AN AGENDA FOR SUSTAINABLE OPERATIONS AND SUPPLY CHAIN MANAGEMENT RESEARCH

Keywords: Sustainability, operations management, supply chain management

The purpose of this paper is to review some of the key challenges to sustainable operations and supply chain management and provide future research directions. While sustainability and sustainable development is defined in many ways, the principles of integrating economic, environmental and social imperatives are well understood. Despite an expanding understanding of the implications of sustainability, research to date, as well as practical implementation, reveals many areas for further development. Sustainability is a particularly difficult subject to research which raises many trade-off situations. As examples, there are conflicts between different environmental factors (energy and waste) when taking a life cycle perspective and burgeoning institutional constraints limit the economic degrees of freedom (limiting opportunities for closed loop solutions). Drawing on past experiences, recent research and current practices, this article provides a framework for future research in sustainable operations and supply chain management.

INTRODUCTION

Sustainable supply chains has been variously described as " the strategic, transparent integration and achievement of an organization's social, systemic coordination of key interorganizational business processes for improving the environmental, and economic goals in the long-term economic performance of the individual company and its supply chains." (Carter and Rogers, by Joe Miemczyk 2008) or "'supplier selection including environmental and social criteria, 'auditing and monitoring of suppliers & certification of suppliers according to environmental and social standards" [START_REF] Seuring | Core issues in sustainable supply chain management -a Delphi study[END_REF]. Overall the integration of the three pillars of sustainability environmental, social and economic -into company operations and supply chains is the main route to sustainability. For the purposes of this paper the majority of the discussion will focus on environmental sustainability, but many of the challenges presented here are equally relevant to social and economic sustainability.

There are many drivers of sustainability in operations and supply chains but these can be broadly grouped into three main areas. These are compliance, risks and opportunities. On the compliance side there have been many developments in legislation over recent years particularly in Europe.

These regulations have variously driven responsibility for the design and manufacturing of products which have lower impacts on the environment, especially in the area of waste generation.

Compliance however is not only a legislative issue but is also driven through standards and codes of practice. More and more companies are imposing standards and codes on their own suppliers in order to drive them to meet new levels of performance related to environmental management and social issues [START_REF] Preuss | Ethical Sourcing Codes of Large UK-Based Corporations: Prevalence, Content, Limitations[END_REF]. A typical approach is for customers to impose suppliers to adopt ISO14001 for example, but of course this only ensures a base level of performance, in a sense leveling the playing field, albeit to a 'higher' common level. The second main driver relates to risks due to not effectively managing operations and supply chains in a responsible manner. On the one hand companies face additional unforeseen costs due to their suppliers not meeting regulations or as a result of accidental pollution or spillages of harmful chemical (clean-up costs or loss of operating licences). On the other hand, and potentially more significant is the damage to reputation if a company is seen as not managing its environmental impacts effectively or allowing product safety to be compromised. In such cases the loss of reputation can immediately affect the bottom line through lost sales as was the case for Gap and Nike in the 1990s due to the use of child labour, and more recently Nestle through the use of palm oil from non-sustainable sources. The last driver is that of opportunity to lower costs or to raise sales income. If companies can avoid waste disposal for example through process redesign, this can avoid disposal charges which in some countries is very significant. On the other hand, products which are seen as green might even command a premium in some customer segments, so that revenues can be increased by demonstrating the product contains recycled materials or avoids hazardous contents harmful to the environment (such as green cleaning products produced and marketed by both Unilever and Procter & Gamble). The combination of drivers tends to depend on the sector and the type of customer and products, but there can be no doubt that the importance of these drivers for sustainable oprations and supply chains is only increasing at present.

KEY CHALLENGES TO SUSTAINABLE OPERATIONS AND SUPPLY CHAINS

While the drivers for sustainability are quite well understood, the way that companies implement responsible operations is less well defined. In particular the specific challenges that companies face are wide and varied and the links between them relatively unexplored. For the purposes of this paper a number of challenges (not all) are discussed with the intention to provide some direction for future research in this field. The following diagram (Figure 1) encapsulates the challenges discussed here. The idea that operations are part of supply chains is shown by including 'produce' as one of the supply chain processes. This framework is loosely based on the SCOR model which has been widely adopted by industry in the last decade or so. The paper will make reference to these challenges with relation to different processes of the supply chain. It is not possible here to provide detail on each of the processes, instead the paper aims to provide some examples of how the challenges can manifest in different parts of the supply chain.

The paper takes each of the four supply chain elements in turn (source, produce, deliver and return) in the following sections.

SOURCING

One of the key issues in sustainable sourcing is the use and imposition of standards whether they be based on legal frameworks or voluntary agreements. In developing countries, this is especially difficult to manage as often the standards vary widely and are difficult to control. One approach that many companies have adopted is this use of codes of conduct for suppliers. Here suppliers are

Responsiv eness & Flexibility

requested to sign the code and demonstrate to the customer how the elements of the code are met.

There are two issues related to this. The first is that compliance is often self reported by the supplier. In some cases the customer will audit a selection of suppliers and even more rarely customers use third parties to verify the supplier compliance to the code. This leads to a great deal of variability in the application of the codes. A second issue is the actual composition of the code.

Recent research has shown that while most codes of conduct request compliance to local laws and environmental protection, it is rare to find codes that consider other elements including life cycle thinking and the protection of biodiversity [START_REF] Preuss | Ethical Sourcing Codes of Large UK-Based Corporations: Prevalence, Content, Limitations[END_REF].

