Maria Castella

Roser Pujol

Elsa Callen

Maria Jose Ramirez

Jose A Casado

Maria Talavera

Teresa Ferro

Arturo Munoz

Julian Sevilla

Luis Madero

Elsa Callén

Maria José Ramírez

José A Casado

Arturo Muñoz

Julián Sevilla

Elena Cela

Cristina Beléndez

Cristina Díaz De Heredia

Teresa Olivé

José Sánchez De Toledo

Isabel Badell

Jesús Estella

Ángeles Dasí

Antonia Rodríguez-Villa

Pedro Gómez

María Tapia

Antonio Molinés

Ángela Figuera

Juan A Bueren

Jordi Surrallés
email: jordi.surralles@uab.es

Prof Jordi Surrallés

Chromosome fragility in Fanconi anemia patients: diagnostic implications and clinical impact

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure, congenital malformations, endocrine dysfunctions and cancer predisposition. It was first described in 1927 by the Swiss pediatrician Guido Fanconi [START_REF] Fanconi | Familiäre infantile perniziosaartige Anämie (perniziöses Blutbild und Konstitution)[END_REF] and its estimated incidence is about 1-5 cases in one million births in the overall population, [START_REF] Joenje | The emerging genetic and molecular basis of Fanconi anaemia[END_REF] or up to less than 1 in 20.000 in some consanguineous ethnic groups. [START_REF] Callen | A common founder mutation in FANCA underlies the world's highest prevalence of Fanconi anemia in Gypsy families from Spain[END_REF][START_REF] Tipping | Molecular and genealogical evidence for a founder effect in Fanconi anemia families of the Afrikaner population of South Africa[END_REF][START_REF] Whitney | A common mutation in the FACC gene causes Fanconi anaemia in Ashkenazi Jews[END_REF] FA is a genetically heterogeneous disease, as 13 different FA complementation groups and corresponding genes have been currently identified (FANC-A, -B, -C, -D1/BRCA2, -D2, -E, -F, -G, -I, -J/BRIP1, -L , -M, and -N/PALB2). [START_REF] Moldovan | How the Fanconi Anemia Pathway Guards the Genome[END_REF][START_REF]Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins[END_REF]8] In the USA, with 681 FA patients subtyped, FA-A is the most frequent complementation group representing 60.5% of the patients; FA-C and FA-G are also frequent, accounting for 16% and 10% of the patients respectively, while the other groups are rare. [START_REF] Joenje | The emerging genetic and molecular basis of Fanconi anaemia[END_REF][START_REF] Auerbach | Fanconi anemia and its diagnosis[END_REF][START_REF] Kennedy | The Fanconi Anemia/BRCA pathway: new faces in the crowd[END_REF] The FA-A subtype is, however, over-represented in some geographical regions, including Mediterranean countries such as Spain, with 4 out of 5 FA patients belonging to this complementation group. [START_REF] Casado | A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network[END_REF] At molecular level, FA proteins are known to function on a common DNA repair pathway, the FA/BRCA pathway, which is focused on the processing of stalled replication forks generated either spontaneously or in response to drug induced DNA insterstrand cross-links (ICLs) and other types of DNA damage. [START_REF] Niedernhofer | Fanconi anemia (cross)linked to DNA repair[END_REF] Genomic instability is, therefore, a hallmark of FA cells. It was first observed in 1966, when Schroeder and co-workers described a high frequency of spontaneously-formed chromosome breaks in cells of FA patients. [START_REF] Schroeder | Cytogenetic and cytologic findings in enzymopenic panmyelopathies and pancytopenias. Familial myelopathy of Fanconi, glutathione-reductase deficiency anemia and megaloblastic B12 deficiency anemia[END_REF] Later, this genomic instability was seen to be highly induced by ICL-inducing agents, such as diepoxybutane (DEB), mitomycin C (MMC) or cis-platin, [START_REF] Schuler | Chromosome studies in Fanconi's anemia][END_REF][START_REF] Sasaki | Is Fanconi's anaemia defective in a process essential to the repair of DNA cross links[END_REF] leading to the development of the first diagnostic test for FA. [START_REF] Auerbach | A test for Fanconi's anemia[END_REF][START_REF] Auerbach | Diagnosis of Fanconi Anemia by Diepoxybutane Analysis[END_REF] Although high sensitivity to ICL-inducing agents is the hallmark of FA cells, an accurate diagnosis is compromised in some cases, especially among mosaic patients, who represent 15-25% of all patients. [START_REF] Soulier | Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway[END_REF][START_REF] Kwee | Unusual response to bifunctional alkylating agents in a case of Fanconi anaemia[END_REF][START_REF] Lo Ten Foe | Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance[END_REF] Somatic mosaicism is produced when one of the pathogenic mutations is reverted in a hematopoietic precursor cell. Owing to an increased proliferative advantage, reverted cells are able to clonally expand and improve patient's blood counts. Depending on the stage of differentiation of the cell in which the gene correction occurred, reversion may affect all hematopoietic cell lineages, leading to a "natural gene therapy". [START_REF] Lo Ten Foe | Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance[END_REF][START_REF] Gross | Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction[END_REF][START_REF] Youssoufian | Natural gene therapy and the Darwinian legacy[END_REF] Alternatively, the reversion may affect only some hematopoietic lineages and therefore, it may not lead to an improvement of a patient's hematological condition. As the diagnostic test of chromosome fragility is usually performed on peripheral blood T-cells, a high proportion of reverted T-cells can lead to a false negative result. On the contrary, some non-FA individuals can have a number of T-cells with chromosome breaks after DEB or MMC treatment and this can be interpreted as mosaicism by non-experienced laboratories leading to false positives. This is due to overlapping values of currently used chromosome fragility indexes (% of cells with breaks or average number of breaks per cell) between non-FA and FA mosaics. [START_REF] Auerbach | Fanconi anemia and its diagnosis[END_REF] The primary cause of death among FA patients is bone marrow failure (BMF), which typically occurs during the first decade of life. [START_REF] Kutler | A 20-year perspective on the International Fanconi Anemia Registry (IFAR)[END_REF] While novel therapeutic strategies to cure BMF are under intensive research, including gene therapy and regenerative medicine based on induced pluripotent stem cells, [24,[START_REF] Raya | Diseasecorrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells[END_REF] the only currently available curative treatment is hematopoietic stem cell transplant (HST) from a compatible donor, which requires a previous conditioning of the patient. Regular conditioning regimens, consisting of chemotherapy and radiation, are highly toxic for FA patients, so milder conditioning regimens must be used. [START_REF] Dufour | Fanconi anaemia: new strategies[END_REF] In this context, a clear and unambiguous diagnosis is, therefore, essential for the survival of the patients after HST.

