestia on B. hebetor efficacy	A.A. AL-Taweel	22
	Mycotoxicological Analysis of stored groundnuts in Southwest of Algeria	H. Meliani & A. Moussaoui	23
	The use of bio-test in the disinfestation of food industries	L. Süss & al.	24
	Sitophilus zeamais: water balance and behavior	N.M. Pinho Guedes	25
	Session 3:

Phytochemicals and semio-chemicals use in stored-product IPM Chairpersons: Catherine Regnault-Roger & Ali A. Isikber

	Study and analysis of Insecticidal & antimicrobial activity of essential oils Effectiveness of Laurus Nobilis and Mentha pullegium on S. zeamais Identification of pure compounds in apricot and apple electrophysiologically A. F. Ndomo Insecticidal effect of ozone against Plodia, F. Mouhouche M.G. Barros Tribolium, Cryptolestes & Oryzaephilus spp. C. Athanassiou et al. Investigations on microsporidian pathogens of P. interpunctella in Turkey K.F. Acar Insecticidal properties of peas homologous proteins (Pisum sativum L.) A. Mebarkia Stored-Product insects natural enemies in Sicilian wheat P. Suma Testing insecticidal efficacy of pure and combined natural substances on S. granarius S. Trdan Effect of fluctuating temperatures on the development of V.canescens S. Andreadis First application in France of heat disinfestation of a large wheat mill B. Vacquer Potential of Anisopterus calandrae and Lariophagus distinguendus as biocontrol agents of Callosobruchus maculatus O. Benkhellat Biological and molecular analysis of an isolated granulovirus Tecia solanivora against potato tuber moths A. Arroyo Natural occurrence of entomopathogenic fungi isolated from stored grain insect species in Punjab W. Wakil Session 5:	38 39 60 61 62 63 64 65 66 67 68

Chemical control in Stored-Product IPM programs Chairpersons: Shlomo Navarro & Yann Ciesla No. Page

	A comparison of efficacy of current insecticides with bio-based and mineral active substances	F. Fleurat-Lessard et al.	82
	Feeding deterrent activity of new alpha-asarone congeners	B. Lozowicka	83
	Weevil x insecticide does 'personality' matter?	R.N.C. Guedes	84
	Phosphine resistance in Cryptolestes ferrugineus from stored wheat in Oklahoma	C. Konemann	85
	Insecticidal effect of chlorantraniliprole against stored cereal product insect pests	N. Kavallieratos	86
	Insecticidal efficacy of thiamethoxam against T. confusum J. du Val	V. Goundy	87
	Determination of hexamethylentetramine in foods by GC-MS	Meehye Kim	88

25 -Sitophilus zeamais: water balance and behavior

  : bio-test, hot air, sulphuryl fluoride, food industries Water availability and temperature are the two most important abiotic variables that influence the distribution and abundance of insects. Insects have physiological and behavioral mechanisms that enable their survival in the absence of water for a long period.

	Abstract type: Poster
	Guedes N.M.P. 1 , Braga L.S. 1 , Silva L.B. 2 , Guedes R.N.C. 1 1 Dep. of Entomology, Federal University of Vicosa (UFV), 36570-000, Vicosa, MG, Brasil 2 Federal University of Piaui, Campus Bom Jesus, PI, Brasil
	E-mail: nmpguedes@gmail.com
	Abstract:

Università degli Studi di Milano-DEFENS, Via Celoria

2, 20133 Milano, Italy 2 Entomologia applicata, Laboratorio di RicercaAgroblu srl, via San Bernardo 35, 20017 Rozzano (Milano), Italy E-mail: guglielmo.cassani@agroblu.com

This year, the 9th

 Bourlaux, CS 20032, F-33882Villenave d'Ornon, France E-mail: francis.fleurat-lessard@bordeaux.inra.fr Abstract: A laboratory test-bench was built up to simulate high-temperature-short-time (HTST) exposure of infested grain samples by hidden stages of Sitophilus granarius or S. oryzae in a fluidized bed in order to model temperature-exposure time relationship. The reduction of emergence of the two weevil species (S. granarius in bread making wheat and S. oryzae in durum wheat) was determined after different exposure times in the fluidized bed exposure chamber crossed by 2 m.s -1 airflow at inlet air temperatures of 60, 90, 120 and 150°C. The influence of a grain moisture content from 14 to 19% w.b. on lethal time 50% (LT 50 ) and lethal time 99% (probit 8 or LT 100 ) was also observed. The main microbiological and technological characteristics checked on grain samples after heat treatment were measured. Higher the moisture content longer the exposure time required for the same mortality rate to be achieved. The "temperature-exposure time products" assessed at LT 50 and LT 100 were modeled as asymptotic negative regression curves. LT 100 was observed in less than 10 s exposure time for temperature at air inlet of 120 and 150°C, whatever the moisture content level. The reduction of fungal colonies was more important with long exposure time at moderately high temperature (90°C) than with short exposure time at very high temperature (150°C) (giving similar levels of insect mortality rate). For bread-making wheat, it was not observed significant changes in alveogramme rheological test parameters processed with white flour extracted from heattreated wheat samples, provided that air temperature and initial grain moisture content are at moderately high level (less than 150°C and 15% m.c.). The HTST treatment of cereal grain is an efficient and rapid method of control of weevil internal feeder stages and may be developed at practical scale for organic cereals or high-added-value use for special healthy cereal food disinfestation.

Keywords: Sitophilus granrius, Sitophilus oryzae, high-temperature-short-time treatment, wheat grain sample, fluidized-bed, quality characteristics changes. Abstract: As a result of high infestation of Dates by Ephestia spp. during its storage period which was for about 3-6 months before manufacturing and marketing it , Iraq used to apply Methyl bromide as fumigant agent to stop its infestation with Ephestia spp ,but because this fumigant agent proved to be carcinogen and ozone depleting agent , a decision was taken to stop using it internationally and nationally. This decision made Iraqi scientist to search for another methods to control Ephestia spp. or to decrease infestation to the lowest percentage. Therefore, Iraqi scientist recently concentrated on using IPM specifically biological control agents (Egg parasitoid, Trichogramma evanescens and larval parasitoid, Bracon hebetor in addition to the pheromone traps for this purpose. The research carried out into two date warehouses, one in Karbala province, while the other in the Babylon province which belong to the Iraqi company for manufacturing and marketing Dates. In these two date warehouses, the biological agents mentioned above were released periodically in addition to hanging pheromone traps. The results revealed that the percentage of preserving date in these two date warehouses were 97.5 and 98.1 % in comparison with 90.5 and 92.5 % into the control dates warehouses in which biological agents and pheromone traps did not used. Furthermore, to prove that this method for controlling Ephestia spp. in date warehouse was succeeded, we examined date from another date warehouse containing about 3250 tons of date packed in polyethylene bags and treated with methyl bromide to protect it from Ephestia spp. infestation. Unfortunately the percentage of preserving dates was only 91.7%. Finally our results showed that the mean number of Ephestia spp. captured by pheromone traps in the control warehouse which treated with methyl bromide was 18 times more than that captured into warehouses treated with biological agents mentioned above and pheromone traps.

-Application of Biological Control Agents and Pheromone Traps For Controlling Ephestia spp. in Date Warehouses of Two Provinces in Iraq

Keywords: Ephestia spp. date warehouses, pheromones traps, capture limitation, biocontrol species, Bracon hebetor 87

-Stored-product insect natural enemies in wheat industry in Sicily

Abstract type: Poster Suma P., Bella S., La Pergola A. & Russo A.

Dept of Agri-food and Environmental Systems Management -Applied Entomology sec. University of Catania, via S. Sofia, 100, 95123 Catania, Italy E-mail: suma@unict.it Abstract: The cultivation of wheat in Sicily (Italy) has its origins in ancient times and today it turns out to be the one that invests the largest areas in the island. Connected to this cultivation, a flourishing industry of primary and secondary processing has been developed and is particularly competitive in the global market thanks to the qualitative features of sicilian durum wheat. In the last decade research activities were undertaken in order to implement quality by studying infesting arthropods and by developing integrated control methods. In this context, during the monitoring activities of the stored grain pests carried out over the past decade in sevral grain industries and warehouses in Sicily, the following natural enemies were collected: Withius piger (Simon) (Pseudoscorpionida; Whiitidae), Xylocoris flavipes (Reuter) (Hemiptera; Anthocoridae), Anisopteromalus calandrae (Howard) (Hymenoptera; Pteromalidae), Habrobracon hebetor Say (Hymenoptera; Braconidae), Theocolax elegans (Westwood) (Hymenoptera; Pteromalidae), Venturia canescens (Gravenhorst) (Hymenoptera; Ichneumonidae), Cephalonomia sp. (Hymenoptera; Bethylidae). More extensive researches are currently in progress for some of these species. Here we discuss their potential use in the framework of the IPM programs.