Another key challenge is the requirement of responsiveness and flexibility in logistics from suppliers. In some industries just in time deliveries are important to responding to customer demand and also the reduction of working capital in the form of inventory. Hence this has driven the need for smaller and smaller delivery lot sizes. As part of a study in the automotive sector we found that there are often missed opportunities to consolidate or pool logistics across suppliers and customers in order to both reduce costs of flexible logistics but also to reduce fuel consumption and carbon emissions. In this case we found in one logistics provider three automotive factories shared 11% of their suppliers and at least two factories had many more suppliers in common, but collections from these suppliers was not shared and coordinated. These kinds of actions have the potential to reduce road transport impacts significantly.

A final issue relating to the sourcing part of the supply chain is the question of what to measure.

Recent research has shown that in the majority of cases the literature focuses on internal processes and how they are organised, and the issues of material, wastes and recycling [START_REF] Miemczyk | Sustainable purchasing and supply management: a structured literature review of definitions and measures at the dyad, chain and network levels[END_REF]. Far less research is focused on the issues of life cycle analysis, risks and other elements of sustainability. Related to this challenge is 'where' to measure and again here the majority of research measures the extent of sustainability in internal processes and direct relationships with direct suppliers. The challenge on the sourcing side of the supply chain is the identification of risks all the way back to raw materials and also consideration of the influence of stakeholders outside of the traditional supply chains such as NGOs. Most research does not take this level of analysis as a basis for studies.

PRODUCTION

Sustainability in operations and production has been a focus of operations management research for a number of years [START_REF] Corbett | Special Issue of Production and Operations Management: Measuring the Impact of Sustainable Operations[END_REF][START_REF] Kleindorfer | Sustainable Operations Management[END_REF]. Research has covered a number of issues from the links between environmental management and production and quality management [START_REF] Hart | Beyond Greening: Strategies for a Sustainable World[END_REF][START_REF] Theyel | Management practices for environmental innovation and performance[END_REF]. Bringing together advanced production systems and sustainability however has been less studied. One focus of this work has been the link between green and lean, with most work showing there to be a positive relationship [START_REF] Florida | Lean and green: the move to environmentally conscious manufacturing[END_REF][START_REF] Mollenkopf | Green, lean, and global supply chains[END_REF][START_REF] Rothenberg | Lean, green , and the quest for superior environmental performance[END_REF]. However as mentioned in the previous section there is not always a positive relationship between lean and green, or in the previous case JIT and green. Other work in the automotive sector also shows that we should be cautious about stating this link.

One area identified as problematic in the lean and green link is the car painting process. Indeed this is viewed as the most environmentally harmful single process element of making cars. Our hypothesis was that if manufacturing was to be truly flexible to respond to actual customer demand then the painting process would need to operate on minimum batch sizeseven a batch size of onehence some of the data collected in the study of the paint process included emissions and waste data in order to estimate this impact. At the time most of the car plants in the study still operated solvent based paint applications. With potentially smaller batch sizes in manufacturing, the changeover frequency would increase and consequently the amount of pollution and waste from the purging process would increase as well (shown in Figure 2). This was published alongside other issues of painting flexibility in 2004 [START_REF] Howard | A strategic approach to automotive painting[END_REF]. Of course, lean is about eliminating waste, so in theory at least lean companies would strive to reduce changeover waste not just relating to time and effort but also environmental pollution. After discussions with industry in an industry conference workshop we proposed a significant painted body store to decouple this process from the end demand and maintain larger batch sizes, in a sense this is a contribution to the decoupling literature providing 'environmental' arguments for where to locate a decoupling point. More general findings based on environmental impacts in the automotive supply chain were published in a book chapter [START_REF] Miemczyk | A car in 3 days! Environmental Implications for the automotive supply chain[END_REF].

Figure 2: Relationship between batch size and environmental impact (based on [START_REF] Howard | A strategic approach to automotive painting[END_REF].

Another issue that was identified in the car manufacturing process was that of unreliability. Here we found that it is not uncommon, especially in the painting process, for cars to require rework either by using offline mini spray booths or for cars to go through the process of painting twice. It is clear that this rework also has implications for pollution and waste which is increased due to quality problems and process unreliability. Hence if we can improve reliability in processes that pollute the environment, we can decrease the associated impactsagain this is an area which is under-researched.

This section on operations and production has focused on two issues flexibility and reliability which are key operations priorities in many industries today. Where there are processes that led to environmental harm these priorities can potentially increase this impact unless they are purposively designed to avoid such effects. One approach could be to re-examine the set-up/die exchange procedure to make sure that increasing changeovers does not increase impacts. The following section examines challenges to the outbound logistics process from manufacturers and distributors towards the end customers.

DELIVERY

Sustainable distribution and delivery has been a focus of research in the logistics field for a number of years now [START_REF] Browne | Assessing transport energy consumption in two product supply chains[END_REF][START_REF] Christensen | The environment and its impact on the supply chain[END_REF][START_REF] De Brito | Towards a sustainable fashion retail supply chain in Europe: Organisation and performance[END_REF][START_REF] Wu | Environmentally responsible Logistics Systems[END_REF].

We examined the impact of the short delivery time on cost and the environment as part of automotive logistics case studies [START_REF] Holweg | Logistics in the 'Three Day Car' Age: Assessing the responsiveness of vehicle distribution logistics in the UK[END_REF]. We found that cost and carbon emissions increased by about 30% overall due to the increased dispersion of delivery points to justify full truck delivery each day, compared with grouping orders to together and delivering when a dealer has 3-4 cars due for delivery to customers. We also found that this varied according to geographic area, being of greater importance when delivery was closer to the point of distribution, as the line haul part of the delivery was a lower proportion of the whole distribution chain. Therefore this is an example of how more responsive delivery (from 3-4 days to 1 day) can lead to greater impacts, especially when undertaking local distribution (Figure 3).

