P De Loor
email: deloor@enib.fr

P Chevaillier

Solving Distributed and Dynamic Constraints Using an Emotional Metaphor : Application to the Timetabling Problem

Keywords: Distributed and Dynamic Problem, Software agent, Metaphorical Assumption, Emotion, Human Behavior Model

This paper presents a method and it's implementation for solving distributed and dynamic constraints satisfaction problem. In order to improve adaptability and performance, our algorithm is based on agents with autonomous behaviors guided by metaphoric assumptions. Our approach can be distinguished by the following points : The metaphor turns on sociological and emotional criterias without negotiation and memorisation. It tries to copy collective and affective human's behavior during a complex decision making. The agent's model include the notions of affective power, intruder and public mood perception. We have applied this method successfully to the timetabling problem. This paper show formalisation, implementation and first results of this work.

Introduction

Our objective is to develop adaptive algorithms for solving distributed and dynamic problem. Recently, some techniques has been developped to solve such king of problem. They are based on distributed operationnal research technics [START_REF] Yokoo | Distributed Constraint Satisfaction[END_REF] or with multi-agent paradigm [START_REF] Durfee | Distributed problem solving and multiagent systems: Comparisons and examples[END_REF], [START_REF] Wooldridge | The cooperative problem-solving process[END_REF]. This two classes of approaches start from different models, mechanism and point of view.

• The firts kind of approaches allow a systematic research. Variables control some constraints and communicate their values with the others. Some Values can be refused depending on the constraints. Some algorithms record the refusals. Consequently, in return for time and memories, this approaches are generales and completes []. However, they present some drawback : for complex problems, as allocation or scheduling, the modelisation of the constraints are a hard stage which imply the definition of numbered variables and constraints []. To optimize performances of such algorithms, somes heuristics are used (which do not keep completness properties). They are based on quantitative criterias whithout particular semantic. However, including qualitative criterias, relative to the problem, seem important to improve the performance of the search.

• The second kind of approaches are based of complex autonomous agent including decision and negociation mechanisms. Usually, they are guided by metaphoric assumption like in [START_REF] Walsh | On market-inspired Solving Distributed and Dynamic Constraints Using an Emotional Metaphor : Application to the Timetabling Problem6 approaches to propositional satisfiability[END_REF] (auction) or [START_REF] Bonabeau | Adaptive task allocation inspired by a model of division of labor in social insects[END_REF] (social insects).

The main advantage is relative to the modelisation of the problem by the way of flexible data structures and implicit constraints rather than by a simple set of data and mathematical constraints. However, the backtracking is generally not explicit or forbiden.

principe

Our approach is clearly agent oriented. It can be distinguished by the following points : the metaphoric assumption guiding the behavior of the agents turns on sociological and emotionnal criterias [START_REF] Sternberg | Models of Intelligence : International Perspectives[END_REF] : It tries to copy collective and affective human's behavior during a complex decision making. Each agent (representing a human) owns somes goals, skills and a variable which mean it's cognitive power. This variable evolves depending on the perception of differents messages : requests, denial, proposal, cancellation (see figure 1). The more important point is that the cognitive power of an agent is altered by messages concerning the agent itself but also by other messages (but with a different strength). Then, each agent perceive the "public mood" of the global system. It is endowed with a self-perception of his part. For example, it can perceive him as an intruder. When it's affective power pass over an emotive threshold, the agent throw a fit : it cancel his commitment (towards other agents) and his affective power is reset. The evolution's rule of the affective power avoid cycle during the problem solving because it's not always the same agent wich throw a fit but rather the next more intruder agent. This algorithm can be compared to the dynamical hierarchie introduced by [START_REF] Hirayama | An approach to overconstrained distributed constraint satisfaction problems: Distributed hierarchical constraint satisfaction[END_REF] but, in our case, this hierarchie is defining for agents by a high level metaphor rather than purely mathematical considerations. Finally, our approaches is dedicated to dynamical's environments. In such environment, variables and constraints can be added or retracted at any time during the search of a solution. The algorithm adapts himself to the current situation. In order to do that, we use two considerations :

• agents don't know the global problem and the set of constraints representing it.

• agent's are unaware of the set (and the number) of the others agents involving in the problem.

In this way, adding agents or constraints implies neither a global restarting of the algorithm nor a modification of it's current mechanisms or structures. Nous prfrons parler de recherche de solution par simulation "anytime", plutot que de rsolution de contraintes.

Algorithm

Models

Emotionnal agent's model

An emotional agent A is formalized by an 8-uplet :

< ψ A , α A s , α A c , ρ A , γ A , S A s , R A g , P A g > • ψ A ∈ [0,1
] is a real meaning the cognitive power.

• α A s ∈ ]0,1[ is a real meaning the self-sensitiveness rate. • α A c ∈ ]0,1[ is a real meaning the collective- sensitiveness rate. • ρ A ∈]0, 1[ is a real meaning the requirement threshold . • γ A ∈ ]0,1[ is a real meaning the crisis threshold (γ A > ρ A ).
• S A s is the set of skills. (symbolic declarations). • R g A is the set of requested goals.

• P g A is the set of personnal goals.

Goal's model

A Goal g is a 3-uplet < P g , S g , A g , V g > :

• P g is a first order predicate which express the goal. This symbolic expression is relative to the problem.

• S g is a skill requiered to achieve or improve the goal (solving P g ).

• A g is a solution to achive or improve the goal. A solution is a data structure depending of the problem (and can be partial).

• V g is a boolean meaning the value of P g .

Message's model

A message M is a 3-uplet < T M , E M , G M > : • T M ∈ { request , proposal , cancellation , ref usal } is the message's type.
• E M is an Agent : the emitter of the message.

• G M is a goal : the topic of the message .

Agent's behavior

default behavior

The default behavior of an agent A is executed as long as no message is received :

∀ goal g ∈ P A g if (V g == f alse) ψ A = ψ A + α A s * (1 -ψ A ) if (ψ A > ρ A ) (requirement) ∀ goal g ∈ P A g if (V g == f alse) broadcasting the message < request , A, g > ψ A = ψ A -(α A s * ψ A ) if (ψ A > γ A ) (crisis) ∀ goal g ∈ R A g broadcasting the message < cancellation , A, g > R A g = φ ψ A = 0 ψ A = ψ A -f (α A s , ψ A ) (default relaxation)
Summary : each unreached personnal goal incrises the cognitive power in proportion to the agent's self-sensitiveness rate (α A s ). If the cognitive power exceeds the requirement threshold, requests concerning personnal goals are sending. In this case, the cognitive power decreases. Such mechanism avoids complex aknowledgments with some answering messages. Indeed, it's the cognitive power which regulate the flow of requests. In fact, some policies can be use. For example,the request can depend on a probability depending on the cognitive power and the requiremen threshold. Only one request for one goal should be broadcasted at one time than requests for all goals. The influence of the policie on the performance of the algorithm must be studied. When the cognitive power exceeds the crisis threshold, the agent cancels all its commitments towards others agents. In order to do that, it broadcast cancellation's messages relative to all the goal included in it's set of requested goals. In this case, the cognitive power is reseted. Therefore, if other agents have a high cognitive power, they should enter in the crisis before the previous one. This mechanism can be seen as a local backtrack which goes among the more awkward agents. This backtrack is based on psychological issues and introduce a dynamic hierachie of intruder. To finish, f is a positive function meaning the natural trend to decrising the cognitive power when no problem occurs. (The definition of this function will be discribing later). It is basically used to evaluate the convergence's algorithm : when each agent's cognitive power decreases, a solution is founded.

messages processing

• processing a message < request , A , g >

if (A = A ) ∧ (S g ∈ S A s ) ψ A = ψ A + α A c * (1 -ψ A ) (public mood) else solving(P g ) if P g is soluble with a solution s A g = s V g = true R A g = R A g ∪ g broadcasting the message < proposal , A, g > ψ A = ψ A -(α A c * ψ A )
summary : if the agent has the skill, it attempts to solve the goal whith a solving method depending of the problem. The success of this method return a solution in abstract solution's goal form (see 3.1.2 and the exemple).

• processing a message < proposal , A , g >

if (A = A ) ∧ {∃g ∈ P A g |P g == P g } analyse(A g ) if ( A g is acceptable) A g = A g V g = true ψ A = ψ A -(α A s * ψ A ) else broadcasting the message < ref usal , A, g > else ψ A = ψ A -(α A c * ψ A ) (public mood)
Summary : an agent A is concerned by a proposal if this one is the result of a request from A. Nevertheless, the affective power af an agent is decreased even if it is not concerned by the proposal. It is a part of the perception of the "public mood". Before accepting the proposal, the agent must analyse it with a problem dependent method.

• processing a message < ref usal , A, g >

if (A = A ) ∧ {∃g ∈ R A g |P g == P g } ψ A = ψ A + (α A s * ψ A ) R A g = R A g -g else ψ A = ψ A + (α A c * ψ A ) (public mood) • processing a message < cancellation , A, g > if {∃g ∈ P A g |P g == P g } ψ A = ψ A -(α A s * ψ A ) V g = f alse else ψ A = ψ A -(α A c * ψ A ) (public mood)

Informal justification

The cognitive power depends on the agents' perception and action. In practical term, perception is the reception of broadcasted messages (figure 1). Actions are the research of a solution for a requested goal.