Keywords: stored grain, predators, parasitoids, Italy.

-Testing the insecticidal efficacy of individual and combined use of four different natural substances against granary weevil (Sitophilus granarius) under laboratory conditions

Abstract type: Poster

Trdan S.

University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, Ljubljana, Slovenia. E-mail: stanislav.trdan@bf.uni-lj.si Abstract: Laboratory experiment was carried out to evaluate the impact of different natural substances on the mortality of the granary weevil (Sitophilus granarius) adults. We tested the insecticidal properties of diatomaceous earth (commercial formulation SilicoSec®), quartz sand of local origin, Neem leaf powder (offered in a local market as fertilizer and plant strengthener), common beech wood ash, combination of SilicoSec® and wood ash, combination of Neem leaf powder and wood ash, combination of quartz sand and wood ash, combination of all 4 substances tested. Natural substances in a form of powder were mixed with grain in different dose rates. Wheat grains were treated with 450 and 900 ppm of SilicoSec® and quartz sand as self-based formulation, wood ash (5 weight (w)% and 2.5 w%) as self-based formulation, Neem leaf dust (1.25 w% and 2.5 w%). Individual treatments were evaluated as mixtures of SilicoSec (450 ppm) and wood ash (2.5 w%), Neem leaf powder (2.5 w%) and wood ash (2.5 w%); quartz sand (450 ppm) and wood ash (2.5 w%); SilicoSec® (225 ppm), wood ash (2.5 w%), Neem leaf powder (0.625 w%) and quartz sand (225 ppm). Control treatment was presented as untreated grain. Specific treatment was composed from 500±0.01 g of wheat grain and concentration of natural substance and it was replicated three times. Mortality of adults was evaluated seventh, fourteenth and twenty-first day after exposure. Insecticidal efficacy was tested at three different temperatures (20, 25, and 30°C) and two different relative humidity levels (55 and 75%). Significantly the highest mortality of adults was evaluated in treatment treated with formulation consisting of wood ash and SilicoSec® (66.38±2.64%), meanwhile wood ash (2.5 w%) provoked 63.35±2.74 % mortality. In general, mortality after 21 days reached 48.52±0.01% and it was also higher at lower humidity level (46.02±1.66%). Interactions between different factors of experiment will be presented, and suggestions for the practical use of the most effective substance will be given.

Keywords: Granary weevil, physical control, diatomaceous earth, quartz sand, neem leaf powder, common beech ash, mortality, efficacy. 90 Abstract: Thermal disinfestations of flour mill structures and equipment were traditionally used before the 1960's, but they have been replaced by methyl bromide fumigation until the international ban of methyl bromide in 2005. During the last decade, heat disinfestation of flour mills was developed in Northern America and in some European countries from the 2000's workshops of heat disinfestation of wheat mills were carried out. In France, the first industrial application of this technology was attempted in 2010 for the disinfestation of a large wheat mill and was carried out by Agronet Company in collaboration with INRA Research Laboratory in Bordeaux. The heating of the flour mill was performed from electric heat generators of Thermonox® technology (Germany). The electric heaters are forced heat air ventilators enabling to reach the target temperature of 50 to 55°C in about 10-12 h in all parts of the mill. Then, temperature is maintained at this level during 24 to 36 h and optimised from the indications of temperature sensors in order to obtain complete mortality of insects, whatever their development stage. Encaged insects (Tribolium castaneum and Rhyzopertha dominica) placed in the mill during mill heating were killed at more than 97%. Very few survivals were only observed on the floor at the ground level of the mill. The reproductive capacity of individuals surviving thermal exposure was severely reduced. This first industrial application of heat disinfestation process to a large wheat mill confirmed the results of laboratory studies performed earlier at INRA Laboratory. The economic evaluation of the heat treatment in the conditions described above revealed that it is competitive compared to fumigation with similar requirements in the preparation of the building before treatment. This technology seems very promising for periodic eradication of insect colonies in cereal processing industries without use of pesticide.

-First application in France of heat disinfestation of a large wheat mill

Keywords: Wheat mill, electric heat generator, Thermonox® technology, heat disinfestation, industrial scale, stored-product insect, complete mortality.

Abstract: Technical audits were achieved in 14 grain elevators in order to record pest management practices and to evaluate the cost of insect control management practices. Stored grain elevators that were selected for this study were representative of the different ways of insect pest prevention and control system in France. The selection of storage sites was based on the results of a previous study led on 95 elevators that identified 6 keyfactors correlated with pest infestation: type of storage bin (vertical silo or 'flat' store), preventive sanitation of empty storage structures, grain cleaning, grain aeration, insecticide treatment on grain, grain temperature monitoring equipment. By combining these 6 factors, 7 modalities of pest management have been described. For each modality, two grain elevators that theoretically apply each modality have been sampled among the 95 elevators surveyed in Leblanc et al. surveys in 2010 and2011. The evaluation carried out after the visit of the selected storage facilities revealed that pest management was mainly based on chemical treatment whatever the kind of insect management system was applied. Although all the visited elevators were equipped with ambient-air aeration systems, the airflow rate and the cooling time were often found inadequate to efficiently cool all stored grain bins. Grain cleaning cannot be made systematically at reception because harvested grain lots needing cleaning are delivered at a much higher rate than the grain cleaner can receive. Pest management costs were assessed in the range 0.15 €/t to 1.40 €/t, according to the 7 modalities. The major variation factor was the type of pesticide used. The economic loss related to price penalties in the case of quality defect at the supply of domestic or export grain markets for cereals are not considered as a driving factor for insect pest management policy adaptation by grain-elevator managers. Bourlaux, CS 20032, 33882 Villenave d'Ornon, France E-mail: marie-pierre.leblanc@franceagrimer.fr Abstract: A survey of stored-wheat bulks in France was carried out during two years in spring season 2010 and 2011 to improve the knowledge about the relationships between grain storage practices, preventive sanitation, grain insecticidal treatment, stored-grain temperature management, and presence of insect species in grain samples. 95 wheat storage sites were surveyed each year (after 8-9 month storage). Besides the quantitative analysis of grain samples for insect presence and residues content, a questionnaire was submitted to grain-store managers to record their storage practices, the characteristics of their equipment for grain quality preservation, and the preventive and corrective means they use for insect control. The data from these two groups of variables were processed in a multidimensional statistical analysis to reveal significant correlations between grain management practices for sanitary quality maintenance and the level of presence of insects in wheat samples. It was observed a strong relation between the kind of storage structure and the frequency of insect presence in wheat samples. Flat storage of large grain bulks was showed the most risky storage structure vs. vertical storage in metal or concrete bins. A relation was observed between insect presence frequency in samples and stored grain temperature level and also, with the lack of temperature measurement equipment installed inside grain bins linked to cooling aeration systems. Preventive sanitation before grain harvest period, i.e. systematic cleaning and insecticide spraying of unfilled bins, was related to 50% reduction of insect presence frequency in corresponding grain samples. The preventive chemical treatment of harvested grain before long-term storage was not related to a limitation of insect presence frequency in samples taken after a 8-9-month storage period. The results of this representative survey give objective arguments for the implementation of good IPM practices and risk prevention for grain sanitary quality preservation during long-term storage periods.

Keywords

Keywords: Grain bulk sampling, grain storage site, national survey, insect presence frequency, insecticide residues content, storage practices, multidimensional data processing.