Figure 3: The impact of one day delivery on kilometres driven to deliver new cars [START_REF] Holweg | Logistics in the 'Three Day Car' Age: Assessing the responsiveness of vehicle distribution logistics in the UK[END_REF].

We also considered ways of mitigating this effect by changing the typical method of delivery such as by using smaller trucks or sharing (mutualising) the delivery stream from more than one vehicle brand.

Another trend in distribution is to use alternative channels to market such as the internet. Instead of having products collected by customers from retail outlets this approach means that customers have their products delivered by truck or van owned by the retailer (or subcontracted by them).

In theory at least this can reduce overall impacts because customers orders can be collected together and delivered in one delivery loop thus saving vehicle kilometres per customer and so reducing carbon emissions. However, linking to the challenge of reliability, if the truck fails to deliver because the customer is absent the resulting impacts of returning to do the delivery again or even the customer driving to the retailer to collect the items, the overall impact can be much greater than for the traditional shopping trip by car.

Another significant issue in delivery is that the majority of logistics operations are carried out by third party logistics providers, who in many cases subcontract part of their transport to other subcontracted transport companies. This means that measuring and monitoring and imposing standards to reduce the environmental impact of transport is increasingly difficult. One example is DHL, the largest logistics contractor (express delivery and also industrial logistics solutions) in the world today. The majority of the carbon emissions related to this company come under what is known as scope 3 (the United Nations definition of external emissions from suppliers). This is because most of the actual transport service is done by DHL's sub-contractors. These are often small family owned businesses who operate on low margins, such that investment in clean engine technology and innovative logistics practices to reduce emissions are often out of reach.

RETURNS

This fourth area of sustainable supply chains has developed significantly in Europe due to new legislation developed in the last 10 years based on the principle of producer responsibility [START_REF] Walther | Impact of WEEE-directive on reverse logisics in Germany[END_REF]. In this context, companies have had to develop strategies for bringing back products at the end of their life at no cost to the end user [START_REF] Jr | The Reverse Supply Chain: Smart Manufacturers Are Designing Efficient Processes for Reusing Their Products[END_REF]. This is particularly challenging because revenue from the reuse and recycling of components and materials is uncertain. Furthermore the cost of doing this whilst taking into account stringent environmental standards (dealing with hazardous waste such as oil and acid from car batteries) can be elevated. Hence it is within this context that companies in Europe have developed new processes and structures to meet newly mandated recycling levels [START_REF] Cairncross | How Europe's Companies Reposition to Recycle[END_REF]. However this is not to say that reverse logistics and product take back systems did not exist before these regulations. In fact companies have been engaged in this area for decades, but with the principle goal of obtaining or recovering value [START_REF] Roy | Successful recycling through value chain collaboration[END_REF]. One case exemplar is Xerox who has been involved in take back and recycling of their photocopiers for many years [START_REF] Maslennikova | Xerox's approach to sustainability[END_REF][START_REF] Mcintyre | Environmental Performance Indicators for Integrated Supply Chains: The Case of Xerox Ltd[END_REF]). In fact this example is one of closed-loop supply chains where recycling materials are reused as raw material for new or at least remanufactured machines. The objective of reusing parts and recycling parts is made easier because not only is the product designed with this in mind but in addition the whole business model based on leasing machines and providing a service is more aligned to achieving product recovery.

As the end user often does not own the copier but instead leases the product, Xerox is able to retain ownership and has control over the 'retirement' process, deciding when it is economically viable to return the product to the remanufacturing site (based on balancing revenue and maintenance cost which rise as the life extends). The process of recovery then follows a number of predefined steps in sorting according to quality then either repairing, refurbishing/reconditioning, remanufacturing (as new) or recycling the constituent parts and materials. Xerox are actually able to save money with this process by having cheaper raw materials and remanufacturing copiers to lease into the market 'as new'.

The following chart (Figure 4) highlights some of the key issues related to the reverse or return supply chain. The first observation to make is that typically there is a complex network of relationships, quite often not formalized, using spot market type transactions for transport and sales of recycled materials. This means that taking a long term, partnership perspective where risks and rewards are shared is difficult to implement. A second major challenge is the problem of both supply and demand uncertainty which means that both flexibility and responsiveness are crucial, but can lead to high cost in an environment where the margins are often quite low. A third difficulty is the lack of standards in the returns process environment. This is driven partly by the high variability in the quality of returned products and also the wide variety of product acquisition routes (from final customers, local authorities and other organizations).

Figure 4: Relationship map of end of life product take back systems (Based on (Miemczyk, 2008)

CONCLUSIONS

There are many challenges, this paper has only mentioned some of these. Yet alongside these challenges come many opportunities for research that fills gaps in theory but also provides impact to companies.

AN AGENDA FOR FUTURE RESEARCH

High supply uncertai nty

High demand uncertai nty

Lack of standa rds

The field of sustainable operations and SCM is still in a developing phase and therefore there are many opportunities for new research. The following non-exhaustive list provides some insights for potential research in this area and is based on the general discussion given in this paper.

 Standards, measurement and monitoring

Both from a research and practice perspective there are many limitations in the measures that are used. In particular research could focus on how companies adopted approaches related to risks, life cycle management and how energy and carbon are managed in supply chains. Another major opportunity is to look at how companies are measuring and monitoring the sustainability performance of their suppliers further up their supply chains past the first tier of supply. This is particularly relevant to identifying and controlling risks but also essential if a life cycle approach is to be taken where often the majority of carbon emissions can be traced back to raw materials.