To further examine chromosome fragility in FA and its diagnostic and clinical implications, we have performed a total of 198 DEB-induced chromosome fragility tests in non-FA individuals and FA patients where information on subtype, cell sensitivity to MMC and clinical data were available. This large series allowed us to quantify the existing variability in ICL-sensitivity among FA patients and normal population and, therefore, we propose a new chromosome fragility index that provides a clear cut-off diagnostic level to unambiguously distinguish FA patients (including mosaics) from non-FA individuals. Spontaneous chromosome fragility and its correlation with DEBinduced fragility is also analyzed and discussed. Finally, the relationship between cell sensitivity to ICLs and the patient's clinical severity markers is evaluated, revealing a direct correlation with congenital malformations, but not with the age of onset of hematological disease.

Materials and methods

Patients and samples

Blood samples from patients with clinical suspicion of FA and controls were collected for chromosome fragility evaluation. Clinical data from FA patients were obtained from their clinicians, including age of onset of hematological disease and number of congenital malformations. Number of congenital malformations was recorded as the well as skeletal, head, gastrintestinal, cardiac, genitourinary system malformations and mental retardation. Thus, the number of congenital malformations ranged from 0 to 10. This study was approved by the Universitat Autònoma de Barcelona Ethical Committee for Human Research and informed consents were obtained according to the Declaration of Helsinki.