Authors of oral presentations (chronological order of presentation)

127 NOTES NOTES


Gerard Barroso
email: gerard.barroso@u-bordeaux2.fr

Cyril Ferandon

Philippe Callac

FROM THE COMPARATIVE ANALYSIS OF FUNGAL MITOCHONDRIAL GENES TO THE DEVELOPMENT OF TAXONOMIC AND PHYLOGENETIC TOOLS

Keywords: Agaricus genus, mitochondria, cox1 gene, group I intron, phylogeny

The complete sequence of the mitochondrial cox1 gene, encoding the largest subunit of the cytochrome oxidase of the Basidiomycota Agaricus bisporus has been achieved. It has the longest cox1 gene (29,902 nt) with the largest number of group I introns (18 group I introns) reported to date in any eukaryote. The group I introns in the A. bisporus cox1 gene are similar to those reported in other Basidiomycetes includeing: 3 of the 4 introns in Agrocybe aegerita, 7 of the 9 introns in Pleurotus ostreatus, 3 of the 6 introns in Moniliophthora perniciosa, and 10 of the 15 introns in Trametes cingulata. constituting 18 of the 23 introns described in all Basidiomycota available genes, Moreover, the A. bisporus cox1 gene possesses two introns specifically reported in this gene (iAbi1 and iAbi14) and one intron (iAbi 18) possessing orthologous sequences only in the Ascomycota phylum but unknown to date in the Basidiomycota. Hence, A. bisporus cox1 gene contains three-quarters (18/24) of the fungal introns described in all the fungal cox1 genes from Dikarya (Ascomycota and Basidiomycota). From the A. bisporus sequence, primers were designed to evaluate the potential of rare and widely distributed introns to act as taxonomic and/or phylogenetic markers of species belonging to the genus Agaricus. Indeed, we found that the rare introns could be specifically recovered in some strains and/or species. Sequences of the widely distributed fungal introns provide information on the phylogeny and spread of introns among distant as well as closely related species.

INTRODUCTION

Although the fungal Dikarya subkingdom (including the Ascomycota and Basidiomycota phyla) contains a broad range of taxa with a great variety of morphologies, ecologies and life cycles, it is sometimes difficult, even for specialized mycologists, to define distinct and unambiguous species boundaries based on their phenotypic differences. In this context, there is an urgent need to obtain improved and cost-effective molecular markers to help discriminate closely related species, especially for species belonging to the same genus and with commercial interests.

To date, molecular identification of fungi relies mostly on nuclear DNA markers, such as the conserved LSU-rDNA (18S), SSU-rDNA (28S) or the frequently studied variable spacers (ITS1 and ITS2) within the nuclear ribosomal gene cluster. Currently, the 18S and 28S are used to discriminate high taxonomic levels such as family and genera while the internal transcribed spacers (ITS) allow the characterization of organisms at the species level [START_REF] Bridges | The impact of molecular data in fungal systematic[END_REF]. However, in recent years, it has been necessary to develop several additional nuclear markers to resolve the inefficiency of ITS to discriminate some well-characterized species or to identify cryptic species in some fungal species complexes, as well as to clarify phylogenetic relationship between related as well as distant species. The β-tubulin gene (BenA) [START_REF] Geiser | The current status of species recognition and identification in Aspergillus[END_REF], the elongation-factor EF-1-α [START_REF] O'donnell | Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex[END_REF], the second largest subunit of RNA polymerase II (RPB2) [START_REF] Ertz | Towards a new classification of the Arthoniales (Ascomycota) based on a three-gene phylogeny focusing on the genus Opegrapha[END_REF] have often been used for these purposes.

Besides these nuclear DNA sequences, the potential use of mitochondrial DNA specific markers has become increasingly common. Indeed, mitochondrial DNA markers possess several interesting features such as their high copy number allowing an easy recovering of the sequences and the paucity of repetitive regions often involved in misleading results. Furthermore, mitochondrial DNA (mtDNA) has been reported to be less affected by genetic recombination, mainly due to a predominantly uniparental heredity, and to show a higher rate of evolution than the nuclear genome. These features make mitochondrial DNA a potentially powerful source of molecular markers to identify species [START_REF] Santamaria | Towards barcode markers in fungi: an intron map of Ascomycota mitochondria[END_REF]. Hence, a 648 nt sequence located at the 5' end of the cox1 gene encoding the subunit I of the "cytochrome c oxydase" (complex IV of the respiratory chain) has been widely used in a successful "DNA barcoding" method in several animal groups such as insects or birds [START_REF] Waugh | DNA barcoding in animal species: progress, potential and pitfalls[END_REF]. With a taxonomic resolution higher than 95% in most of the Metazoa group, this mitochondrial region was proposed as the core of global bio-identification systems for eukaryotes [START_REF] Hebert | Biological identification through DNA barcodes[END_REF].

In this way, in the Metazoa related kingdom of fungi, the cox1 gene was recently shown suitable for discriminating fungal species in the taxonomically challenging genus Penicillium [START_REF] Seifert | Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case[END_REF]. However, in most genera of the fungal kingdom, this approach is hampered by the presence of several large group I introns, frequently occurring in mitochondrial coding sequences and, especially in the cox1 gene which is the mitochondrial gene showing the highest number of introns [START_REF] Ferandon | The Agaricus bisporus cox1 gene : the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns[END_REF]. Owing to this wealth of introns, few fungal complete cox1 gene sequences are available in database, especially for species belonging to the Basidiomycetes where only six sequences have been reported and correctly annotated to date (Moniliophtora perniciosa, Pleurotus ostreatus, Agrocybe aegerita, Schyzophyllum commune, Agaricus bisporus and Trametes cingulata) [START_REF] Ferandon | The Agaricus bisporus cox1 gene : the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns[END_REF].

Here, we report the analysis of three types of mitochondrial sequences with the aim to define molecular markers suitable as taxonomic and/or phylogenetic tools at different taxonomic ranks and/or for strain fingerprinting: (i) Variables domains of the SSU-rDNA of the mitoribosome, (ii) the sequences of a" rare" group I intron, and (iii) a widely distributed intron carried by the cox1 gene. The taxonomic and phylogenetic potentials of these mitochondrial markers will be discussed by comparing with the sequences of the conventional nuclear ribosomal cluster.

The comparative analysis was carried out with six strains representing five Agaricus species (A. boisseletti, A. gennadii, A. arvensis, A. subrufescens and A. bisporus) belonging to four different taxonomic sections of this genus. Two A. subrufescens strains from two geographical origins (France and Brazil) were included in the analysis for comparison.

MATERIALS AND METHODS

Agaricus species sampling and determination. Sporophores representing six Agaricus strains (Table 1) were collected and morphologically identified. Sequences of the nuclear ribosomal unit obtained from these stains were established and compared with sequences available in the GenBank (data not shown). In vitro DNA manipulation and sequencing. Sequences used as molecular markers were obtained by conventional procedures from cloned PCR products. Total DNA of fungal strains were extracted from 0.1 g of dried carpophores after grinding in liquid nitrogen to generate a fine powder. Nucleic acids were extracted according to the Ncethyl-NNN-trimethyl ammonium bromide (CTAB) procedure adapted to small quantities of basidiomycete mycelia by [START_REF] Barroso | A miniprep method for RFLP analysis and dsRNAs detection perfected in the cultivated fungus Agrocybe aegerita. In: Science and Cultivation of Edible Fungi[END_REF]. DNA (OD 260 ) was quantified using a NanoDrop spectrophotometer (NanoDrop ND-1000, Nanodrop technologie, DE, USA), diluted in deionized sterilized Milli-Q water (Milli-Q water system production, Millipore, Saint-Quentin en Yveline, France) and stored at -20°C.

PCR amplifications were carried out using the Go Taq polymerase from Promega Corp. (Madison, Wis, USA) and with corresponding primer pairs synthesized by Eurofins MWG Operon (Germany). PCR were performed in a Programmable Thermal Cycler PTC 200 (MJ Research Inc., Watertown, Mass., USA). Each reaction contained 10 to 100 ng of fungal genomic DNA, 1 μM of both primers, 200 μΜ of each dNTP, 1 unit of Taq DNA polymerase, in a final volume of 50 μl of the appropriate buffer. Reactions were run for 30 cycles at 95 ° C for 30s, then two degrees below the lowest Tm of both oligonucleotides for 30s, 72° C for 1 to 2 min, and one final cycle at 72° C for 5 min. An aliquot of 10 µl of each PCR product was analysed by agarose (1%, w/v) gel electrophoresis containing 200ng/ml of ethidium bromide, in TEB buffer [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF].