 Responsiveness and flexibility

A key trend in supply chains is the need for responsiveness and the required flexibility to achieve this. In sectors where there is more and more product proliferation such as in the automotive sector [START_REF] Stäblein | Theoretical versus actual product variety: how much customisation do customers really demand?[END_REF], being able to respond to end customer demand is essential. However despite research and advanced practices, there is very little research in this area. Although research has explored to limited extent the link between lean and green, supply strategies such as agile and leagility warrant further examination in the context of sustainability. In addition, the huge challenges of product take back are further hindered by product proliferation and therefore research can also look to address how design and manufacturing techniques such as modularity can help this process of material and component recovery.

 Reliability

Rework, expediting, non-productive actions are all defined as wastes in the lean production paradigm. Equally this activities can cause environmental pollution and wastes over and above that which is 'planned' within production processes. Furthermore, logistics systems which fail to deliver as planned also require 'rework' and are non-valued added. Hence there is a need to assess the total costs of failure including the costs to society due to the release of harmful chemicals or the generation of excessive wastes, providing further arguments for prevention actions and right first time.

 Life cycle thinking

The final opportunity suggested in this paper is the adoption of life cycle thinking in the delivery of products and services. As in the adoption of total cost thinking, life cycle thinking aims at drawing a picture of the total impacts of products from the extraction of raw materials to the disposal and return of the end of life products. Life cycle management will never be achievable without effective supply chains, so a possible research question could be are well organized and integrated supply chains more able to adopt life cycle management, than disparate, uncoordinated supply chains.

One other promising area is the development of product service systems -service dominant logic whereby instead on a focus on manufactured product companies concentrate on the solving the customers problems and needs [START_REF] Vargo | Evolving a New dominant Logic for Marketing[END_REF]. Such an approach moves companies away from simply managing products to providing innovative solutions which no longer requires buying and selling physical objects. This approach is providing new ways of thinking about the customer offer which could lead to more leasing type models such as in the Xerox case example.

This view could provide further drivers to maximize asset utilization and extension of product life times and hence reduce waste from product disposal.

A final comment to these conclusions reflects on the relevance of these challenges to sustainable operations and supply chains in Brazil. If we consider how companies and organizations respond to stakeholders, a useful concept is that of stakeholder salience [START_REF] Mitchell | Toward a theory of stakeholder identification and salience: defining the principle of who and what really counts[END_REF]. According to this theory, organizations view stakeholders (and their associated issues) as salient when there is power, legitimacy and urgency. There can be no doubt that stakeholders in the domain of sustainability are becoming more powerful, public bodies more legitimate and the sense of urgency is growing in a world of constrained resources and an environment under pressure. Before legislators create constraining rules and additional costs, now is the time to act and develop innovative solutions which reduce risks and provide opportunities for sustainable operations and supply chains.

Figure 1 :

 1 Figure 1: A framework for sustainable operations and supply chain challenges.

The impact of batch size on emissions

			0.60
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	Purge solvent	emissions -kg/car	0.10 0.20 0.30 0.40
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			1	2	3	4	5	6	7	8	9 10 11 12 13 14
						Average batch size


Maxime R Hervé
email: mx.herve@gmail.com

Nathan Garcia

Marie Trabalon

Anne Le Ralec

Régine Delourme

Anne Marie Cortesero

Oviposition behavior of the pollen beetle (Meligethes aeneus): a functional study

Keywords: Oilseed rape (Brassica napus), Insect pest, Plant acceptability, Behavioral sequence, Scanning Electron Microscopy

The recognition by female phytophagous insects of a plant as a 'good' or 'bad' host for egg laying is based on a variety of cues (either visual, physical or chemical). Specific cues are often looked for during stereotypic oviposition behaviors, composed of several phases having their own function(s). In this study the oviposition behavior of the pollen beetle Meligethes aeneus, a pest which lays eggs in flower buds of only some brassicaceous plants, was described in detail on five oilseed rape (Brassica napus) genotypes. In parallel, setae borne by the ovipositor were characterized by scanning electron microscopy. Observations showed that the stereotypic oviposition sequence is functionally divided into three independent phases: external inspection, internal inspection and egg laying. The ovipositor plays a role in all phases by gaining information about external and internal bud parts. This role appears to be only physical since all the setae it bears are mechanoreceptors. Despite the fact that the pollen beetle is a specialist for oviposition, important variations in secondary metabolites that are typical of its host plant family (i.e. glucosinolates) on the bud did not influence clutch size.

The crucial phase in the oviposition sequence seems to be the external inspection, during which poor and high-quality host plants are probably discriminated. Chemical information on bud surface is likely to be determinant in this process.

Introduction

The classical 'preference -performance' hypothesis (e.g. Thompson 1988) predicts that female insects should be selected to lay eggs preferentially on oviposition sites that are favorable for offspring development. In phytophagous species, this hypothesis has been supported by a recent meta-analysis [START_REF] Gripenberg | A meta-analysis of preferenceperformance relationships in phytophagous insects[END_REF]. The recognition of a plant as a host or non-host, and more precisely of the quality of a host plant, is based on cues (i.e. plant traits) used by females. These can be either chemical and/or visual, acting at distance (e.g. volatile compounds, size or architecture) and/or at contact (e.g. primary and secondary metabolites inside and on surface of plant tissues, trichome density, tissue thickness or toughness).