Chromosome fragility test

Chromosome fragility tests on peripheral blood lymphocytes were performed basically as described by Auerbach and co-workers, [START_REF] Auerbach | Diagnosis of Fanconi Anemia by Diepoxybutane Analysis[END_REF] with some minor modifications. Three blood cultures were prepared for each patient, including 0.5ml of blood in heparin and 4.5ml of culture consisting of 15% fetal bovine serum, 1% antibiotics, 1% L-Glutamine and 1% phytohemaglutinin in RPMI (all reagents form Gibco). Twenty four hours after culture set-up, two cultures were treated with DEB at a final concentration of 0.1µg/ml (Sigma, Cat.No.202533), and the remaining culture was left untreated for spontaneous chromosome fragility evaluation. 46h after DEB treatment, colcemid was added at a final concentration of 0.1µg/ml. Cultures were harvested 2h later when metaphase spreads were obtained following standard cytogenetic methods and finally stained with Giemsa. For chromosome fragility evaluation, 25-50 metaphases with 46 [START_REF] Fanconi | Familiäre infantile perniziosaartige Anämie (perniziöses Blutbild und Konstitution)[END_REF] centromeres were analyzed for each culture. The microscope analysis was performed with a Leitz Aristoplan microscope and, later with a Zeiss Imager M1 microscope coupled to a computer assisted metaphase finder (Metasystems, Germany). The main criteria for the determination of chromosome fragility were as follows: gaps were not counted as chromosome breaks and figures were converted to the minimum number of breaks necessary to form each figure. DEB stock was routinely replaced every 6 months. Before using a new lot, a control fragility assay was performed using a FA lymphoblastoid cell line to ensure that there was no significant variation between lots.

Analysis of cell survival to MMC

The survival of FA patient T cells to MMC was calculated from data obtained during the subtyping studies described in a previous report. [START_REF] Casado | A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network[END_REF] Briefly, mononuclear cells from peripheral blood were stimulated in plates coated with anti-human CD3 (OKT3 Ortho Biotech) and anti-CD28 (CD28 Pharmingen, San Diego, CA) monoclonal antibodies. Five days later, the proliferating T cells were collected and exposed to increasing concentrations of MMC (0 to 1000nM) during additional five days in the presence of IL-2 (100u/ml, Proleukin, Chiron Corp, CA). Finally, the cells were resuspended in PBS-BSA (0.05%) containing 0.5μg/ml propidium iodide (PI; Sigma) and incubated for 10 min at 4ºC. Cell viability was determined by flow cytometry based on the PI exclusion test. In our previous study we observed that 33nM is the concentration of MMC that better discriminates between MMC resistant and sensitive T cells. [START_REF] Casado | A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network[END_REF] Therefore, survivals obtained after exposure to this concentration of MMC were routinely used to discriminate between T cells with a differential response to MMC.

Statistics

Correlations between variables were analyzed using Pearson's test when both variables were normally distributed, or Spearman's test if not. To compare means between several groups, ANOVA and Tamhane for post-hoc analysis were used. All statistical analyses were performed using SPSS software package.

Results

DEB-induced chromosome fragility: discriminating between non-FA, FA mosaics and FA non-mosaic patients.