• positives informations : it decreases the cognitive power, and can be devided as following :

-le : l'agent a russi rsoudre un (ou des) but(s), personnel ou requis.

-la dcharge de responsabilit : l'agent fait une requte car il ne peut rsoudre un but personnel. Dans ce cas, il se repose sur la communaut. Prcisons que contrairement certains protocoles de type contract net protocol, l'agent n'opre cette dcharge que pour ses buts personnels et non sur des buts requis.

-le succs coopratif : un agent lui a fait une proposition qui permet de rsoudre un de ses buts personnels.

-la crise : l'agent annule toutes les solutions qu'il avait auparavant proposes. La crise est un vnement particulier qui va provoquer la remise zro de la charge cognitive de l'agent et provoquer une remise en question de la solution en cours de recherche.

• les informations ngatives : elles augmentent la charge cognitive d'un agent. Elles peuvent tre dcomposes de la faon suivante :

-les requtes : toute requte est un signe que "quelque chose n'est pas rsolu au sein du systme multi-agents" et va augmenter la charge cognitive. Si l'agent peut rpondre cette requte, le succs personnel conscutif (voir les informations positives) diminue la charge cognitive. S'il n'est pas comptent pour rpondre cette requte, il ne fait rien. S'il est comptent mais qu'il ne peut rsoudre le problme li la requte, il en rsulte un chec personnel.

-les checs personnels : l'agent est incapable de rsoudre un problme, qu'il lui soit propre ou pos par un autre agent.

-les refus : l'agent peroit les refus diffuss par les autres agents. Il distingue les refus le concernant directement (annulant une proposition qu'il a faite) et qui accrossent davantage sa charge cognitive que les refus ne le concernant pas. Cependant le fait qu'elle croisse reflte la perception de la "mauvaise ambiance" gnrale au sein du systme multi-agents. Cette proposition part de la constatation que la communication indirecte peut tre un mcanisme fondamental lors de la rsolution de problmes [START_REF] Dugale | A pragmatic development of a computer simulation of an emergency call centre[END_REF].

Implementation

Agent

Application to the timetabling problem

We applie algorithme au problme de la gnration des emplois du temps rput difficile [START_REF] Werra | An introduction to timetabling[END_REF]. Notre solution se diffrencie de celles proposes classiquement [START_REF] Terashima | Combinations of gas and csp strategies for solving the examination timetabling problem[END_REF], [START_REF] Staudte | Producing and improving time tables by means of constraint and multi-agent systems[END_REF] par le fait que la recherche est distribue, compltement asynchrone et adaptative. Par contre,contrairement [START_REF] Staudte | Producing and improving time tables by means of constraint and multi-agent systems[END_REF] nous ne proposons pas pour l'instant de dmarche d'optimisation qui fait l'objet de nos recherches actuelles. L'article montrera la formalisation de l'algorithme et les rsultats obtenus sur cet exemple implement l'aide du langage oRis [START_REF] Harrouet | oRis : un environnement de simulation interactive multi-agents[END_REF]. Elaboration d'un langage plus formel de ces changent en broadcast un peu comme propos par [START_REF] Torroni | Extending a logic based onto-toone negotiation framework to one-tomany negociation[END_REF]

Figure 1 :

 1 Figure 1: principe.

Figure 2

 2 Figure2summarizes the agent's cyclic behavior. Each message processing depends of it's nature. The cognitive power is affected according to the message or the result of the processing. It acts upon the reaction of the agent as a broadcasting of some messages.

Figure 2 :

 2 Figure 2: emotionnal agent newel loop.

Figure 3 :

 3 Figure 3: typical cognitive power evolution.

Figure 4 :

 4 Figure 4: emotional Agent's class.

Results

This section will describe the result we obtain. It can include grphical reprsenting the convergence time relative to the problem complexity.

conclusion and futur work


PhD Guido A Van Wingen
email: guido.vanwingen@donders.ru.nl

PhD Elbert Geuze

MD, PhD Eric Vermetten

MD, PhD Guillén Fernández

Perceived threat predicts the neural sequelae of combat stress Running title: Neural sequelae of combat stress

Keywords: Stress, combat, PTSD, fMRI, amygdala, ACC

Exposure to severe stressors increases the risk for psychiatric disorders in vulnerable individuals, but can lead to positive outcomes for others. However, it remains unknown how severe stress affects neural functioning in humans and what factors mediate individual differences in the neural sequelae of stress. The amygdala is a key brain region involved in threat detection and fear regulation, and previous animal studies have suggested that stress sensitizes amygdala responsivity and reduces its regulation by the prefrontal cortex. Here, we used a prospective study design to investigate the consequences of severe stress in soldiers before and after deployment to a combat zone.

We found that combat stress increased amygdala and insula reactivity to biologically salient stimuli across the group of combat exposed individuals. In contrast, its influence on amygdala coupling with the insula and dorsal anterior cingulate cortex (dACC) was dependent on perceived threat, rather than actual exposure, suggesting that threat appraisal affects interoceptive awareness and amygdala regulation. Our results demonstrate that combat stress has sustained consequences on neural responsivity, and suggest a key role for the appraisal of threat on an amygdala-centered neural network in the aftermath of severe stress.

INTRODUCTION

Highly stressful experiences such as natural disaster, terrorism, assault, or military combat are significant events in people's lives. Such stressful life events have large impact on the exposed individual, but its influence on subsequent psychological wellbeing is highly variable between individuals. Whereas traumatic experiences lead to positive changes for some [START_REF] Helgeson | A meta-analytic review of benefit finding and growth[END_REF][START_REF] Linley | Positive change following trauma and adversity: a review[END_REF] , they increase the risk for posttraumatic stress disorder (PTSD) and depression for others [START_REF] Galea | Psychological sequelae of the September 11 terrorist attacks in New York City[END_REF][START_REF] Hoge | Combat duty in Iraq and Afghanistan, mental health problems, and barriers to care[END_REF] , which suggests that the impact of stress is highly variable between individuals. However, the neural consequence of severe stress on neural functioning in humans remains unknown. Patients with PTSD show exaggerated amygdala responses and deficient prefrontal cortex function, in particular in the anterior cingulate cortex and ventromedial prefrontal cortex [START_REF] Rauch | Exaggerated amygdala response to masked facial stimuli in posttraumatic stress disorder: a functional MRI study[END_REF][START_REF] Shin | An fMRI study of anterior cingulate function in posttraumatic stress disorder[END_REF][START_REF] Shin | A functional magnetic resonance imaging study of amygdala and medial prefrontal cortex responses to overtly presented fearful faces in posttraumatic stress disorder[END_REF][START_REF] Etkin | Functional neuroimaging of anxiety: a meta-analysis of emotional processing in PTSD, social anxiety disorder, and specific phobia[END_REF] . The amygdala and prefrontal cortex are key brain regions involved in threat detection and fear regulation [START_REF] Davis | The amygdala: vigilance and emotion[END_REF][START_REF] Quirk | Prefrontal involvement in the regulation of emotion: convergence of rat and human studies[END_REF][START_REF] Stein | A validated network of effective amygdala connectivity[END_REF] . These neural alterations in PTSD presumably result from a combination of stress exposure and stress vulnerability [START_REF] True | A twin study of genetic and environmental contributions to liability for posttraumatic stress symptoms[END_REF][START_REF] Admon | Human vulnerability to stress depends on amygdala's predisposition and hippocampal plasticity[END_REF][START_REF] Shin | Resting metabolic activity in the cingulate cortex and vulnerability to posttraumatic stress disorder[END_REF] . Nevertheless, animal studies suggest that severe stress has lasting influences on neural functioning [START_REF] Pavlides | Effects of chronic stress on hippocampal long-term potentiation[END_REF]16 . Specifically, recent animal studies have shown that prolonged severe stress sensitizes amygdala responsivity and reduces its regulation by the prefrontal cortex [START_REF] Correll | Chronic cold stress alters prefrontal cortical modulation of amygdala neuronal activity in rats[END_REF][START_REF] Rosenkranz | Chronic stress causes amygdala hyperexcitability in rodents[END_REF] . Furthermore, acute stress exposure increases amygdala reactivity in humans [START_REF] Van Marle | From specificity to sensitivity: how acute stress affects amygdala processing of biologically salient stimuli[END_REF] . To investigate whether prolonged severe stress also affects amygdala functioning in humans, even in the absence of acute stress, we used a prospective study design. We assessed amygdala functioning in soldiers before and after deployment to a combat zone, which is typically associated with severe stress exposure. In addition, we included a group of soldiers who were never deployed to control for repeated testing and unspecific time effects.