In many insect species, females show a stereotypic oviposition behavior (e.g. the cabbage root fly Delia radicum [START_REF] Städler | Oviposition behavior of the cabbage root fly, Delia radicum (L.), influenced by host plant extracts[END_REF], the mustard leaf beetle Phaedon cochleariae [START_REF] Müller | Different oviposition behaviour in Chrysomelid beetles: characterization of the interface between oviposition secretion and the plant surface[END_REF] or the cabbage seedpod weevil Ceutorhynchus obstrictus (Ulmer and Dosdall 2006)). Such behavioral sequence is often divided into several phases, each of them having one or several precise function(s). Specific host cues are detected during these phases, by means of sensory organs such as mechano-or chemoreceptors borne by antennae, tarsi or the ovipositor. Describing stereotypic oviposition behaviors, deciphering their function(s) and characterizing sensory organs that are used by females is the first step before identifying specific cues used by females to evaluate plant quality.

The pollen beetle (Meligethes aeneus F.; Coleoptera: Nitidulidae) is one of the major insect pests of oilseed rape (Brassica napus L.; Brassicaceae) (OSR) crops. Females lay eggs only on brassicaceous plants [START_REF] Ekbom | Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species[END_REF][START_REF] Free | The responses of the pollen beetle, Meligethes aeneus, and the seed weevil, Ceutorhynchus assimilis, to oil-seed rape, Brassica napus, and other plants[END_REF]. Adults colonize OSR fields after their winter diapause, when plants are at the bud stage. They destroy flower buds to reach the pollen inside, from which they feed. This destruction sometimes leads to important yield losses [START_REF] Nilsson | Yield losses in summer rape caused by pollen beetles (Meligethes spp.)[END_REF]. Mating occurs on the plant and females oviposit inside buds, after having made a small hole at its base. Reproduction goes on until death of the individuals, in summer. Buds are not destroyed during oviposition and usually continue their normal development. Larvae hatch inside the bud and feed from the pollen contained in anthers during their first instar. Transition to the second (and last) instar occurs approximately as the flower opens. Second-instar larvae move from one flower to another, still feeding on pollen. They finally drop from the plant at the end of their development and pupate in the soil (Williams 2010). Not all brassicaceous plants are accepted for oviposition by females even when they are attractive at distance [START_REF] Cook | Do pollen beetles need pollen? The effect of pollen on oviposition, survival, and development of a flower-feeding herbivore[END_REF]Kaasik et al. 2014aKaasik et al. , 2014b;;Veromann et al. 2012).

Cues present on the buds and acting upon contact play a key role in determining oviposition.

White mustard Sinapis alba is, for example, especially known to be a low-quality host for the pollen beetle [START_REF] Ekbom | Clutch size and larval performance of pollen beetles on different host plants[END_REF][START_REF] Ekbom | Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species[END_REF]Hopkins andEkbom 1996, 1999;[START_REF] Hopkins | Glucosinolate content and susceptibility for insect attack of three populations of Sinapis alba[END_REF]. [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] characterized for the first time the oviposition behavior of pollen beetle females and proved that S. alba is of inferior acceptability (in the strict sense of [START_REF] Singer | Reducing ambiguity in describing plant-insect interactions: "preference", "acceptability" and "electivity[END_REF]) compared to Brassica spp. They showed that the flower bud is inspected in a stereotypic sequence before oviposition, and that this inspection can lead to females stopping the sequence and leaving the plant. Although contact cues appear essential in determining plant acceptability, these authors were not able to identify the cues females use to make their decision.

The aims of this study were (i) to describe in more detail the oviposition behavior of the pollen beetle, (ii) to give a functional interpretation of the different steps of the behavioral sequence, and (iii) to identify the critical step(s) of this sequence determining the acceptance of the plant. For this purpose, five OSR genotypes for which the biochemical composition of buds is known (Hervé et al. 2014a) were compared in a no-choice experiment, as precisely as possible. As the ovipositor is known to bear setae in its distal part in the Meligethinae subfamily [START_REF] Audisio | Preliminary re-examination of genus-level taxonomy of the pollen beetle subfamily Meligethinae (Coleoptera: Nitidulidae)[END_REF]), morphology of these setae was also characterized by scanning electron microscopy (SEM) to understand their function.

Material and methods

Plants

All genotypes used in this study were lines from the INRA OSR collection (BraCySol Center for Genetic Resources, INRA, Le Rheu, France). Both winter (genotypes 'Darmor', 'Express' and 'Mar') and spring (genotypes 'Liho' and 'Yudal') OSR genotypes were used. Plants were produced in controlled conditions as described in Hervé et al. (2014a) and used at BBCH stage 55-57 [START_REF] Lancashire | A uniform decimal code for growth-stages of crops and weeds[END_REF], i.e. the 'green bud stage'. To keep the interaction natural, entire intact plants were used.

Insects

Overwintered pollen beetle females were collected from an unsprayed winter OSR crop near Le Rheu (Brittany, France), in April-May. Females were identified by observing mating behavior. Experiments took place within 3 h after field collection.

Oviposition behavior characterization

One female was placed on the main inflorescence of an intact OSR plant, in a plastic pot (diameter 6.5 cm, height 9 cm) isolating this inflorescence from the rest of the plant. As described by [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF], the beginning of the oviposition sequence is discernible when, after walking on several buds, a female walks circuitously on the same bud.

Observations were carried out by constantly following the female with a hand magnifier (x 5), recording the sequence and the duration of each behavior with a handheld recorder, and transcribing it later with the interface SequenceR [START_REF] Hervé | SequenceR : une interface d'encodage de séquences comportementales[END_REF]. Based on the previous characterization of [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] and our preliminary observations, six behaviors were considered (Table 1). After the end of the sequence, the bud in which the female oviposited was measured and dissected to count the number of eggs laid. A different plant was used for each female. Thirty different individuals were recorded per OSR genotype.