DNA Sequencing. PCR products were purified with the Wizard SV gel and PCR Clean-Up System (Promega Corp.Madison, WI, USA) before they were sequenced by the primer walking methods using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Courtaboeuf, France) and corresponding primers used for the initial PCR amplifications. Sequence reactions were carried out, according to the supplier recommendations, in a final volume of 10 μl containing 100 ng of PCR product and 0.5 µM of the specific primer. Sequence reactions were conducted in a thermocycler by applying an initial denaturation step at 95°C for 1 minute; 27 cycles each composed of the three following steps: 96°C for 10 s, 50°C for 5 s, 60°C for 4 min. The reaction products were ethanol precipitated, dried then separated by capillary electrophoresis (on an automated sequencer ABI 3130x1, ABI Prism Corp., France) at the genomic platform of the University Bordeaux Segalen (France). Sequencing profiles were edited and corrected using the BioEdit sequence alignment editor v7.0.9 free software (Ibis Biosciences Carlsbad, CA, USA).

Sequence analyses.

Comparisons with sequences of the GenBank and EMBL databases were performed using the search algorithm BLAST [START_REF] Altschul | Basic local alignment search tool[END_REF]. Multi-alignments of nucleic acid and/or proteins were performed using Clustal W algorithm [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice[END_REF] or Muscle [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] for multiple alignment and Gblocks for automatic alignment curation [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF]. For phylogenic analyses, the sequence data were aligned and checked for accuracy manually. Pairwise evolutionary distances based on unambiguous nucleotides were computed using the dnadist (Jukes and Cantor option) and neighbor-joining programs in the PHYLIP suite. Seqboot was used for Bootstrap analysis [START_REF] Felsenstein | PHYLIP (Phylogeny Inference Package) version 3.5c. Distributed by the Author[END_REF], using algorithm version 3.572c. One thousand Bootstrap replicates were employed to determine confidence in the branches order. (The phylogenetic softwares used were part of the PHYLIP package version 3.572 Mac executables [START_REF] Chevenet | TreeDyn: towards dynamic graphics and annotations for analyses of trees[END_REF].

The alignments were also submitted to the PhyML tree building program [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] and TreeDyn for tree drawing [START_REF] Chevenet | TreeDyn: towards dynamic graphics and annotations for analyses of trees[END_REF]. PhyML was run with the aLRT statistical test of branch support. These programs were obtained on line at: http://www.phylogeny.fr/ [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the non-specialist[END_REF][START_REF] Dereeper | BLAST-EXPLORER helps you building datasets for phylogenetic analysis[END_REF]. In this case, confidence in the branches order was measured by the ratio test developed by [START_REF] Anisimova | Approximate likelihood ratio test for branchs: A fast, accurate and powerful alternative[END_REF] working with the PhyML tree building program at the phylogeny site (http://www.phylogeny.fr/) [START_REF] Dereeper | Phylogeny.fr: robust phylogenetic analysis for the non-specialist[END_REF][START_REF] Dereeper | BLAST-EXPLORER helps you building datasets for phylogenetic analysis[END_REF].

RESULTS AND DISCUSSION

Comparison of the molecular organization of the cox1 gene in A. bisporus (section Bivelares) and in other Basidiomycete species

The complete sequence of the mitochondrial cox1 gene of Agaricus bisporus was achieved. This gene is both the longest mitochondrial gene (29,902 nt) and the largest intron reservoir reported to date in an eukaryote [START_REF] Ferandon | The Agaricus bisporus cox1 gene : the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns[END_REF]. It possesses 18 group I and one group II introns. An exhaustive analysis of the group I introns available in cox1 genes shows that they are ancestral mobile genetic elements, whose frequent events of loss (according to the "late theory") and gain by lateral transfer ("early theory") would combine to obtain the observed wide and patchy distribution extending on several kingdoms [START_REF] Lambowitz | Introns as mobile genetic elements[END_REF]. Its distributions are consistent with both the "early" and "late" paradigms, which are still matters of debate [START_REF] Cusimano | Reevaluation of the cox1 group I intron in Araceae and angiosperms indicates a history dominated by loss rather than horizontal transfer[END_REF][START_REF] Sanchez-Puerta | Frequent, phylogenetically local horizontal transfer of the cox1 group I Intron in flowering plant mitochondria[END_REF]. However, the overview of the intron distribution in eukaryotes indicates that they are mainly evolving towards elimination and, in such a landscape of eroded and lost intron sequences, the A. bisporus largest intron reservoir, by its singular dynamics of intron keeping and catching, would constitute the most fitted relic of an early split gene [START_REF] Ferandon | The Agaricus bisporus cox1 gene : the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns[END_REF]. However, the analysis was carried out on phylogenetically distant organisms extending on several kingdoms.

When the analysis was limited to the Dikarya, results show that the Abi cox1 gene possesses most of the group I introns available in other Basidiomycete cox1 genes: it contains 3 of the 4 introns in Agrocybe aegerita, 7 of the 9 introns in Pleurotus ostreatus, 3 of the 6 introns in Moniliophthora perniciosa, 10 of the 15 introns in Trametes cingulata, and 18 of the 23 introns described in all basidiomycota available genes (Fig. 1). Moreover, the A. bisporus cox1 gene possesses two introns only reported in this organism (iAbi1 and iAbi14) and one intron (iAbi 18) possessing orthologous sequences found only in the Ascomycota phylum so far but unknown to date in the Basidiomycota. Hence, A. bisporus cox1 gene contains three quarters(18/24) of the fungal introns described in all the fungal cox1 genes from Dikarya, i. e. from all the available Ascomycota and Basidiomycota cox1 genic sequences [START_REF] Ferandon | The Agaricus bisporus cox1 gene : the longest mitochondrial gene and the largest reservoir of mitochondrial group I introns[END_REF]. In other to investigate the evolution (loss and/or gain), the ancestral feature and the correlated potentiality of cox1 group I introns to act as phylogenetic markers, we have studied the occurrence and phylogenetic relationships of two group I introns reported in the Abi cox1 gene (strain BS518) in five additional Agaricus species belonging to four different sections. The evolution of these intronic sequences was compared with that of the nuclear ribosomal unit and with that of variable domains of another mitochondrial gene, namely the SSU-rDNA, encoding the small rRNA (16S) of the mitoribosome.

Moniliophthora perniciosa (i5) Pleurotus ostreatus (i5)

GrII I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 I14 I15 I16 I17 I13 E1 E19 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E14 E15 E16 E17 E13 E2 E18 E20 I18
Occurrence and phylogenetic analysis of the widely distributed iAbi7 group I intron. The first intron studied was iAbi7 (1207 nt) which is the most widely distributed group I intron in eukaryotes (present in all the divisions of the fungal kingdom and also in the Viridiplantae kingdom). All the available Basidiomycete cox1 genes reported to date possess orthologous sequences of the iAbi7 intron, with the exception of Schizophyllum commune whose mitochondrial genome does not possess any intron.

From the A. bisporus cox1 gene, a couple of specific primers located in the upstream (primer U7: 5'ACAGGGTGGACGGTA3') and downstream (primer R7: 5'GATTCCTGATAAAGGAGG3' ) exon regions flanking iAbi7 were defined and used in PCR to amplify six Agaricus strains as matrix. As shown in Table 1, all the studied strains generated a PCR product of a large size around 1,200 nt. For each strain, this PCR product was purified and sequenced. The resulting sequences confirmed the presence in the cox1 gene of each strain of an iAbi7 orthologous sequence. Moreover, all these orthologous introns possess a large ORF corresponding to the heg encoding a putatively functional Homing Endonuclease (HE) involved in the transfer and site-specific integration (homing) of the mobile intron. These results suggest that the iAbi7 intron is a mobile genetic element with likely conserved functions in the Agaricus genus.

A phylogenetic tree was constructed by two methods: a distance (Neighbor-Joining) method (Fig. 2C) and the maximum likelihood method. The trees were obtained from the MUSCLE alignment of a 754 nt sequence read on both strands and located in the central part of the intron sequence and in the central part of the heg (Homing Endonuclease Gene) carried by these orthologous introns.