MATERIALS AND METHODS

Participants

The combat stress group consisted of thirty-three healthy soldiers who were deployed for 4 months to Afghanistan as part of the NATO International Security Assistance Force (ISAF) peacekeeping operation. We investigated them before and on average 1.5 (SD = 0.8) months after their first military deployment. They were recruited from a larger prospective study on the development of stress-related disorders following military deployment in the Dutch armed forces. Their duties included combat patrols, clearing or searching homes and buildings, participation in demining operations, and transportation across enemy territory. They were exposed to typical war-zone stressors such as enemy fire, armed combat, and seeing seriously injured fellow soldiers and civilians (including women and children). The control group consisted of twenty-six soldiers who were recruited from training bases and army divisions currently not involved in combat missions. One soldier of the control group scored above a clinical threshold for PTSD symptoms on a self-report questionnaire (see below) at baseline, and one soldier of the combat group scored above the threshold after deployment. Both participants were therefore excluded from the analyses. The groups did not differ significantly in sex ratio, age, and intelligence quotient (see Table 1). Furthermore, we investigated both groups at the same test-retest interval (mean ± SD; 6.9 ± 1.6 months; t(55) = 0.5, P = 0.65).

Because the number of females in the study was small, we also conducted all analyses with males only, which did not alter the pattern of results (data not shown). The study was in accordance with the declaration of Helsinki and institutional guidelines of the local ethics committee (CMO Regio Arnhem-Nijmegen, The Netherlands), and all participants provided written informed consent after written and oral description of the study.

Questionnaires

To evaluate the influence of severe stress exposure on PTSD symptoms, mood, and anxiety, we used three questionnaires. The short version of a previously validated Dutch questionnaire, the Self-rating Inventory for PTSD, was used to assess self-reported PTSD symptoms. We excluded individuals with possible PTSD from all analyses, as defined by the cut-off score of 52. [START_REF] Hovens | The development of the Self-Rating Inventory for Posttraumatic Stress Disorder[END_REF] The Dutch version of the Positive and Negative Affect Schedule (PANAS) was used to assess positive and negative mood [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF] , and Dutch state version of the State Trait Anxiety Inventory (STAI) was administered to assess state anxiety [START_REF] Spielberger | STAI manual for the State Trait Anxiety Inventory[END_REF] . To quantify individual differences in stress exposure, we measured combat exposure and perceived threat during deployment using the Combat experiences and Perceived threat scales of the Deployment Risk and Resilience Inventory [START_REF] King | Deployment risk and resilience inventory: a collection of measures for studying deployment-related experiences of military personnel and veterans[END_REF] . The scores on these scales were not significantly correlated (r = 0.10, P = 0.58), suggesting that the amount of perceived threat during the time of the military deployment was not directly related to the number of actual combat experiences.

Behavioral task

The experimental paradigm that was performed during functional MRI scanning consisted of a blocked design, including an emotion condition with angry and fearful face stimuli (http://www.macbrain.org) and a visuo-motor control condition with scrambled ellipse stimuli [24][START_REF] Van Wingen | Progesterone selectively increases amygdala reactivity in women[END_REF][START_REF] Van Wingen | Testosterone increases amygdala reactivity in middle-aged women to a young adulthood level[END_REF] . Two emotion blocks were interleaved with three control blocks, and each 30 s block consisted of six 5 s trials. Each trial consisted of three simultaneously presented stimuli, with the cue stimulus presented above the target and distractor. In the emotion condition, an angry or fearful face was presented on top as cue, and participants had to indicate by an appropriate button press which of the bottom two faces (one angry and one fearful) matched the cue in emotional expression. In the control condition, a horizontally or vertically oriented ellipse was presented as cue above two ellipses (one vertical and one horizontal), and participants had to select the identically oriented ellipse.

MRI data acquisition

MR data were acquired with a 1.5 T Siemens (Erlangen, Germany) Avanto MR scanner, equipped with a standard head coil. T2*-weighted BOLD images were acquired using EPI with an echo time of 35 ms to reduce signal dropout in the medial temporal lobes.

Each image volume consisted of 32 axial slices (3.5 mm, 0.35 mm slice-gap, TR = 2.340 s, 64 × 64 matrix, FOV = 212 mm, FA = 90°). In addition, a high resolution T1-weighted structural MR image with optimized gray/white matter contrast was acquired (3D MP-RAGE, 1 × 1 × 1 mm 3 voxels, TR = 2.730 s, TE = 2.95 ms, TI = 1000 ms, FOV = 256 mm, FA = 7°).

FMRI data analysis

Image analysis was performed with SPM5 (http://www.fil.ion.ucl.ac.uk/spm). The first five EPI-volumes were discarded and the remaining images were realigned to the first volume. Images were then coregistered to the anatomical scan, corrected for differences in slice acquisition time, spatially normalized to the MNI T1 template, resampled into 2 × 2 × 2 mm 3 voxels, and spatially smoothed (8 mm FWHM). Statistical analysis was performed within the framework of the general linear model 27 . To assess the influence of combat stress on neural responsivity, the two experimental conditions were modelled as box-car regressors convolved with the canonical hemodynamic response function of SPM5. In addition, the realignment parameters were included to model potential movement artefacts, as well as a constant. Furthermore, a high-pass filter (cut-off 1/128 Hz) was included, and temporal autocorrelation was modelled with an AR(1) process.

Contrast images subtracting the visuo-motor control condition from the emotion condition were obtained, and analyzed in subsequent random effects models.

Functional connectivity

To assess the influence of severe stress on the amygdala network, we performed an additional functional connectivity (psychophysiological interaction) analysis using SPM5. The time-course of amygdala activity was obtained for each scanning session. The first eigenvariate of a sphere with 5 mm radius around the peak group × time interaction in the right amygdala (24, -4, -18) was extracted and entered as an additional regressor to the original fMRI model, as well as the interaction between the experimental conditions and amygdala time-course [START_REF] Friston | Psychophysiological and modulatory interactions in neuroimaging[END_REF] . Psychophysiological interaction (time-course × condition) images were obtained, and analyzed in subsequent random effects models.

Voxel-based morphometry

To assess the influence of severe stress on regional gray matter volume, we performed voxel-based morphometry using SPM5 with standard procedures [START_REF] Ashburner | Unified segmentation[END_REF] and default parameters of the VBM5 toolbox (http://dbm.neuro.uni-jena.de/vbm.html) [START_REF] Luders | The underlying anatomical correlates of long-term meditation: larger hippocampal and frontal volumes of gray matter[END_REF] . Analyses were performed on gray matter segmentations, which were multiplied by the nonlinear components derived from the normalization matrix in order to preserve local gray matter volumes (i.e., the modulated images) and spatially smoothed (12 mm FWHM).

Statistical testing

Mixed model ANOVAs with the factors group and time were used to test whether changes in brain structure and function over time were different between the combat stress and control groups. To test whether stress-induced changes were related to individual differences in combat exposure and perceived threat, additional correlation analyses were performed. Statistical tests were family-wise error (FWE) rate corrected (P < 0.05) for multiple comparisons across the entire brain, or for the search volume for regions of interest using a small volume correction (SVC) [START_REF] Worsley | A unified statistical approach for determining significant signals in images of cerebral activation[END_REF] . The search volumes for the amygdala, insula, and anterior cingulate cortex (Brodmann areas 24 and 32) were anatomically defined using the WFU Pickatlas toolbox implemented in SPM5 [START_REF] Maldjian | An automated method for neuroanatomic and cytoarchitectonic atlas-based interrogation of fMRI data sets[END_REF] .

RESULTS

Questionnaires

We observed no significant differences in self-reported PTSD symptoms, state anxiety, positive affect, and negative affect scores at baseline, nor different changes in these variables between the combat stress group and control group (see Table 1), indicating that changes in neural activity did not reflect increases in symptomatology. In addition, we measured combat exposure and perceived threat during deployment to quantify the individual differences in stress exposure. The average score for combat exposure (mean ± SD; 5.0 ± 2.5) was higher than that of a previously reported reference population of Gulf War veterans (4.0 ± 3.2) [START_REF] King | Deployment risk and resilience inventory: a collection of measures for studying deployment-related experiences of military personnel and veterans[END_REF] , confirming that the combat group was exposed to typical combat zone stressors such as armed combat, combat patrols, and exposure to enemy fire, as well as modern warfare with a risk of exposure to improvised explosive devises (IEDs). To explore whether there was a delayed onset of PTSD symptoms, we contacted the deployed individuals again six months after their deployment, but observed no significant changes in PTSD symptoms over time in those individuals that completed all three questionnaires (n = 16; F(2,14) = 1.3, P = 0.31).

Behavioral performance

Across both groups and measurements, task accuracy was higher in the emotion than control condition (Z = -2.0, P = 0.04; median ± IQR; emotion condition: 100.0 ± 4.2%, control condition: 97.2 ± 5.6%) but reaction times were slower (F(1,55) = 171.1, P < 0.001; mean ± SD; emotion condition: 1863 ± 346 ms, control condition: 1141 ± 391 ms). However, no significant changes in accuracy (emotion condition: U = 362.0, Z = -0.73, P = 0.47; control condition: U = 387.0, Z = -0.22, P = 0.83) or reaction times (F < 1) over time between the combat stress and control groups were observed, which suggests that the imaging results are unlikely explained by differences in behavioral performance.

Neural responsivity

To verify that the task indeed activated the amygdala, we compared the emotion condition with the control condition across both groups and measurements. As expected, the task increased activity in the amygdala, as well as in the insula, dorsal anterior cingulate cortex (dACC), occipitotemporal cortex, hippocampus, orbitofrontal cortex, inferior and middle frontal gyri, thalamus, precuneus, angular gyrus, and cerebellum (all P cor < 0.05). These activation patterns were not significantly different between groups at baseline (P cor > 0.05), suggesting that neural functioning was comparable before stress exposure.