Replicates were conducted randomly through time (total study period: about one month) and experiments took place at 20 °C.

Scanning electron microscopy of the ovipositor

About 40 ovipositors were dissected, dehydrated by successive alcohol-bath (70 %, 80 %, 90 %, 96 % and 100 %), critical-point dried and coated with gold-paladium. Observations were then performed with a JSM-7100F (Jeol) microscope.

Statistical analysis

All statistical analyses were performed using R software (R Core Team 2013). The proportion of females completing their oviposition sequence was compared among genotypes using a likelihood ratio test on a Generalized Linear Model (GLM) (distribution: binomial, link function: logit). Only females that completed their sequence were included in subsequent analyses. Number of eggs laid was analyzed using a likelihood ratio test on a GLM (distribution: Poisson, link function: log) taking into account OSR genotype, size of the bud in which oviposition took place and duration of each behavior. ANOVAs were used to compare genotypes for the size of the bud in which the female oviposited, the duration of each behavior (durations of 'Walking with ovipositor' and 'Resting' had to be log-transformed for a better model fit) and the total duration of the sequence. When needed, pairwise comparisons of Least Squares Means were performed using the function 'lsmeans' (package 'lsmeans' [START_REF] Lenth | lsmeans: Least-squares means. R package version 1[END_REF]) and the False Discovery Rate (FDR) correction for P-values [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. Pearson's correlation tests were used to assess the relationship between duration of all pairs of behaviors. The P-value of each test was adjusted with the FDR correction.

Results

Oviposition behavior characterization

The mean length (± SE) of the bud in which pollen beetle females laid eggs was 4.0 (± 0.07) mm. Although the size of all buds forming an OSR raceme are quite variable, ranging between less than 1 mm and 7-8 mm long, only a narrow size range was used by females (90 % of chosen buds had a length between 3 and 5.5 mm). No difference was observed among the five OSR genotypes for the length of the chosen bud (F 4,128 = 0.80, P = 0.525).

The observed sequences were consistent with the description of [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF], but we recorded finer details during two particular steps. Firstly, [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] described a behavior of "walking with the abdomen touching the bud surface". Our observations showed that during this step, the abdomen is very close to the bud surface but does not touch it, and the ovipositor is partly extruded, tapping for a few seconds with its distal end on the bud surface. Secondly, [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] described a behavior consisting of "placing the abdomen over the bite hole". Our observations showed that the abdomen is not only placed over the hole, but that the ovipositor is fully extruded and inserted inside the hole, tapping on bud organs we were not able to identify. Finally, we add that antennae were constantly used to tap the bud surface throughout the 'Walking' and 'Walking with ovipositor' behaviors.

No difference was found among OSR genotypes for the duration of 'Walking', 'Walking with ovipositor', 'Resting' and 'Oviposition' (Table 2). On the contrary, the mean duration of 'Biting' and 'Ovipositor inside hole' was significantly longer in genotypes 'Darmor' and 'Mar' compared to 'Express', 'Liho' and 'Yudal'. Consequently, the mean total time of the sequence was greater in 'Darmor' and 'Mar' than in the three other genotypes. Pairwise correlations between durations of each behavior (Table 3) revealed that all durations were independent, except for two pairs. First, the time spent walking was highly positively correlated to the time spent walking with the ovipositor tapping on the bud surface. Secondly, the time spent biting the perianth was highly positively correlated to the time spent with the ovipositor tapping inside the bud.

A high proportion of females completed their oviposition sequence (overall proportion [95 % CI]: 0.89 [0.82 -0.93]). This proportion was not statistically different among the OSR genotypes (Table 2). Altogether, 17 females left the bud before laying eggs. Six left it before biting any hole, after a mean time (± SE) of 561.2 (± 94.0) s (26.6 % of the mean total time of completed sequences). The other eleven females left the bud after at least starting biting, after a mean time of 1,250.4 (± 269.2) s (59.4 % of the mean total time of completed sequences).

Between one-six eggs were laid, with a mean (± SE) of 2.88 (± 0.11). The number of eggs laid was not influenced by any variable except the duration of the 'Oviposition' behavior (Genotype: χ² = 3.07, df = 4, P = 0.547; Size of the bud in which eggs were laid: χ² = 0.02, df = 1, P = 0.890; 'Walking': χ² = 0.001, df = 1, P = 0.971; 'Walking with abdomen': χ² = 0.88, df = 1, P = 0.349; 'Resting': χ² = 0.03, df = 1, P = 0.866; 'Biting': χ² = 0.24, df = 1, P = 0.625; 'Abdomen over hole': χ² = 0.50, df = 1, P = 0.481; 'Oviposition': χ² = 4.37, df = 1, P = 0.037). The more time a female spent laying eggs, the more eggs were laid.

Scanning electron microscopy of the ovipositor

Sixteen setae were found on each half of the ovipositor. They were located at the end of the gonostyloid (i.e. the distal end of the gonocoxites), for one part on the gonostyloid themselves and for the other part on two cylindrical styli (Fig. 1b,c). Two types of sensilla were observed: long trichoid sensilla (between 10-15 µm long; Fig. 1d) and short basiconic sensilla (2-3 µm long; Fig. 1e,f). Both types were strictly aporous.