These trees confirm the close relationship between the six iAbi7 orthologs. The deduced relationship among the strains are in agreement with those deduced from the trees based either on the nuclear ribosomal cluster (figure 2A) or on the compiled sequences of two variable domains (V6 and V9) of the mitochondrial SSU-rDNA (figure 2B). Particularly, sequences of the intron 7 of both strains of A. subrufescens (CA454 from Brazil and CA516 from France) were identical to each other and to the A. Arvensis strain CA640 which belongs to the same section Arvenses. When these sequences were compared with iAbi7 sequence of A. bisporus which belongs to the second clade of the tree, they showed 82,6 % of nt identity.

The agreement between the iAbi7 phylogeny and those based on the nuclear ribosomal gene cluster (figure 2A) or based on the compiled sequences of two variable domains (V6 and V9) of the mitochondrial SSU-rDNA (figure 2B) argues for an ancestral feature of the iAbi7 intron. This result, along with the high conservation of the i7 intron within the cox1 genes, and the wide distribution of heg within the i7 intron represent a powerful tool for phylogenetic studies of the genus Agaricus, and more particularly at the section level.

Occurrence and phylogenetic analysis of the rare iAbi18 group I intron. The second studied intron is iAbi18 (1148 nt), a rare intron only reported in A. bisporus in the Basidiomycota and two species in the Ascomycota, Gibberella zea and Penicillium marneffei.

From the A. bisporus cox1 gene, a couple of specific primers located in the upstream (primer U18: 5'TGCAGGTTTCTATTATTGG3') and downstream (primer R18: 5'AAGTGTTGAGGGAAAAATG3' ) exon regions flanking iAbi18 were defined and used in PCR to amplify the six Agaricus strains. As shown in table 1, only one of the six strains, A. boisseletti CA123 did not possess orthologous sequence to iAbi18. In this case, the PCR product with a size of 120 nt corresponded to the size of the COX1 CDS located between both primers. The exon nature of the PCR product was verified by sequencing. This confirms that the presence of a group I intron in a strain or species is optional. However, the study of several A. boisseletti strains will be needed to determine if the observed intron loss concerns the strain level or has to be extended to the species level.

The large size PCR product (around 1,300 nt) obtained with the five other strains was sequenced. The resulting sequences confirmed the presence in the cox1 gene of each strain of an iAbi18 orthologous sequence, carrying a putative functional heg.

Moreover, the two orthologs carried by both A. subrufescens strains from Brazil and France were identical, with a 100% of nt sequence identity.

Similar to the analyses for iAbi7 sequences above, trees were constructed by the Distance and PhyML programs. The trees (figure 2D) were obtained from the MUSCLE alignment of a 896 nt sequence read on both strands, and located in the central part of the intron sequence and in the central part of the heg (Homing Endonuclease Gene) carried by the intron. These trees confirmed the close relationships between the five iAbi18 orthologs. It is to be noted that the iAbi18 ortholog harboured by the A. Arvensis strain (iAarv18) follows the phylogenetic relationships deduced from the trees based on the nuclear ribosomal unit and on the mitochondrial variable domains as well as on the iAbi7 orthologous sequences. However, in the Arvenses section, the sequence of A. arvensis was highly diverged from the two sequences of A. subrufescens. Indeed, iAarv18 possesses 91.4 % nt identity with iAbi18 and 92.3% with iAsub18, although A. arvensis and A. subrufescens are two phylogenetically closely related species (belonging to the same Arvenses section).

Section: Diversity and Taxonomy

CONCLUSION

This report focused on three different mitochondrial molecular markers and six strains representing five species and four sections of the Agaricus genus. From the preliminary results, two types of mitochondrial sequences appear as potentially suitable tools to add phylogenetic and/or taxonomic information to the well-established nuclear ribosomal units. The first one is the compiled sequences of two variable domains (V6 and V9) of the SSU-rDNA, encoding the 16S RNA of the small-subunit of the mito-ribosome. Indeed, these domains mainly evolve by length mutations involving indel (insertion/deletion) sequences and, consequently, can easily lead to CAPS markers for species determination.

The second kind of sequences is constituted by the orthologs of the iAbi7 intron. Indeed, this group I intron appears widely distributed in the eukaryote kingdom, but also in the Agaricus genus. Moreover, this mobile genetic element carries a structural gene, encoding a Homing Endonuclease (HE) which seems to have maintained its function during evolution, and consequently can constitute a permanent phylogenetic marker, to replace the "barcoding region " of the cox1 gene which is split too much by several large group I introns in the fungi, and especially in the Agaricus genus (data not shown).

The third studied sequence, the "rare" group I intron iAbi18, was shown to be frequent but not universally distributed in the Agaricus genus. Additionally, one of its orthologous sequences described in an A. arvensis strain reveals an unexpected phylogenetic behavior, suggesting that its evolution might not strictly follow evolution by descent but could involve lateral gene transfer between a distantly related species. This behavior does not allow to consider it as an easy phylogenetic marker but opens the way to the discovery of still unknown transfers of mitochondrial sequences.

Figure 1 :

 1 Figure 1: Molecular organization of the A. bisporus strain BS518 cox1 gene. The position of orthologous (red) and not orthologous (blue) introns reported in other Basidiomycota are indicated below and above the A. bisporus cox1 gene, respectively. Blue boxes represent exons E1 to E20. The names of the introns are indicated in the empty boxes. The putative functional heg in group I introns are shown by green boxes; the eroded reverse transcriptase gene carried by the iAbi 2 group II intron by an orange box.

Figure 2 :

 2 Figure 2: Distance (Neighbor) cladograms based on the nuclear ribosomal unit (A), a compilation of the V6 and V9 variable domains (B), the orthologs of the iAbi7 intron (C) and iAbi18 intron (D). The Bootstraps values indicated above branches were obtained with 1,000 replicates. The cladograms based PhyML program (maximum likelihood) led to similar trees with branches supported by comparable SH-like Branch supports.

Table 1 : Agaricus strains and sequences Section Agaricus species strain N°ITS Acc N°V6 domain V9 domain intron i7 intron i18

 1

	Sanguinolenti
	A. boisseletti	CA369	CA369	nd	CA369	0
	A. boisseletti	CA123	DQ182531
	Chitonioides
	A. gennadii	CA387	CA387	CA387	CA387	CA387
	A. gennadii	Gn17	AF432881
	Arvenses
	A. arvensis	CA640	CA640	CA640	CA640	CA640
	A. arvensis	strain 15	AJ887993
	A. subrufescens	CA516	CA516	CA516	CA516	CA516
	A. subrufescens	CA454	CA454	CA454	CA454	CA454
	A. subrufescens	I_101_S1	AY818660
	Bivelares
	A. bisporus	BS518	BS518	BS518	BS518	BS518
	A. bisporus	ATCC MYA-4626	GU327642

Section: Diversity and Taxonomy

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011


est destinée au dépôt

Insulin resistance (IR) is one of the main components of metabolic syndrome. However, the early phases of its development such as the first metabolic alterations preceding its installation remain unclear. In order to analyse the early processes over time from the gene to the metabolite levels, we have pair-fed two groups of rats with either a control (65% starch) or a high-fructose (65% fructose) diets for 45 days. The final phenotype of the fructose-fed group consisted in fasting hyperglycaemia, hyperinsulinemia, and IR at the muscle level. Several major changes were also noticed over time before the IR installation: at the molecular level, the liver expression profile (90 genes related to lipid and glucose metabolism and insulin signalling) was strongly modified at D5. This drift was quickly compensated and the differences were no longer visible between D12 and D30. Nevertheless, this phenotypic flexibility was limited over time, and after 45 days of fructose feeding, the observed profile was identical as at D5, suggesting a final drift of the molecular phenotype probably linked to IR installation. This hypothesis was further supported by the metabolic trajectory observed in urine metabolomics analysis of the fructose-fed rats, which was completely different from that of the control group. This trajectory, together with the identified metabolites between D30 and D45 (when the IR was installed) allowed us to better characterize the metabolic flexibility of the fructose-fed rats. Finally, at the metabolic level, the fructose-fed phenotype was characterized by a rapid (D5) increase in the lipogenic and gluconeogenic potentials (enzyme activities and gene expression). All together, the events preceding the fructose-induced IR were characterized by an early (D5) shift of the metabolic phenotype (at the molecular and metabolite levels) mainly reflected in a highly-induced hepatic lipogenesis. While the global phenotype partially recovered the initial profile in the middle of the trial (D12-30, concomitantly with hyperglycaemia), a final shift was observed at D45 when IR was fully installed. This final phenotype was characterized (despite fasting hyperinsulinemia) by uncontrolled beta-oxidation and gluconeogenesis and impaired insulin signalling.