Next, we compared the change in amygdala reactivity over time between groups.

The increase in amygdala reactivity was significantly larger in the combat stress group than the control group (peak Montreal Neurological Institute coordinate (x, y, z); (24, -4, -18), Z = 3.1, P cor = 0.037). Subsequent tests showed that combat stress exposure increased amygdala reactivity ((30, 0, -18), Z = 3.1, P cor = 0.044; see Fig. 1a), whereas no significant change in activity was observed in the control group (P cor > 0.05). A similar group × time interaction was also observed in the anterior insula ((32, 24, 2), Z = 3.8, P cor = 0.037; see Fig. 1b), which is a brain region involved in interoceptive awareness and thought to signal internal body states [START_REF] Craig | How do you feel? Interoception: the sense of the physiological condition of the body[END_REF][START_REF] Paulus | An insular view of anxiety[END_REF] . Subsequent tests showed that combat stress exposure also increased insula reactivity (P = 0.002), whereas insula reactivity decreased with repeated testing in the control group (P < 0.001), but these additional tests did not remain significant after correction for multiple comparisons. These results demonstrate that severe and long-term stress exposure sensitized amygdala and insula reactivity to biologically salient stimuli in humans. Next, to evaluate whether these activity changes over time were related to individual differences in stress exposure, we performed additional correlation analyses. However, we observed no significant correlations between activity changes and combat experiences or perceived threat (P cor > 0.05).

Amygdala connectivity

To investigate whether the influence of severe stress on amygdala reactivity also affected the neural network that is centered around the amygdala [START_REF] Stein | A validated network of effective amygdala connectivity[END_REF][START_REF] Ghashghaei | Sequence of information processing for emotions based on the anatomic dialogue between prefrontal cortex and amygdala[END_REF] , we performed a functional connectivity analysis. Across groups and investigations, the amygdala was negatively coupled to cingulate cortex areas (see Fig. 2a) including the dorsal anterior cingulate cortex (dACC; (6, 8, 44), Z = 4.1, P cor = 0.017), pregenual ACC ((6, 48, 2), Z = 3.8, P cor = 0.040, middle cingulate cortex ((4, -20, 42), Z = 4.3, P cor = 0.006), and posterior cingulate cortex (-2, -36, 26), Z = 5.9, P cor < 0.001). These connectivity patterns were not significantly different between groups at baseline (P cor > 0.05), suggesting that amygdala coupling was comparable before stress exposure. Changes in functional connectivity over time were not significantly different between the groups (P cor > 0.05), but were related to the amount of perceived threat in stress exposed individuals. Importantly, the strength of amygdala coupling with the dACC was positively related to perceived threat ((-10, 0, 42), Z = 4.5, P cor = 0.003; see Fig. 2b-c). Thus, negative coupling of the amygdala with the dACC was enhanced in individuals that perceived little threat, but reduced in individuals that perceived much threat. In addition, perceived threat also enhanced amygdala coupling with the insula ((42, 16, -2), Z = 3.8, P cor = 0.039; see Fig. 3). In contrast, no significant correlations between actual combat exposure and functional connectivity changes were observed (P cor > 0.05). Moreover, the associations with perceived threat remained significant after correcting for combat exposure, suggesting that perceived threat rather than actual exposure influenced amygdala coupling.

Brain structure

To investigate whether the stress induced changes in neural activity and connectivity were related to changes in brain structure, we compared regional brain volumes using voxel-based morphometry. We observed no significant volume differences at baseline, no significant changes over time between the combat stress group and control group, and no significant correlations between combat experiences or perceived threat and regional volume changes (P cor > 0.05).

DISCUSSION

To study the neural consequences of severe stress exposure, we investigated soldiers before and after deployment to a combat zone. Our results demonstrate that combat stress increases amygdala and insula reactivity to biologically salient stimuli. These neural changes were not observed in the control group and occurred in the absence of selfreported changes in posttraumatic stress symptoms. This suggests that sustained enhanced reactivity to biologically salient stimuli is a common adaptive response to prolonged environmental threat. The amygdala is a crucial brain region for the detection of threat and the enhancement of vigilance, and both the amygdala and insula are part of a larger salience network [START_REF] Davis | The amygdala: vigilance and emotion[END_REF][START_REF] Seeley | Dissociable intrinsic connectivity networks for salience processing and executive control[END_REF] . These adaptations may therefore be beneficial to maintain sustained vigilance in continuously dangerous situations, such as the combat zone the soldiers were deployed to. But as heightened amygdala and insula reactivity is thought to increase the risk for mood and anxiety disorders [START_REF] Stein | Increased amygdala and insula activation during emotion processing in anxiety-prone subjects[END_REF][START_REF] Wolfensberger | Amygdala responses to emotional faces in twins discordant or concordant for the risk for anxiety and depression[END_REF] , these alterations may not be adaptive for safe environments. Although our second follow-up assessment suggests that the combat group did not develop PTSD symptoms within half a year after deployment, it remains possible that stress symptoms do evolve later on. Furthermore, the amygdala and insula sensitization may also increase the vulnerability to future stressors.

To investigate whether the influence of severe stress on amygdala reactivity also affected the neural network that is centered around the amygdala, we performed an additional functional connectivity analysis. Our results show that the influence of severe stress on amygdala coupling with the dACC and insula was dependent on individual differences in perceived threat. Individuals that perceived little threat during deployment showed enhanced negative amygdala-dACC coupling, whereas individuals that perceived much threat showed reduced coupling. Given that the dACC is thought to regulate amygdala activity [START_REF] Quirk | Prefrontal involvement in the regulation of emotion: convergence of rat and human studies[END_REF][START_REF] Stein | A validated network of effective amygdala connectivity[END_REF] , this finding suggests that the perception of threat during severe stress exposure alters amygdala regulation thereafter. In addition, perceived threat also enhanced amygdala-insula coupling. The insula is involved in interoceptive awareness and is thought to signal internal body states [START_REF] Craig | How do you feel? Interoception: the sense of the physiological condition of the body[END_REF][START_REF] Paulus | An insular view of anxiety[END_REF] , which suggests that this may reflect increased bodily awareness in those individuals that perceive the most threat.

The impact of perceived threat rather than actual combat exposure on the amygdala network is in line with appraisal theory, which posits that the cognitive appraisal of threat rather than the actual environmental stressor determines the impact of stress exposure [START_REF] Lazarus | Stress, appraisal, and coping[END_REF][START_REF] Solomon | Locus of control and combat-related post-traumatic stress disorder: the intervening role of battle intensity, threat appraisal and coping[END_REF] . Thus, our results suggest that cognitive appraisal also has a critical role in determining the impact of severe stress on the amygdala network. Interestingly, perceived threat also appears a better predictor for PTSD symptoms than actual combat exposure [START_REF] King | Deployment risk and resilience inventory: a collection of measures for studying deployment-related experiences of military personnel and veterans[END_REF] . Even though previous studies have demonstrated a close relation between combat experiences and the prevalence of PTSD [START_REF] Hoge | Combat duty in Iraq and Afghanistan, mental health problems, and barriers to care[END_REF] , path analyses suggest that the influences of combat exposure on PTSD symptoms is mediated by its influence on perceived threat [START_REF] King | Alternative representations of war zone stressors: relationships to posttraumatic stress disorder in male and female Vietnam veterans[END_REF] .

The divergent influences of severe stress on the amygdala network may help to explain why some individuals are vulnerable to stress whereas others are stress resilient.

Our results show opposite effects on amygdala-dACC and amygdala-insula coupling depending on how the stressful experience is perceived. In turn, this may lead to opposite effects on amygdala regulation and interoceptive awareness, with better emotion regulation in those individuals that perceive little threat but worse emotion regulation in those that experience much threat. These individual differences may explain in part why we did not observe consistent changes in symptomatology across the group of combat exposed individuals, even though their amygdala and insula reactivity had increased, and suggest a neural mechanism by which traumatic experiences could lead to highly variable outcomes [START_REF] Helgeson | A meta-analytic review of benefit finding and growth[END_REF][START_REF] Linley | Positive change following trauma and adversity: a review[END_REF][START_REF] Galea | Psychological sequelae of the September 11 terrorist attacks in New York City[END_REF][START_REF] Hoge | Combat duty in Iraq and Afghanistan, mental health problems, and barriers to care[END_REF] .