Discussion

Functional organization of the oviposition sequence [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] made the first, broad description of the oviposition behavior of the pollen beetle. Based on our results on the correlation and the transitional frequencies between behaviors, we were able to go further and draw a general functional view of the oviposition sequence of this species (Fig. 2). This sequence is divided into three independent steps: the first comprising alternate walking and walking with the ovipositor tapping on the bud surface. This probably represents 'external inspection' of the bud. If the female did not leave the bud during this first step, the second step started. This comprised alternate biting of the oviposition hole and, after a U-turn, placing the ovipositor inside this hole and tapping on internal organs of the bud. We called this phase 'internal inspection'. Finally, if the female did not leave the bud during the second step, the third and final step (consisting of laying eggs) started.

External inspection

It is very likely that external inspection of the bud surface has several functions. Pollen beetle females oviposited only in a narrow bud size range, which supports previous results [START_REF] Ekbom | Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species[END_REF][START_REF] Ferguson | Activity-temperature relationships in Meligethes aeneus: implications for pest management[END_REF][START_REF] Nilsson | The pollen beetle (M. aeneus F.) in winter and spring rape at Alnarp 1976-1978. II. Oviposition[END_REF]). This bud selection is considered as an adaptive compromise between the protection of larvae against natural enemies until bud opening, and the amount of food (i.e. pollen) available for these larvae during the first part of their development [START_REF] Ekbom | Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species[END_REF]. The external inspection, as it is the first step of the oviposition sequence, probably plays a crucial role in evaluating the size of the bud. The process by which the female assesses this size is unknown, but it might be based on a comparison with its own size.

The majority of females oviposited on the five studied OSR genotypes, confirming that OSR is a host species of high acceptability. [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] showed that during the external inspection, S. alba, a known low-quality host plant species for the pollen beetle [START_REF] Ekbom | Clutch size and larval performance of pollen beetles on different host plants[END_REF][START_REF] Ekbom | Pollen beetle (Meligethes aeneus) oviposition and feeding preference on different host plant species[END_REF]Hopkins andEkbom 1996, 1999) was systematically rejected after a very short walk on the bud surface. A second function of this phase of the oviposition sequence could hence be to discriminate between poor and high-quality host plants. Cues that are used by the female to perform such discrimination are likely to be multiple. Indeed, phytophagous insects and plant surfaces interact in a complex manner, as both physical and chemical parameters can influence insect behavior (reviewed in [START_REF] Müller | Plant surface properties in chemical ecology[END_REF]). However, as the pollen beetle oviposits only on certain brassicaceous plant species, it is likely that chemical cues (e.g. surface metabolites), which are more specific than physical ones (e.g. trichome density), are of primary importance. Interestingly, we observed that female's antennae are constantly used to tap the bud surface throughout the external inspection. This suggests that surface compounds are sampled during this phase of the oviposition sequence.

Our results revealed that the female's ovipositor plays an active role in the external inspection, by tapping on the bud surface. This organ is known to bear sensilla at its distal end [START_REF] Audisio | Preliminary re-examination of genus-level taxonomy of the pollen beetle subfamily Meligethinae (Coleoptera: Nitidulidae)[END_REF]), although their nature remains unknown. Our observations showed that all of them are totally aporous, clearly indicating that they have a sole, mechanosensory role [START_REF] Chapman | Mechanoreception[END_REF]. The function of the behavior 'Walking with ovipositor' is probably therefore to get physical information from the bud. It may either be the toughness or the thickness of the perianth which has to be pierced to bite the oviposition hole.

Internal inspection

The internal inspection consists of alternating biting of the oviposition hole and tapping inside the bud with the ovipositor. During biting, it is possible that the female would be influenced by cues from the perianth, which could either be physical or chemical. Although often neglected, plant toughness can negatively influence chewing insects by reducing consumption rate [START_REF] Clissold | Gross vs. net income : how plant toughness affects performance of an insect herbivore[END_REF]). On the other hand, mouth parts of insects are a 'hot spot' of chemosensory receptors [START_REF] Chapman | Mechanoreception[END_REF] and the pollen beetle is not an exception [START_REF] Błażejewicz-Zawadziska | Sensory organs of mouth parts of selected species of the genus Meligethes Stephens (Coleoptera: Nitidulidae)[END_REF]. Interestingly, clear differences appeared among OSR genotypes, dividing them into two groups. The biochemical composition of the perianth of all of these genotypes has previously been characterized by Hervé et al. (2014a) and showed two clear results. Firstly, 'Yudal' is much more concentrated in total glucosinolates -secondary metabolites typical of a few plant families including Brassicaceae [START_REF] Fahey | The chemical diversity and distribution of glucosinolates and isothiocyanates among plants[END_REF]) -than the other genotypes (in descending order and relatively to 'Yudal': 'Express' 0.22, 'Darmor' 0.11, 'Liho' 0.04 and 'Mar' 0.04). Glucosinolate profiles are similar; differences are essentially quantitative. Biting duration being equivalent on 'Yudal', 'Express' and 'Liho', this suggests that there is no link between glucosinolate content of the perianth and biting duration (Fig. 3a). Moreover, the number of eggs laid was not different among genotypes, which also indicates the absence of link between glucosinolate content of the perianth and clutch size (Fig. 3b). These results confirm those of Hervé et al. (2014b) that an important increase in glucosinolate content (maximum -minimum ratio: 23.4) does not further stimulate oviposition of the pollen beetle. Such a pattern was previously reported for another insect specialized on brassicaceous plants, the cabbage seedpod weevil Ceutorhynchus obstrictus (Ulmer and Dosdall 2006). This conclusion is rather unexpected, as increased amounts of glucosinolates generally stimulate feeding and oviposition of phytophagous insects specialized on brassicaceous plants (reviewed in [START_REF] Hopkins | Role of glucosinolates in insect-plant relationships and multitrophic interactions[END_REF]). It has to be noted that since we did not use glucosinolate-free plants, it cannot be concluded that these compounds do not stimulate oviposition at all. Secondly, Hervé et al. (2014a) showed a gradient of feeding stimulation among the same five genotypes: 'Express' is the most stimulant, 'Liho' the least and the three others are intermediate. Again, biting duration does not seem to be linked with perianth biochemistry. All of these results suggest that biting duration could be independent of the biochemical composition of the perianth and is possibly influenced only by its structural characteristics. Finally, since no difference in the acceptability or in the number of eggs laid has been observed among the five OSR genotypes, it seems that oviposition is not determined by cues present in the perianth of this host plant.