  Metabolic and molecular trajectories reveal the loss of metabolic flexibility in the early processes of fructose-induced insulin resistance Sergio Polakof, Dominique Dardevet, Mélanie Larquier, Charlotte Joly, Estelle Pujos-Guillot, Jean-Louis J.-L. Sébédio, Blandine Comte To cite this version: Sergio Polakof, Dominique Dardevet, Mélanie Larquier, Charlotte Joly, Estelle Pujos-Guillot, et al.. Metabolic and molecular trajectories reveal the loss of metabolic flexibility in the early processes of fructose-induced insulin resistance. 9. Annual conference of the Metabolomics Society, Jul 2013, Glasgow, United Kingdom. 2013. hal-02745511

P11-27 METABOLIC AND MOLECULAR TRAJECTORIES REVEAL THE LOSS OF METABOLIC FLEXIBILITY IN THE EARLY PROCESSES OF FRUCTOSE-INDUCED INSULIN RESISTANCE

	Sergio Polakof Guillot 3 , Jean-Louis Sébédio 1,2 , Dominique Dardevet 1,2 , Mélanie Larquier 1,2 , Blandine Comte 1,2	1,2 , Charlotte Joly 3 , Estelle Pujos-
	1 INRA, UMR 1019, UNH, CRNH Auvergne, F-63000 Clermont-Ferrand, France, Université, Université d'Auvergne, Unité de Nutrition Humaine, BP 10448, F-63000 Clermont-2 Clermont Ferrand, France, 3 INRA, UMR 1019, Plateforme d'Exploration du Métabolisme, UNH, F-63000
	Clermont-Ferrand, France


domain: sdv sde

text:
Jean-Michel Savoie
email: savoie@bordeaux.inra.fr

R Vedier

Frederic Blanc

Nathalie Minvielle

T Rousseaut

Jean-Philippe Delgenès
email: delgenes@supagro.inra.fr

J.-M Savoie

Biomethane digestate from horse manure, a new waste usable in compost for growing the button mushroom , Agaricus bisporus ?

Keywords: composting, button mushroom, horse manure, methanisation

INTRODUCTION

Because many natural resources are limited, sustainability becomes an important concept in maintaining the human population, health, and environment. Mushroom cultivation is a direct utilization of their ecological role in the bioconversion of solid wastes generated from industry and agriculture into edible biomass, which could also be regarded as a functional food or as a source of drugs and pharmaceuticals. In Europe, mushroom industry mainly focuses in the cultivation of only few species under standardised cultivation conditions, the button mushroom, Agaricus bisporus, being largely dominant. Today, wheat straw and horse or poultry manures are the main wastes used for the button mushroom cultivation, but changes in climatic conditions and agricultural practices may results locally in difficulties for supplying, with the limitation of transportation as an objective. Concerning the integrated management of wastes streams in the future, significant changes are expected due to the use of plant biomass for biofuel and energy production and other non-food crops. On the one hand they may use the same wastes than those allowing mushroom cultivation and problems of competition for supplying may occur. On the other hand these new activities will generate new wastes to be treated and valorise by mushroom industry.

In the present investigation, the possibility to develop an integrated process of biogas production and mushroom cultivation from horse manure was studied. The objective is to state how Anaerobic Digestion of horse manures can be used to produce both renewable energy as methane and an edible biomass as button mushroom.

MATERIALS AND METHODS

Anaerobic Digestion of horse manure. Horse manure on wheat straw was purchased by a racing stable of horses. The optimal conditions for methane productions were studied in a pilot reactor of 30 L filled with 90 % of Horse manure and 10 % of garden wastes and load at a rate of 5.5 g organic matter per L per day during the process. The inoculum was from two reactors working with horse manures on wheat straw and corn. A process of thermophilic (55°C) dry (moisture content of less than 75%) anaerobic fermentation was used to produce biogas. Digestate was collected every week from the reactors and stored at 4°C before being used for growing A. bisporus.

Composting. Digestate collected every week in 5L or 30L reactors were stored at 4°C before being used for composting experiments. Composting was performed in reactors for aerobic solid state fermentation containing approximately 3 kg of raw materials in which temperature is managed [START_REF] Savoie | Stimulation of environmentally controlled mushroom composting by polysaccharidases[END_REF]. The studied parameters of composting were the composition of the mixture raw ingredients: digestates, wheat, straw, horse manure, mushroom compost ready to be spawned, wheat bran, gypsum. The microbial quality of the obtained composts was estimated by measuring the metabolic activity through the hydrolysing activity of fluorescein diacetate and the number of fungal and bacterial colony forming units at both 28°C and 45 °C as in Libmond & Savoie [START_REF] Libmond | Degradation of wheat straw by a microbial communitystimulation by a polysaccharidase complex[END_REF]. Analyses of the chemical composition of composts were performed by a private laboratory with normalised methods. The efficiency of the produced compost as cultivation substrate for A. bisporus was evaluated by measurement of mycelial growth rates in Petri dishes and small boxes inoculated with commercial spawn. After the optimisation of the formulation in the reactors, an experiment was performed at a larger scale with a conventional process of composting in two phases for estimation of mushroom yield in 0.09 m² trays filled with 8 Kg of compost spawned at 0.8 % as in previous works [START_REF] Largeteau | Effect of the fungal pathogen Verticillium fungicola on fruiting initiation of its host, Agaricus bisporus[END_REF].

A commercial substrate (Renaud Champignon, Pons, France) obtained with wheat straw and horse manure as main ingredients was used as control.

Digestates as supplement in button mushroom cultivation. Phase II mushroom compost was obtained from France Champignon Substrate Company for all experiments. It was compost made from wheat straw and horse manure. The plots consisted of plastic trays (0.09 m²) filled with 9 kg of pasteurized compost (100 kg/m 2 ) set in a randomized block experimental design with six replicates. Hybrid white strain of Agaricus bisporus (Sylvan A15) was inoculated at the rate of 0.8 %. The compost temperature during spawn-run (13 days) was maintained around 25°C. The casing soil was made of 50 % limestone and 50 % peat. Digestates collected from INRA-LBE Narbonne reactors were stored at 4°C before being used for compost and casing experiments. They were mixed manually with compost or casing at the different rates used in experiments. Mushrooms were grown under French standards of culture with controlled environmental conditions in CTC facilities. Mushrooms were harvested (without trimming the stipe) daily, at market maturity i.e. veil unbroken and gills not exposed, for the crop duration of three weeks, and this for each plot. In this way, the weight of mushrooms was registered daily. The yield was expressed as kg/m 2 .

RESULTS AND DISCUSSION

Anaerobic Digestion of horse manure. The average production of biogas containing 50 % of CH 4 was 80 + /-20 L.j -1 . The transformation yield of the dry matter to biogas was of 40 %.

Composting. Digestates may not be used directly as cultivation substrate for A. bisporus. There microbial and chemical compositions resulting from anaerobic digestion are not favourable to the mushroom. Mycelium inoculated directly in digestates does not grow from the spawn grain.

During various experiments of composting in laboratory scale reactors we observed that one of the difficulties with this material used as main component was the production of ammonia that necessitated long time of composting. Consequently we tested mixtures of digestate and wheat straw in which digestate did not represent more than 60 % (dry weight) of the raw ingredients and where commercial compost was used as an inoculum. Based on the mycelial growth rate tests, analyses of the microbial quality, pH and organic matter measurements and ammonia odour, the selected mixture was (dry weight basis): 45 % wheat straw, 30 % digestate, 11 % gypsum, 9 % wheat bran, 5 % mushroom compost. Composting of this formula at laboratory scale for 20 days followed the temperature patterns presented in Fig 1 , and allowed to obtain compost on which the mycelial growth rate was at mean 2/3 that measured in commercial compost used as control. Table 1 shows their chemical compositions and that of the initial mixture before composting. The differences in mineral composition may be noticed and is due to the high quantity of gypsum we used. The values of the other parameters of the chemical compositions are close.