The stress-induced changes in the amygdala network did not lead to consistent changes in mood and anxiety as assessed here. We have measured the changes in neural reactivity during affective stimulation. But in addition to increased reactivity to biologically salient stimuli, combat exposure may also have altered the manner in which the brain restores after stress exposure. For example, the release of cortisol after stress exposure normalizes amygdala reactivity [START_REF] Henckens | Time-dependent effects of corticosteroids on human amygdala processing[END_REF] , and adaptive changes in this process may prevent the development of symptoms. Interestingly, patients with PTSD have abnormalities in the hypothalamic-pituitary-adrenal axis [START_REF] Yehuda | Response variation following trauma: a translational neuroscience approach to understanding PTSD[END_REF] , and soldiers that develop PTSD symptoms after combat exposure have a high preexisting number of glucocorticoid receptors [START_REF] Van Zuiden | Pre-existing high glucocorticoid receptor number predicts development of posttraumatic stress symptoms after military deployment[END_REF] . This suggests that PTSD symptoms could result from unsuccessful normalization after exposure to consecutive stressors [START_REF] De Kloet | Stress and the brain: from adaptation to disease[END_REF][START_REF] Karst | Metaplasticity of amygdalar responses to the stress hormone corticosterone[END_REF] . Therefore, we propose that whereas the stress-induced changes in neural reactivity appear a common adaptive response to prolonged environmental threat, maladaptive normalization of neural hyperactivity may lead to the development of stress symptoms, which could be addressed in future studies.

The neural sequelae of severe stress exposure were remarkably similar to the neural alterations in PTSD, which include amygdala and insula hyperactivity and dACC hypoactivity [START_REF] Etkin | Functional neuroimaging of anxiety: a meta-analysis of emotional processing in PTSD, social anxiety disorder, and specific phobia[END_REF]47 . This is in line with the idea that PTSD reflects the upper end of a stress response continuum [START_REF] Ruscio | The latent structure of posttraumatic stress disorder: a taxometric investigation of reactions to extreme stress[END_REF] . However, the neural basis of PTSD presumably results from a combination of stress exposure and stress vulnerability [START_REF] True | A twin study of genetic and environmental contributions to liability for posttraumatic stress symptoms[END_REF][START_REF] Gilbertson | Smaller hippocampal volume predicts pathologic vulnerability to psychological trauma[END_REF] . Previous studies have shown that high amygdala reactivity to masked stimuli (which prevents conscious awareness) and dACC metabolism prior to stress exposure increase the risk for subsequent stress symptoms [START_REF] Admon | Human vulnerability to stress depends on amygdala's predisposition and hippocampal plasticity[END_REF][START_REF] Shin | Resting metabolic activity in the cingulate cortex and vulnerability to posttraumatic stress disorder[END_REF] . Our results now suggest that sustained increases in amygdala and insula reactivity to biologically salient stimuli reflect a consequence of stress exposure, which may be additive to preexisting vulnerability factors. In contrast, the individual differences in altered amygdala coupling suggest that the impact of stress on the amygdala network is a consequence of the interaction between stress vulnerability and stress exposure.

The similarity between the neural consequences of stress exposure and PTSD pathophysiology has important implications for studies investigating the neural basis of PTSD. Previous neuroimaging studies with PTSD patients have included a control group of trauma exposed individuals that did not develop PTSD, to control for trauma exposure (for example, see ref 5). Our results underscore the importance for this procedure, by demonstrating that trauma exposure by itself also influences brain functioning.

Comparing PTSD patients to non-trauma exposed individuals may result in misattribution of group differences to PTSD pathophysiology.

One of the thirty-three soldiers (3%) scored above the clinical threshold for PTSD symptoms on a self-report questionnaire after their four month deployment to Afghanistan. This is comparable to PTSD prevalence rates for Dutch and UK soldiers that have been deployed to Iraq or Afghanistan [START_REF] Hotopf | The health of UK military personnel who deployed to the 2003 Iraq war: a cohort study[END_REF][START_REF] Engelhard | Deployment-related stress and trauma in Dutch soldiers returning from Iraq. Prospective study[END_REF] , but lower than the estimate for US soldiers [START_REF] Hoge | Combat duty in Iraq and Afghanistan, mental health problems, and barriers to care[END_REF] . Differences in these prevalence rates may have various causes, including differences in study populations, level of combat exposure, and screening methods [START_REF] Sundin | PTSD after deployment to Iraq: conflicting rates, conflicting claims[END_REF] .

Although our sample appears representative for the military population, it may not be representative for the general population. For example, the process of self-selection to serve in the military may lead to a relatively resilient population. To address this issue, future studies may investigate the consequences of severe and traumatic stress in nonmilitary samples.

In conclusion, our results demonstrate that severe stress exposure sensitizes amygdala and insula reactivity. In addition, individual differences in threat perception predicted divergent influences on the amygdala network, which may explain why some individuals are vulnerable to stress whereas others are stress resilient. Long-term followup studies are required to determine whether these stress-induced neural changes indeed represent resilience or vulnerability factors. The scatter plot illustrates the correlation in panel a at the peak voxel.

Figure 1 .

 1 Figure 1. Severe stress exposure increases amygdala and insula reactivity to biologically

Figure 2 .

 2 Figure 2. Individual differences in perceived threat during military deployment affects

Figure 3 .

 3 Figure 3. Individual differences in perceived threat during military deployment affects

ACKNOWLEDGEMENTS

We thank A. Beekman and M. Groenewald for assistance with data collection and P.

Gaalman for technical support. This work was supported by a grant (918.66.613) from the Netherlands Organization for Scientific Research (NWO) and the Dutch Ministry of Defense. The sponsors had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The authors have no potential conflicts of interest.


J J Beukema

R Dekker

Increasing species richness of the macrozoobenthic fauna on tidal flats of the Wadden Sea by local range expansion and invasion of exotic species

Keywords: long-term changes, species per unit area, macrozoobenthos, intertidal, soft sediment, introduced species, temperature change

A 40-y series of consistently collected samples (15 fixed sampling sites, constant sampled area of 15 x 0.95 m 2 , annual sampling only in late-winter/early spring seasons, and consistent sieving and sorting procedures; restriction to 50 easily-recognizable species) of macrozoobenthos on Balgzand, a tidal-flat area in the westernmost part of the Wadden Sea (The Netherlands), revealed significantly increasing trends of species richness. Total numbers of species annually encountered increased from ~28 to ~38. Mean species density (number of species found per sampling site) increased from ~13 to ~18 per 0.95 m 2 . During the 40 y of the 1970-2009 period of observation, 4 exotic species invaded the area: (in order of first appearance) Ensis directus, Marenzelleria viridis, Crassostrea gigas, and Hemigrapsus takanoi. Another 5 species recently moved to Balgzand from nearby (subtidal) locations. Together, these 9 new species on the tidal flats explained by far most of the increase in total species numbers, but accounted for only one third of the observed increase in species density (as a consequence of the restricted distribution of most of them). Species density increased particularly by a substantial number of species that showed increasing trends in the numbers of tidal-flat sites they occupied. Most of these wider-spreading species were found to suffer from cold winters. During the 40-y period of observation, winter temperatures rose by about 2 o C and cold winters became less frequent.

The mean number of cold-sensitive species found per site significantly increased by almost 2 per 0.95 m 2 . Among the other species (not sensitive to low winter temperatures), 6 showed a rising and 2 a declining trend in number of occupied sites, resulting in a net long-term increase 2 of species density amounting to another gain of 1.6 per 0.95 m 2 . Half of the 50 studied species did not show such long-term trend, nor were invaders. Thus, each of 3 groups (local or alien invaders/ winter-sensitive species/ other increasing species) contributed to a roughly similar extent to the overall increase in species density.

INTRODUCTION

Though there is widespread and right concern on global-wide declining biodiversity, reports of increasing species richness in specific areas have been reported as well. Such increases of species numbers in local areas can arise from introductions of invaders from distant locations (exceeding numbers of extinctions) or from changes in environmental conditions (when these changes promote more species than they do harm). Both types of causes can be directly influenced by man: invasions by increased shipping [START_REF] Reise | Introduced marine species of the North Sea coasts[END_REF]) and changes in habitat diversity and suitability by management (protection/ restoration) and disturbance. A special class of changing environmental conditions affecting species richness is the ongoing climate change by which some species are locally promoted and others put at a disadvantage.

For invertebrates in the marine environment, few homogeneous data series of sufficient length appear to be available to reliably judge long-term trends in species richness and relate possible trends to underlying causes. There are several reports on successful introductions of exotic species leading to increases in species numbers, as displacement of autochtonous species appear to be a rare phenomenon [START_REF] Reise | Introduced marine species of the North Sea coasts[END_REF][START_REF] Wolff | Causes of extirpation in the Wadden Sea, an estuarine area in the Netherlands[END_REF]. A warming climate generally results in pole-ward shifts of distribution areas, southern species increasing in abundance and northern species declining in the northern hemisphere [START_REF] Sagarin | Climate-related change in an intertidal community over short and long time scales[END_REF]). As species richness generally declines with increasing latitude, species gains tend to exceed species losses in areas at higher latitudes [START_REF] Hawkins | Consequences of climate-driven biodiversity changes for ecosystems functioning of North European rocky shores[END_REF].