Further studies on a greater number of genotypes are needed to confirm this hypothesis.

During the internal inspection, pollen beetle females tap inside the flower bud with their ovipositor. In a study comparing two OSR genotypes producing male-fertile or male-sterile flowers [START_REF] Cook | Do pollen beetles need pollen? The effect of pollen on oviposition, survival, and development of a flower-feeding herbivore[END_REF], pollen beetle females made the same number of oviposition holes on buds from the two types of plants. However, oviposition took place more often on malefertile plants (i.e. the likelihood of laying eggs after having making a hole was greater in pollen-containing buds). These results, combined with our observations on the mechanosensory function of ovipositor's sensilla, suggest that the behavior 'Ovipositor inside hole' is likely to be no more than a simple assessment of the presence (and possibly the size) of anthers.

Finally, it seems that during the second phase of the oviposition sequence of the pollen beetle, chemical (i.e. specific) information provided by the bud does not play an important role. It suggests that this phase is not decisive in the assessment of host quality. This is concurrent with the results of [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] who found that even on the low-quality host S. alba, once females started this phase they did not stop the sequence until its end. In our study, it is likely that the few interruptions that occurred after females started biting their oviposition hole were accidental and not purposeful. Some disturbance in our experimental conditions may have occurred.

Oviposition

The third and last phase of the oviposition sequence consists of laying eggs. We found no difference among OSR genotypes in terms of number of eggs laid. [START_REF] Borg | Characteristics of oviposition behaviour of the pollen beetle, Meligethes aeneus on four different host plants[END_REF] showed that for females completing their sequence, there was no difference among host species differing in their quality. In the same manner, [START_REF] Cook | Do pollen beetles need pollen? The effect of pollen on oviposition, survival, and development of a flower-feeding herbivore[END_REF] showed that for females having oviposited, the same number of eggs was laid in male-fertile and male-sterile buds. These results suggest that clutch size of the pollen beetle is not influenced by immediate cues obtained during the oviposition sequence. The daily egg load of individual pollen beetle females has previously been shown to be between one and five eggs [START_REF] Ekbom | Field oviposition rates and egg load dynamics of pollen beetles (Meligethes aeneus Fab.) (Coleoptera: Nitidulidae)[END_REF][START_REF] Ferguson | Activity-temperature relationships in Meligethes aeneus: implications for pest management[END_REF][START_REF] Hopkins | Low oviposition stimuli reduce egg production in the pollen beetle Meligethes aeneus[END_REF]. Our results suggest that, if a host plant is accepted, all available mature eggs are laid.

Conclusion

Our observations have resulted in a more detailed characterization of the behavioral sequence of oviposition in pollen beetle, and highlighted the importance of females' ovipositor in gaining information from the oviposition site. Combined with results of previous studies, we were able to draw a functional scheme of the oviposition sequence, divided into three phases.

Oviposition starts with an external inspection of the flower bud, in which the acceptability of the plant is assessed. This assessment is likely to be influenced mainly by chemical information, i.e. surface metabolites. This is likely to be the critical step, discriminating low and high-quality host plants. The second phase is an internal inspection of the bud, probably essentially influenced by structural parameters of the perianth and during which the presence (and possibly the size) of anthers (i.e. food for larval development) is assessed. Finally, the third phase consists of laying probably all the mature eggs the female is carrying.

Fig. 1 Fig. 2 Fig. 3

 123 Fig. 1 Stereomicroscope observation: a -pollen beetle female (M. aeneus) with fully extruded

20 Table 1 .

 201 Behaviors used to characterize the oviposition sequence of M. aeneus

	Behavior	Description
	Walking	All locomotion except for "Walking with ovipositor"
	Walking with ovipositor	Walking with ovipositor tapping on the bud surface
	Resting	No locomotion
	Biting	Female stays at the same place and chews on the bud; once a hole is
		initiated all biting behavior occurs at the same location
	Ovipositor inside hole	Female stays at the same place, inserts her ovipositor inside the hole
		bitten and taps on internal bud organs. The female is wiggling and
		antennae are constantly agitated
	Oviposition	Ovipositor is inserted into the hole and egg(s) laid. The female is
		completely immobile, including antennae

Table 2 .

 2 Results of (i) ANOVAs comparing durations of each behavior and total oviposition sequence of M. aeneus on oilseed rape; (ii) likelihood ratio tests comparing number of eggs and number of females having completed their oviposition sequence. Mean (SE) values are given. For significant ANOVAs, different letters show statistically different means. P-values less than α = 0.05 are represented in bold

	Genotype

Acknowledgments

We are very grateful to Jo Le Lannic, Francis Gouttefangeas, Loïc Joanny and Maryline Guilloux-Viry for their help with the SEM and to the UMR IGEPP glasshouse team for taking care of the plants used in this study. Maxime Hervé was supported by a CJS grant from the French National Institute of Agronomical Research.

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