The above mixture of raw ingredients was used for the production of 50 Kg of compost with a conventional composting process performed in trays. The recorded mean temperature during composting is reported in Fig 2 . The yields of mushroom for 5 weeks of harvest were 25.9 ± 5.68 and 15.2 ± 1.64 Kg/m² (means of 6 replicates) for the control (commercial mushroom compost) and the compost from digestate respectively. The mushroom mean weights were not different in the treatments. The present work shows that digestates from dry anaerobic digestion of horse manure for production of biogas might be used as raw ingredients of composting in place of horse manure for the cultivation of A. bisporus. However it is necessary to add an inoculum of aerobic thermophilic microbial communities. This inoculum can be obtained by introducing mushroom compost obtained with conventional ingredients or by inoculation of pure cultures of useful microorganisms either bacteria [START_REF] Ahlawat | Potential of thermophilic bacteria as inoculant for commercial scale white button mushroom (Agaricus bisporus) compost production[END_REF] or fungi [START_REF] Coello-Castillo | Production of Agaricus bisporus on substrates pre-colonized by Scytalidium thermophilum and supplemented at casing with protein-rich supplements[END_REF]. We only obtained half of the yield of a commercial cultivation substrate, but the simple composting process used here could be optimised.

Digestates as supplement in button mushroom cultivation. Incorporation of digestates into the compost at spawning is compared to the effect of a proteinic supplementation. In that case, compost was supplemented with Promycel 600 at spawning at the rate of 0.8 %. One trial was conducted to investigate the effect of two rates of digestates (2% and 5%). Non-supplemented compost produced 23.48 kg/m² of mushrooms (Fig. 3). With commercial supplementation, yield increased very significantly to 33.38 kg/m² (+ 42 %). In the same time lower increases in yield were observed with addition of digestates to the compost (+ 12 % to + 21 %). Digestates can't be a substitute to commercial supplementation to improve mushroom yield, but might be used with some beneficial effects. Incorporation of digestates into the standard casing soil was made at different rates (5-10-15 and 20%). Compost cased with commercial casing (control) produced 32.79 kg/m² of mushrooms (Fig. 4). On casing with digestates, we noted the same level of mushroom production compared to the control, despite the presence of "black" area in the casing soil. Only the highest rate of digestates (20%) gave a significant lower yield (28.24 kg/m²). In our conditions, we can conclude that digestates may be added in casing soil, but without beneficial effect on A. bisporus mushroom production.

Figure 1 :

 1 Figure 1: Temperature recorded in the substrate during composting at laboratory scale in fermenters.

Figure 2 :

 2 Figure 2: Temperature recorded in the substrate during conventional composting at pilot scale

Figure 3 :

 3 Figure 3 : Effect of 2 rates of digestates, used as supplementation at spawning, on total yield

Figure 4 :

 4 Figure 4: Effect of 4 rates of digestates incorporated into casing on total yield. Error bars are standard deviations

Table 1 :

 1 Chemical composition of the mixture of raw ingredients including digestate, of obtained compost, and commercial compost as control

		mixture of raw	compost	commercial
		ingredients	from digestate	compost
	Minerals (g Kg-1)	288.6	345.5	303.3
	K	25.9	35.5	32.1
	Mg	5.5	7.11	6.4
	Ca	66.4	80.1	55.8
	Na	1.8	2.3	2.8
	S	65.5	84.4	35.8
	Organic C	356	323	348
	N (Kjeldahl)	17.8	21.5	23.1
	C/N	20.0	15.0	15.1
	water soluble OM (% of total OM)	29.5	29.8	32.5
	% of OM non soluble in acid detergent	45.8	52.1	48.8
	Hemicelluloses (% of total OM)	24.7	18.1	18.7
	Cellulose	5.1	5.1	2.9
	lignin and cutin	40.7	47.1	45.9

Section Posters

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011

ACKNOWLEDGEMENT

The authors want to acknowledge research funding from Ministère de l'Agriculture, de l'Alimentation, de la Pèche, de la ruralité et de l'Aménagement du Territoire, Cas Dar n°8068. We are grateful to Ludovic Devaux for pilot scale composting and mushroom cultivation.

CONCLUSION

Dry and thermophilic anaerobic digestion of horse manure on wheat straw is efficient for the production of Methane and digestate produced as waste of this process proved to be a raw material that can be used in mixture with others for the composting process aiming at producing a cultivation substrate for the button mushroom A. bisporus. However mushroom yields obtained in this study were lower than those obtained with compost used in Europe and further improvements are needed. The use of digestate as supplement, without composting was less efficient for the mushroom production.

Integrated management of horse manure for biomethane and mushroom production might have an economic interest if it is used in parallel with the current way of compost production from horse manure.


text:
J C Motte

E Trably

R Escudié

N Bernet

J-P Delgenes

J-P Steyer

C Dumas

IMPACT OF WATER CONTENT ON BIOHYDROGEN PRODUCTION FROM LIGNOCELLULOSIC RESIDUES

Biohydrogen production from lignocellulosic residues is restricted by several factors that limit industrial application of this process. Among them, the reactor size reduces the economical feasibility of the process. Dry ananerobic processes operates with low water quantity and could allow treating more waste. This study investigates the hydrogen production from wheat straw under several dryness conditions (sixtotal solid (TS) content). The results showed a hydrogen production of 14 NmlH2.gVS-1 in semi dry consitions (9.5-14.2 %TS). For dry medium (18.8 to 28.1 %TS), the hydrogen production decreases to lower value of 6-7 NmlH2.gVS-1. Finally, in highly dry conditions (28.1 to 33.2%) the hydrogen production is reduced to 3-4 NmlH2.gVS-1. The study reveals that hydrogen can be produced in semi-dry conditions in similar proportion than saturated conditions. However, dry and particularly highlydry conditions are not favourable to hydrogen production.

INTRODUCTION

It is well established that bioenergy production from solid waste can be an attractive alternative to fossil fuels. Biogas, such as methane or hydrogen, can be produced by anaerobic digestion from lignocellulosic substrates. At industrial scale, anaerobic digestion processes treating municipal and agricultural residues are widely used in dry conditions [START_REF] De Baere | Anaerobic digestion of solid waste: state-of-the-art[END_REF]. This technology presents many advantages such as compact reactor, high solid concentration in the inlet, less energy for heating and less water addition. So far, hydrogen production by dark fermentation has usually been investigated under saturated water conditions [START_REF] Guo | Hydrogen production from agricultural waste by dark fermentation: A review[END_REF][START_REF] Nasirian | Development of a method for biohydrogen production from wheat straw by dark fermentation[END_REF][START_REF] Ren | Bioconversion of lignocellulosic biomass to hydrogen: Potential and challenges[END_REF]. All the advantages of dry processes could overcome the low productivity of biohydrogen production. The objective of this study is to investigate the impact of water content on fermentation of lignocellulosic biomass.

MATERIAL AND METHOD

Wheat straw (Triticum aestivum) harvested in summer 2010 was grinded to 1 mm. This wheat straw contains 95.6 % of total solids and 88.9 % of organic matter. It was used as substrate to be andWaste, Venise, ITA (2012-11-12 -2012-11-15). Padova, ITA : CISA Publisher.

Venice 2012, Fourth International Symposium on Energy from Biomass and Waste degraded in mesophilic conditions under limited water content conditions. The range of total solid (TS) content covered the semi-dry (9.5 and 14.2 % TS) and dry fermentation conditions (18.8 to 33.2 % TS). Four replicates were performed for each operating condition. A protocol for the assessment of biohydrogen production was adapted to supply the different dryness conditions [START_REF] Fang | Acidophilic biohydrogen production from rice slurry[END_REF]: same quantity of susbstrate, trace element and MES buffer (2-(Nmorpholino)ethanesulfonic acid) substrate fixed to inoculum ratio of 20 on volatil solid basis. Biogas volume and composition were analyzed automatically by pressure measurement and gas microchromatography (VARIAN 580). Solid and liquid samples were analyzed at the beginning and the end of the experiment.