The present study is based on a 40-y series of consistently collected quantitative data on macrozoobenthos in a vast area in the Dutch Wadden Sea. The Wadden Sea is an estuarine area of geologically-young age and was seriously human-altered for the last centuries (Lotze et al. 2005). Such areas appear to be particularly vulnerable for exotic invaders [START_REF] Wolff | Exotic invaders of the meso-oligohaline zone of estuaries in the Netherlands: why are there so many?[END_REF]. The Wadden Sea is characterized by vast tidal mud and sand flats which are inhabited by a fauna with high biomass and low species diversity [START_REF] Beukema | Biomass and species richness of the macrobenthic animals living on the tidal flats of the Dutch Wadden Sea[END_REF][START_REF] Beukema | Local differences on macrozoobenthic response to enhanced food supply caused by mild eutrophication in a Wadden Sea area: food is only locally a limiting factor[END_REF]. Part of the species suffer from severe winters [START_REF] Beukema | Biomass and species richness of the macrobenthic animals living on a tidal flat area in the Dutch Wadden Sea: effects of a severe winter[END_REF][START_REF] Beukema | Expected effects of changes in winter temperatures on benthic animals living in soft sediments in coastal North Sea areas[END_REF] and may profit from the warming climate [START_REF] Beukema | Expected changes in the Wadden Sea benthos in a warmer world: lessons from periods with mild winters[END_REF]. Data series reviewed by [START_REF] Van Der Graaf | Common Wadden Sea Secretariat, Wilhelmshaven Von Cosel R (2009) The razor shells of the eastern Atlantic, part 2. Pharidae II: the genus Ensis Schumacher, 1817 (Bivalvia, Solenoidea)[END_REF] point to increasing species numbers in various parts of the Wadden Sea for recent decades. However, for methodological reasons, these trends should be considered with caution. Avoiding these methodological problems by a restriction to a delimited group of conspicuous species, we analyze changes in total species numbers as well as in species density (number of species per unit area) and relate these changes to some possible underlying processes, such as invader introductions, climate change and human disturbance.

METHODS

Since around 1970, data on macrozoobenthos have been collected twice annually at 15 permanent (marked) sampling stations ("sites") located on Balgzand, a 50-km 2 tidal flat area in the westernmost part of the Wadden Sea. For the present study we used only data collected in late-winter/early spring, mostly in March, when numbers are at an annual minimum. Most (12) sites were 1-km long transects where 50 bottom samples of nearly 2 dm 2 each were taken at distance intervals of 20 m, together 0.95 m 2 per site. The 3 other sites were squares of 900 m 2 where 9 samples of nearly 0.1 m 2 each were taken at randomly chosen places. The sites covered the entire intertidal range and nearly the full range of sediment types on Balgzand [START_REF] Beukema | Local differences on macrozoobenthic response to enhanced food supply caused by mild eutrophication in a Wadden Sea area: food is only locally a limiting factor[END_REF]. There was little change in these abiotic conditions during the 40-y period of observation. Hard substrates such as dikes were not sampled, but animals attached to shells of mussels or oysters were included. The samples were sieved (1-mm square meshes) in the field and sorted alive in the laboratory. All living animals were picked out, sorted to species level as much as possible, and counted. Further details on the sampling area, the sampling stations, and the methods can be found in [START_REF] Beukema | Local differences on macrozoobenthic response to enhanced food supply caused by mild eutrophication in a Wadden Sea area: food is only locally a limiting factor[END_REF].

Temperature data were taken from [START_REF] Van Aken | Variability of the water temperature in the western Wadden Sea on tidal to centennial scales[END_REF]. We used monthly means of surface water temperatures in the nearby Marsdiep tidal channnel and averaged for each winter the means of December, January and February to obtain a mean winter water temperature.

Sources of error

Thin worms will be underestimated or completely missed, as a high proportion of them will pass the 1-mm sieve. Such species were completely excluded (e.g. nematodes, oligochaetes, tiny spionid polychaetes such as Pygospio sp.). Inevitably, sorting sieved samples with lots of debris (peat, empty shells) is imperfect: some small and inconspicuous specimens will at least partly be overlooked, even by experienced researchers. We standardized sorting and counting procedures as much as possible. Another problem arises in identification: some closely related species are so similar in external appearance that they cannot routinely be counted separately in macroscopic procedures. To reduce these errors, we decided to limit the present study to a list of 50 species that are sufficiently conspicuous and easily identifiable. To this end, we treated resembling species as one species, e.g. Corophium volutator and C. arenarium were recorded as C.sp., all barnacles as balanoids, and all sea anemones as Anthozoa. The full list of taxa can be found at the bottom of Table 1.

In fact, many more than these 50 macrozoobenthic species are present on Wadden Sea tidal flats and some tens of them were regularly or occasionally found in our samples. Some hundreds of benthic invertebrate species have been reported from the tidal plus subtidal parts of the international Wadden Sea, most of them small-sized [START_REF] Wolff | Note-1: Macoma balthica, Mya arenaria, Mytilus edulis, Scrobicularia plana, Hydrobia ulvae, Retusa obtusa, Phyllodoce sp. (mostly Ph. mucosa), Arenicola marina, Bylgides sarsi, Heteromastus filiformis, Hediste diversicolor, Scoloplos armiger, Bathyporeia sp., Corophium sp., Anthozoa (mostly Metridium senile and Sagartia troglodytes). Note-2: Abra alba, Petricola pholadiformis, Spisula subtruncata, Tellina (Angulus) fabula, Littorina obtusata, Crepidula fornicata, Pectinaria koreni, Neomysis / Praunus sp[END_REF][START_REF] Petersen | Red list of macrofaunal benthic invertebrates of the Wadden Sea[END_REF]. By reducing a full list to 50 selected items, we are convinced that we anticipated a possible positive bias in the species-richness trend due to a growing skill in recognizing species.

RESULTS

Long-term trends in species numbers and temperature

During the 1970-2009 period, the annual benthos samples taken showed significantly increasing numbers of macrozoobenthic species. Total numbers observed at 15 x 0.95 = 14.25 m 2 increased from ~28 in the early-1970s to ~38 in the late-2000s (solid points in Fig. 1b). The mean rate of increase amounted to 0.21 + 0.04 (mean and 1 s.e.) species y -1 . Likewise, the 15site means of species number per sampling site of 0.95 m 2 increased from ~12 to ~18 (open points in Fig. 1b), at a mean rate of 0.13 + 0.02 y -1 . Long-term means of species numbers amounted to 32.4 + 0.6 and 15.2 + 0.3 for sampled areas of 14.25 and 0.95 m 2 , respectively.

Note that these numbers are underestimates of the true numbers of macrozoobenthic species present, because they are out of a maximal number of 50 listed species (considered as easilyrecognizable and not to be overlooked).

In accordance with the recent trend of rising temperatures which is particularly strong in Dutch coastal waters and occurs in all seasons [START_REF] Van Aken | Variability of the water temperature in the western Wadden Sea on tidal to centennial scales[END_REF], 2010), annual mean water temperatures rose by a mean rate of ~0.05 o C y -1 during the 1970-2009 period of observation (Fig. 1a). Annual means increased from about 10 to almost 12 o C (correlation with year number r = 0.63, n = 39, p<0.0001); fluctuations in winter (Dec-Jan-Feb periods) temperatures were stronger and more irregular (open points in Fig. 1a), but nevertheless winter water temperatures showed a significantly positive correlation with year number (r = 0.36, n = 39, p<0.05), again at a mean rate of increase of ~0.05 o C y -1 . Among the 40 winters of the period of observation, 8 were particularly cold (mean winter water temperature <3 o C) : 1970, 1979, 1982, 1985, 1986, 1987, 1996, and 1997 (see open points of Figs 1a and1c). Such severe winters appear to occur less frequently during the last than the first half of the 1970-2009 period.

Species numbers observed after severe winters were usually lower than after neighbouring mild winters, compare open and solid points in Fig. 1c. The influence of winter temperature on species richness can be removed by addition or subtraction a number of 1.7 (totals per 14.25 m 2 ) or 1.0 (means per 0.95 m 2 ) species (values taken from Fig. 3a) for each o C the water temperature of the preceding winter was lower or higher, respectively, than the long-term mean of 4.2 o C. The thus temperature-corrected values for species numbers still showed a statistically significant increasing trend in the course of the period of observation (Fig. 1d).

In early 1991, the species numbers were particularly low (crosses in Fig. 1c). In that year, not any mussel bed was present on Balgzand as a consequence of heavy fishing in the summer of 1990 [START_REF] Beukema | Increased mortality in alternative bivalve prey during a period when the tidal flats of the Dutch Wadden Sea were devoid of mussels[END_REF][START_REF] Beukema | Consequences of the sudden removal of nearly all mussels and cockles from the Dutch Wadden Sea[END_REF]. In 1991, the number of species was not only reduced by the absence of the one species Mytilus edulis, but also by the absence of species that use mussel shells as a hard substrate such as barnacles and periwinkles.

Effects of winter temperatures

Several zoobenthic species living on Wadden Sea tidal flats are sensitive to low winter temperatures, i.e. they show significantly reduced survival rates in severe winters [START_REF] Beukema | Biomass and species richness of the macrobenthic animals living on a tidal flat area in the Dutch Wadden Sea: effects of a severe winter[END_REF]). Some of these species completely or almost completely disappear from the intertidal after the coldest winters, either at all intertidal zones [START_REF] Beukema | Expected effects of changes in winter temperatures on benthic animals living in soft sediments in coastal North Sea areas[END_REF]) or only at the sampling sites at high intertidal levels [START_REF] Beukema | Zoobenthos survival during severe winters on high and low tidal flats in the Dutch Wadden Sea[END_REF]. [START_REF] Beukema | Expected effects of changes in winter temperatures on benthic animals living in soft sediments in coastal North Sea areas[END_REF] listed 11 of such winter-sensitive species of benthic animals on Balgzand. They are all included in the present 50-species list, and 10 of them showed a significantly positive relationship between winter temperature and number of sites subsequently occupied: the bivalves Cerastoderma edule (Fig. 2d), Abra tenuis (Fig. 2b), Tellina tenuis (Fig. 2b) and Mysella bidentata (Fig. 2c), the polychaete worms Lanice conchilega (Fig. 2a) and its commensal Malmgreniella lunulata (Fig. 2a) and Nephtys hombergii (Fig. 2c), the group of Nemerteans (Fig. 2d), and the crustaceans Crangon crangon (Fig. 2e) and Carcinus maenas (Fig. 2f). The 11 th species (Bylgides sarsi) that was reported to show reduced survival in cold winters did not show a significant relationship as shown in Fig. 2, nor did it show a long-term trend in numbers of site occupied.

As to be expected from the substantial share of winter-sensitive species among the macrozoobenthic fauna on Balgzand, the total numbers of species found were higher after mild than after cold winters, both for 14.25 and 0.95 m 2 sampling areas (solid and open points of Fig. 3a). To allow for the interaction between year number and winter temperatures, the species numbers were de-trended, i.e. they were augmented by 0.21 (number per 14.25 m 2 ) or 0.13 (number per 0.95 m 2 ) for each year before 1990 and reduced by the same amount for each year after 1990 (correction values taken from Fig. 1b). The thus year-influence-corrected species numbers were still significantly positively correlated with preceding-winter water temperatures (Fig. 3b), increasing by ~1.1 (14.25 m 2 ) and ~0.6 (0.95 m 2 ) species per 1 o C temperature rise.

At least 6 frequently observed species were completely absent after the coldest winters (zero points in Fig. 2 at the lowest temperatures) and some of them needed a few years after a severe winter to recover and thus remained absent for another 1 or 2 y (most of the zero points in Fig. 2 at winter temperatures of >4 o C). Some other species (e.g. N. hombergii and C. edule, Figs 2c and 2d) occurred at reduced numbers of sites after the coldest winters, but were never completely absent. Complete absence affects the total species number per 14.25 m 2 as well as the mean number per 0.95 m 2 , whereas absence at part of the sampling sites only reduces the mean species number per 0.95 m 2 .

After most of the mild winters, all of the above 10 winter-sensitive species were present at one or more sites. In these years, the group contributed a number of 10 to the total (per 14.25 m 2 ) species number (solid squares in Fig. 1c). After cold winters, only about half of these species were present at one or more sites, resulting in a contribution of ~5 winter-sensitive species to total species numbers (open squares for 1970, 1979, 198, 1985, 1986, 1987, 1996, and 1997 in Fig. 1c). Whenever a severe winter had occurred at least 3 years ago (as in the 1974-1978, 1991-1995, and 2000-2009 periods), the contributions of winter-sensitive species were 10 or close to 10 species (solid squares of Fig. 1c). Likewise, but less pronounced, mean contributions of winter-sensitive species per 0.95 m 2 were higher after mild than after cold winters (solid and open circles in Fig. 1c) and were particularly high in years long after a severe winter.

Introduced species

In the course of the 1970-2009 period of observation, at least 4 non-native species turned up in the samples. The first to appear was the American jack-knife clam Ensis directus in 1982, about 5 y after its first find in the Wadden Sea and Europe [START_REF] Beukema | Dynamics and growth of a recent invader into European coastal waters: the American razor clam, Ensis directus[END_REF]Von Cosel 2009). The species was observed in the Balgzand samples in nearly all years after 1982 (solid squares in Fig. 5). The next invader was the American spionid worm Marenzelleria viridis) in 1989, 7 and 6 y after its first records in the North Sea and Wadden Sea, respectively [START_REF] Essink | Dispersal and development of Marenzelleria spp. (Polychaeta, Spionidae) populations in NW Europe and The Netherlands[END_REF][START_REF] Essink | General patterns in invasive ecology tested in the Dutch Wadden Sea: the case of a brackish-marine polychaetous worm[END_REF] as M. cf. wireni, but see Sikorsky and Bick (2004)). It rapidly spread over the entire Balgzand area, occurring at 13 to 15 out of the 15 sampling sites ever since 1996 (open circles in Fig. 5). The third exotic invader was Crassostrea gigas, appearing in the samples in 2001, about 40 and 30 y after its first deliberate introduction from East Asia into Europe and the Wadden Sea [START_REF] Wolff | Non-indigenous marine and estuarine species in The Netherlands[END_REF]. Since 2003, it was regularly found at 2 or 3 sites (crosses in Fig. 5). Finally, the Asian decapod Hemigrapsus takanoi (see [START_REF] Asakura | Hemigrapsus takanoi, new species, a sibling species of the common Japanese intertidal crab H. penicillatus (Decapoda: Brachyura: Grapsoidea)[END_REF] turned up in the samples in 2006, more than 20 y after its first find in Europe [START_REF] Gollasch | The Asian decapod Hemigrapsus penicillatus (De Haan, 1835) (Grapsidae, Decapoda) introduced in European waters: status quo and future perspective[END_REF]. It was subsequently found every year at 1 to 3 sites (triangles in Fig. 5).

Apart from the above 4 invasive species that recently arrived in Europe, 5 more species (that were already present in the North Sea area for at least decades) were only recently observed on the Balgzand tidal flats: Crepidula fornicata, Glycera alba, Lepidochitona cinerea, Echinocardium cordatum, and Ophiura ophiura. This small group of newcomers on the Balgzand tidal flats was observed in small numbers (mostly only 1 specimen) at few sites (mostly only 1 site), thus contributing little to species numbers per 0.95 m 2 (open points in Fig. 4b).

Other species

Among the remaining 31 species (50 -10 -4 -5), 6 showed a statistically significant upward and 2 a significant downward trend in the numbers of sites where they were encountered in the successive years of the 1970-2009 period of observation. Together, the 6 species with a positive trend (Littorina littorea, Nereis spp. (mostly Alitta succinea, but also including some A. virens and Eunereis longissima), Eteone spp., Urothoe poseidonis, Gammarus locusta, and the group of Balanoids) showed an increase of mean numbers of species per site from ~0.7 to ~2.7 species per 0.95 m 2 (Fig. 4c). The 2 species with a negative trend (Magelona johnstoni, Scolelepis foliosa) showed a decline from ~0.7 to ~0.3 species per 0.95 m 2 (Fig. 4c). Thus, the net contribution to species density of the 8 species with a significant trend amounted to a positive value of 2.0 -0.4 = 1.6 species per 0.95 m 2 . The remaining 24 species did not contribute (neither positively nor negatively) to species density (Fig. 4d).

DISCUSSION

A long period of consistent sampling of the macrozoobenthic fauna on the Balgzand tidal flats revealed a gradual increase of species richness. Expressed as total number of species encountered, the increase amounted to about 35% (~28 to ~38) over the entire 40-y period of observation. Expressed as a mean number per sampling site, the increase was of a similar magnitude (~13 to ~18 per 0.95 m 2 ). As a measure of species richness, we consider the latter estimate of species density superior to total species number, because the total number found in any year strongly depends on finds of just one individual of a rare species. Each of such finds would add about 3% to total species number, but only about 0.5% to the number per site (1 / 15 = 0.07, being 0.5% of 14.2, the long-term mean number of species per 0.95 m 2 ).

The above numbers refer to a limited group of 50 species, all easily identifiable and relatively conspicuous. This limitation was thought imperative to exclude a possibly serious influence of growing experience of the observers in sorting samples and/or recognizing rare or similar-looking species, which would lead to undeservedly increasing estimates of numbers of species found during the long period of observation.

We identified various groups of species to contribute to the increasing trend in species richness. The 10 winter-sensitive species (mentioned in Fig. 2) contributed together ~1.8 species per 0.95 m 2 to the increase in species density (Fig. 4a), in accordance with the increasing trend of winter temperatures and the declining frequency of occurrence of cold winters (Fig. 1a). The species belonging to this group hardly affected the total species numbers, because they were all present at one or more sites both at the start and at the end of the 40-y period. The appearance of 8 invaders, either of exotic or local origin, explained almost completely the long-term increase in total species number (amounting to 10 species). The contribution to the long-term increase in species density of exotic and local newcomers amounted to 1.5 and 0.2 species per 0.95 m 2 , respectively (Fig. 4b). A substantial share of the observed increase in species density was contributed by species that showed a long-term positive or negative trend in the numbers of sites occupied, but without an apparent coupling to a known change in environmental conditions. This contribution amounted to +2.0 and -0.4 (net +1.6) species per 0.95 m 2 (Fig. 4c). Taken together, the above groups contributed 1.8 + 1.7 + 1.6 = 5.1 species per 0.95 m 2 and this is close to the observed overall increase in species density of 5.2 species per 0.95 m 2 (Table 1). Note that not just a few species accounted for the long-term changes in species density, but that more than half (26 / 50) of the studied species were in some way involved in the explanation of this increase. On the other hand, the one 8-species group of newcomers (including 4 truly exotic species and 4 showing only local range extension) could almost completely explain the observed increase in total species number.