RESULT AND DISCUSSION

The results indicate a strong influence of the TS content on biohydrogen production from wheat straw. The figure 1 represents the cumulative biogas production among the experiment. The table 1 shows the characteristic values concerning the hydrogen production.

Figure 1. Biogas production over experiment time At 9.5 and 14.2 % TS, the biogas production was fast and the hydrogen yield equalled the standard value obtained in saturated water conditions (≈ 14 Nml H 2 / gVS) [START_REF] Quéméneur | Effect of enzyme addition on fermentative hydrogen production from wheat straw[END_REF]. For higher TS contents (18.8 and 24.0 % TS), the biogas production remained similar to the previous ones, but the hydrogen productivity decreased significantly (> 6 Nml H 2 / gVS). At 30% TS, two different behaviours were observed on the four replicates. Two replicates, named group 30a, had a rapid hydrogen production (3.7 day to reach 90% of maximal H 2 value); while the two other replicates, named group 30b, had a slow hydrogen production (8.0 day to reach the mawimal biohydrogen production). Moreover, in these two different groups the maximal hydrogen production was 7.2 and 4.3 Nml H2/ gVS respectively. The higher TS content tested (35 % TS) showed a slow profile of biogas production (8.4 day to reach 90% of maximal H 2 production) with a low hydrogen yield (2.6 Nml H2/ gVS). This observation is consistent with hydrolysis rates previously reported under similar conditions [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF]. 9.5% 14.5 ± 1.4 2.6 ± 0.5 14.2%

13.9 ± 0.6 2.5 ± 0.4 18.8% 6.5 ± 0.3 2.5 ± 0.1 24.0% 6.4 ± 0.2 3.6 ± 0.6 28.1%a 7.2 ± 0.1 3.7 ± 0.1 28.1%b 4.3 ± 0.9 8.0 ± 0.1 33.2%

2.6 ± 0.6 8.4 ± 0.7

CONCLUSIONS

As a conclusion, this work demonstrated that dry processes could be applied for hydrogen production from lignocellulosic biomass. Furthermore, these results open the perspective of a two stage dry process coupling hydrogen and methane production. Further studies are in progress to better understand the microbial processes involved in this kind of ecosystems.

  Comment citer ce document : Motte, J.-C., Trably, E., Escudié, R., Delgenès, J.-P., Bernet, N., Dumas, C. (2012). Impact of water content on the hydrogen production from lignocellulosic residues by dark fermentation. In: Proceedings Venice 2012. Presented at Venice 2012 -4. International Symposium on Energy from Biomass

  Comment citer ce document : Motte, J.-C., Trably, E., Escudié, R., Delgenès, J.-P., Bernet, N., Dumas, C. (2012). Impact of water content on the hydrogen production from lignocellulosic residues by dark fermentation. In: Proceedings Venice 2012. Presented at Venice 2012 -4. International Symposium on Energy from Biomass and Waste, Venise, ITA (2012-11-12 -2012-11-15). Padova, ITA : CISA Publisher. Venice 2012, Fourth International Symposium on Energy from Biomass and Waste

Table 1 .

 1 Hydrogen production from wheat straw at various TS contents (based on four replicates)

	Condition (% of TS)	Hydrogen production (Nml H 2 / gVS)	Time to reach 90 % of the total H 2 production (days)


Marie-Christine Daunay

Anne-Laure Laure Girard

From the 8ench to Innovative Applications UNIVERSITÉ DE NEUCHÂTEL

HAL is a

African eggplants and nightshades: an overview of indigenous vegetables

INRA, Génétique et Amélioration des Fruits & légumes, UR 1052, Montfavet, France Several Solanaceae species are found among the many indigeneous African vegetables. They belong to the wide genus Solanum, and are part either of the Leptostemonum subgenus (e.g. S. aethiopicum, S. macrocarpon) to which the common eggplant S. me/ongena belongs to, or to the subgenus Solanum members of which are often referred to as nightshades (e.g. S. scabrum, S. vil-/osum). Statistical data of surfaces or tonnage for these fruit and/or leafy vegetables are scarce. Most of the material cultivated nowadays still consist of local land races displaying a wide phenotypic diversity. For most of the species, geneflows occur between the wild native compartment and the cultivated one, with as a result a loose llrnit between both. Cultivated germplasm and wild relatives, collected as early as in the 1980s under the umbrella of IBPGR, and much more recently by AVRDC, is presently maintained by a few germplasm holders. The main scientific breakthroughs concem (i) the taxonomie treatment of these species, (ii) the investigation of their wild relatives, (iii) inheritance studies and more recently (iv) some molecular insights of their diversity. Breeding targets are numerous and the potential of genetic progress is high given the wide diversity of these species. Genomic knowledge of betler known Solanaeeous crops can benefit to the breeding of these indigenous vegetables, although the low seed priee African peasants can yet afford might limit its use.


est destinée au dépôt

Insulin resistance (IR) is one of the main components of metabolic syndrome. However, the early phases of its development such as the first metabolic alterations preceding its installation remain unclear. In order to analyse the early processes over time from the gene to the metabolite levels, we have pair-fed two groups of rats with either a control (65% starch) or a high-fructose (65% fructose) diets for 45 days. The final phenotype of the fructose-fed group consisted in fasting hyperglycaemia, hyperinsulinemia, and IR at the muscle level. Several major changes were also noticed over time before the IR installation: at the molecular level, the liver expression profile (90 genes related to lipid and glucose metabolism and insulin signalling) was strongly modified at D5. This drift was quickly compensated and the differences were no longer visible between D12 and D30. Nevertheless, this phenotypic flexibility was limited over time, and after 45 days of fructose feeding, the observed profile was identical as at D5, suggesting a final drift of the molecular phenotype probably linked to IR installation. This hypothesis was further supported by the metabolic trajectory observed in urine metabolomics analysis of the fructose-fed rats, which was completely different from that of the control group. This trajectory, together with the identified metabolites between day 30 and 45 (when the IR was installed) allowed us to better characterise the metabolic flexibility of the fructose-fed rats. Finally, at the metabolic level, the fructose-fed phenotype was characterized by a rapid (D5) increase in the lipogenic and gluconeogenic potentials (enzyme activities and gene expression). All together, the events preceding the fructose-induced IR were characterized by an early (D5) shift of the metabolic phenotype (at the molecular and metabolite levels) mainly reflected in a highlyinduced hepatic lipogenesis. While the global phenotype partially recovered the initial profile in the middle of the trial (D12-30, concomitantly with hyperglycaemia), a final shift was observed at D45 when IR was fully installed. This final phenotype was characterized (despite fasting hyperinsulinemia) by uncontrolled beta-oxidation and gluconeogenesis and impaired insulin signalling.

N°

  Metabolic and molecular trajectories in the fructose-fed rat model provides new information about the loss of metabolic flexibility during the early insulin resistance installation Sergio Polakof, Dominique Dardevet, Catherine Besson, Mélanie Larquier, Charlotte Joly, Estelle Pujos-Guillot, Jean-Louis J.-L. Sébédio, Blandine Comte To cite this version: Sergio Polakof, Dominique Dardevet, Catherine Besson, Mélanie Larquier, Charlotte Joly, et al.. Metabolic and molecular trajectories in the fructose-fed rat model provides new information about the loss of metabolic flexibility during the early insulin resistance installation. Phenotypic flexibility Symposium, Nutrigenomics Organisation (NuGO). NLD., Feb 2013, Madrid, Spain. hal-02745563 , INRA, Clermont-Ferrand, France Besson, CB , INRA, Clermont-Ferrand, France Larquier, ML , INRA, Clermont-Ferrand, France Joly, CJ , Plateforme d'Exploration du Métabolisme, Clermont-Ferrand, France Pujos-Guillot, EPG , Plateforme d'Exploration du Métabolisme, Clermont-Ferrand, France Sebedio, JLS , INRA, Clermont-Ferrand, France Comte, BC , INRA, Clermont-Ferrand, France