The observed increases in macrozoobenthic species richness may not be limited to the westernmost part of the Wadden Sea. A recent survey of the macrozoobenthos of the Wadden Sea [START_REF] Van Der Graaf | Common Wadden Sea Secretariat, Wilhelmshaven Von Cosel R (2009) The razor shells of the eastern Atlantic, part 2. Pharidae II: the genus Ensis Schumacher, 1817 (Bivalvia, Solenoidea)[END_REF] indicates increasing trends of species numbers at all 3 sites (Balgzand, Groninger Wad, Norderney) that were continuously sampled ever since the 1970s.

However, as the authors rightly remark, part of these increases in species numbers will have been caused by methodological reasons (viz. the above discussed improved species identification). In the north Frisian Wadden Sea near the island of Sylt, [START_REF] Reise | Historical changes in the benthos of the Wadden Sea around the island of Sylt in the North Sea[END_REF] report increases in numbers of infauna species, particularly in polychaetes, between the 1930s and 1980s, resulting in enhanced numbers of species per 0.2 m 2 (compare figure 8b of Lotze 2005).

Van der Graaf et al. ( 2009) could not confirm a suggested "polychaete take-over". In the central western North Sea, a 33-y data series by [START_REF] Frid | Observing change in a North Sea benthic system: a 33 year time series[END_REF] revealed a significant increase in numbers of genera found per 0.5 m 2 from ~100 in the 1970s to ~120 in the 1990s and early-2000s.

Though the trends in species richness may usually be upward, species losses have been reported from the Wadden Sea. The main causes of (local or total) species extinction in the Wadden Sea appear to be habitat change or destruction and exploitation (compare figure 3 of Lotze et al. 2005). Several cases of habitat destruction were caused by fishery [START_REF] Reise | Historical changes in the benthos of the Wadden Sea around the island of Sylt in the North Sea[END_REF][START_REF] Piersma | Long-term indirect effects of mechanical cockle-dredging on intertidal bivalve stocks in the Wadden Sea[END_REF], including disturbance of the upper bottom layer and complete removal of oyster and mussel banks. Mussel (and oyster) beds are usually richer in species than their surroundings, as these structures offer locally hard substrates to species that cannot live on the surrounding soft sediments (Buschbaum et al. 2009). The few sampling sites on Balgzand that usually included mussel beds showed higher species densities than other sites. The removal of all mussel beds in 1990 [START_REF] Beukema | Consequences of the sudden removal of nearly all mussels and cockles from the Dutch Wadden Sea[END_REF] resulted in exceptionally low estimates of species richness at the next sampling occasion in early-1991 (see + and x points in Figs 1c, 1d, and3b), but species richness and mussel numbers had largely recovered already by 1992, due to highly successful reproduction in many species (including mussels) in the summer of 1991.

As in other areas, part of the increasing species richness on Balgzand was due to invaders. In Wadden Sea invertebrates, the numbers of newly introduced species exceeded those of species that became extinct (compare figure 2 of Lotze et al. 2005). No cases appear to be known of extinction caused by an invasive species in the Wadden Sea [START_REF] Wolff | Causes of extirpation in the Wadden Sea, an estuarine area in the Netherlands[END_REF]Lotze et al. 2005) and examples of invaders reducing abundance of other species appear to be few. An example of such negative interaction may be the local declines of biomass of Nereis spec. and other species simultaneously with the rapid increase of biomass of Marenzelleria viridis after their introduction [START_REF] Essink | Long-term changes (1977-1994) in intertidal macozoobenthos of the Dollard (Ems estuary) and effects of introduction of the North American spionid polychaete Marenzelleria cf. wireni[END_REF][START_REF] Essink | General patterns in invasive ecology tested in the Dutch Wadden Sea: the case of a brackish-marine polychaetous worm[END_REF]. [START_REF] Markert | Recently established Crassostrea-reefs versus native Mytilus-beds: differences in ecosystem engineering affects the macrofaunal communities (Wadden Sea of Lower Saxony, southern German Bight)[END_REF][START_REF] Troost | Causes and effects of a highly successful marine invasion: case-study of the introduced Pacific oyster Crassostrea gigas in continental NW European estuaries[END_REF] found no indication of a suppression of indigenous species by the recent establishment of reefs of Pacific oysters Crassostrea gigas in the German and Dutch Wadden Sea, respectively.

A substantial part of the observed increase in species density could be attributed to the warming climate: the 10 winter-sensitive species (Fig. 2) became more abundant and were found at an increasing number of sampling sites (Table 1; Fig. 4a). Declines in numbers of occupied sites were observed in a few species, but these declines appeared to be unrelated to temperature change. A few species, notably the bivalve Macoma balthica do suffer from the warming climate and showed declining trends in abundance for recent decades [START_REF] Beukema | Some like it cold: populations of the tellinid bivalve Macoma balthica (L.) suffer in various ways from a warming climate[END_REF]. So far, however, this did not result in declining numbers of sites were they were found.

Thus, the long-term warming trends resulted in increasing species richness on the Balgzand tidal flats. This conclusion is in accordance with that of [START_REF] Hawkins | Consequences of climate-driven biodiversity changes for ecosystems functioning of North European rocky shores[END_REF] for British rockyshore fauna, that more southern (warm-water) species have been recorded advancing than northern (cold-water) species retreating. A similar conclusion was reached by [START_REF] Sagarin | Climate-related change in an intertidal community over short and long time scales[END_REF] for the Californian rocky intertidal.

The two processes (invasions and advancement of species, mostly by climate change) that caused the increase in species richness on Balgzand tidal flats are still in progress and may be expected to continue. Immigration of alien species by a.o. transport in ballast water, attachment to ship hulls or to imported organisms (such as oysters) cannot completely be prevented. Brackish estuaries and other coastal areas appear to offer plenty of open niches for newcomers [START_REF] Wolff | Exotic invaders of the meso-oligohaline zone of estuaries in the Netherlands: why are there so many?[END_REF][START_REF] Reise | Introduced marine species of the North Sea coasts[END_REF]. Further spread of lower-latitude species into a pole-ward direction (in fact largely a re-colonization process ever since the Ice Ages) will rapidly continue as long as global warming continues. So far, there are no signs of extirpation of species in the study area as a consequence of warming. Warming promoted the distribution of winter-sensitive species that were already present in the area. Occasional cold winters caused an immediate but temporary reduction in species number and species density, but recovery went rapidly. In conclusion: we expect further increases of species numbers and species-density values from all processes underlying the observed increase in species richness. LEGENDS Fig. 1. Long-time (1970Long-time ( -2009) ) Table 1. Summary of contributions by various groups of species to long-term changes in macrozoobenthic species richness on Balgzand, expressed in (changes of) mean number of species per site (n, per 0.95 m 2 ). N is number of species allotted to each group.

  Fig. 1. Long-time (1970[START_REF] Hawkins | Consequences of climate-driven biodiversity changes for ecosystems functioning of North European rocky shores[END_REF] series of data on:

Fig. 2 .

 2 Fig. 2. Relationships between mean water temperature (T in o C) in the preceding winter(temperatures as open points in Fig.1a) and number N of sampling sites (among a total of 15) where at least 1 specimen was found in the subsequent late-winter/early-spring sampling of 10 winter-sensitive species:

Fig. 3 .

 3 Fig. 3. Relationships between mean water temperature (T in o C) in the preceding winter (as open points in Fig.1a) and (a) actual numbers N of species (among a list of 50 easily recognizable macrozoobenthic species) found in late-winter/early spring in (solid points) all samples together (15 sites x 0.95 m 2 ) and (open points) mean number per sampling site (0.95 m 2 ). In (b) these numbers of species have been de-trended for the influence of year number (see text). The numbers found in 1991 are indicated separately by crosses (+ for 14.25 and x for 0.95 m 2 , respectively). Best linear fits:

Fig. 4 .

 4 Fig.4.Long-term (1970Long-term ( -2009) ) changes in the contributions to total species richness (n = mean number of species per 0.95 m 2 ) of 4 groups of species:

Fig. 5 .

 5 Fig. 5. Numbers of sampling sites (among a total of 15) where at least 1 specimen was found in the samples taken in late-winter/early spring in the indicated year of 4 newly-introduced species. (a) (solid squares) Ensis directus and (open circles) Hemigrapsus takanoi; (b) (solid squares) Marenzelleria viridis and (open circles) Crassostrea gigas.

*

  Crepidula fornicata, Glycera alba, Lepidochiton cinerea, Echinocardium cordatum, Ophiura ophiura. **Littorina littorea, Nereis spp. (mostly Alitta succinea), Eteone spp., Urothoe poseidonis, Gammarus locusta, Balanoids (Balanus crenatus, Semibalanus balanoides, Austrominius modestus). ***Magelona johnstoni, Scolelepis foliosa.

  Figure

Acknowledgements. The present study is part of a long-term monitoring program on macrozoobenthos in the Wadden Sea executed by NIOZ and financially supported by the Dutch Ministry of Transport, Public Works and Water Management, Water Service (RWS-Waterdienst).