Chromosome fragility was evaluated in 105 non-FA individuals and 93 FA patients using exactly the same conditions. Of the 93 patients included in this analysis, 7 were of unknown complementation group and 86 were successfully subtyped. Of these, 70 FA patients were found to belong to complementation group FA-A, while other complementation groups were rare. A summary of the results obtained on spontaneous and DEB-induced fragility is provided in Table 1. Chromosome fragility is usually reported as "breaks/cell" and "% of aberrant cells". As shown in Figure 1a andb, the number of "breaks/cell" is more than 10 times higher in the FA population when compared to the non-FA population, while "% of aberrant cells" increases 60 times in the FA group. However, the level of variability between FA patients is very high (see panels below Figures 1a and1b) and, in some cases, chromosome fragility values in FA patients overlap with those observed in non-FA patients. Part of this high variability in the FA patients is due to the existence of a subgroup of FA patients who have lower values of "% of aberrant cells" and "breaks/cell" (Figure 1c and1d). This subgroup corresponds to FA patients with a T-cell mosaicism. In this study, FA patients with less than 40% of aberrant cells are considered mosaic, while those with ≥60% of cells are considered non-mosaic FA patients. FA patients with a proportion of aberrant cells between 40 and 60% are considered possible mosaics, while waiting for additional evidence of mosaicism. This distinction is based on parallel data on cell sensitivity to MMC, mutational data (pathogenic mutations were identified in 12 out of 17 mosaics), repeated DEB tests over time, ICL-sensitivity of all primary fibroblasts available from mosaic patients, and the reverted nature of 7 out 8 lymphoblastoid cell lines generated from mosaic FA patients. All mosaic patients could be subtyped by retroviral complementation, either in blood lymphocytes, when they were sensitive enough to MMC or in fibroblasts when they were resistant. In the latter case, 5 out of 5 established fibroblast cell lines showed the characteristic, MMC-induced G2/M phase cell cycle arrest and, therefore, were able to be subtyped by retroviral complementation, confirming the mosaic nature of the patients (see below and data not shown).

Discrimination between FA mosaic patients and non-FA is the principal difficulty found when making an FA diagnosis. As shown in Figure 2a, the mean level of "breaks/cell" in FA-mosaics is close to that observed for the non-FA population. However, when considering only "breaks/multiaberrant cell" (breaks observed in cells with 2 or more breaks) (Figure 2b), chromosome fragility level of mosaic patients is equivalent to that observed in FA non-mosaics, even if a few cells with 2 or more breaks can be found in non-FA individuals (Figure 2b, lower panel). Therefore, neither "% aberrant cells" nor "breaks/cells" or "breaks/multiaberrant cell" indexes alone are enough to discriminate between non-FA and FA-mosaic patients. However, when combining "% of aberrant cells" and "breaks/cell" or "breaks/multiaberrant cell" in the same graph (Figures 2c and2d, respectively), a better separation between patient subgroups is obtained. Thus, the data graphically presented in Figure 2d were transformed into a new index that we called "Chromosome Fragility Index" (CFI), resulting from multiplying the other two indexes. As shown in Figure 2e, CFI allowed establishing a cut-off value to clearly distinguish the non-FA from the FA population, including mosaics, without overlapping. The results of our study showed that non-FA patients have CFI values below 40, while all FA patients, including mosaics, have a CFI above 55.

The presence of tri-or tetra-radial figures is a characteristic feature of FA cells upon DEB treatment. In FA individuals, 1.373 multiradial figures were found in a total of 4.011 metafases studied. Among all non-FA patients, 5.250 metaphases have been studied and 3 cells with one multiradial figure were found in 3 different individuals (frequency of 0.057% or 1 in 1750 metaphases). Therefore, even though FA patients have a 600-fold increase in radial figures upon DEB treatment, the presence of figures among the non-FA population is rare, but possible, and should not be interpreted as a positive diagnosis of FA mosaicism.

Spontaneous chromosome fragility

Spontaneous chromosome fragility has also been analyzed. Results obtained are described in Table 1. Overall higher spontaneous chromosome fragility is observed in FA patients (Figure 3a and 3b): 5.2 fold in "% aberrant cells" and 8.3 fold in "breaks/cell", when compared with non-FA patients. However, the variability in spontaneous chromosome fragility is very high in all three groups, and no statistically significant differences could be detected between the non-FA and FA-mosaic groups.

As shown in Figure 3a and 3b lower panels, spontaneous chromosome fragility interval values overlap in all 3 populations. Quantification of the proportion of FA patients (excluding mosaics) that present a level of spontaneous chromosome fragility in the range of non-FA population (Figure 3c), revealed that 54.4% of FA patients had spontaneous chromosome fragility within the normal range. In this graph, spontaneous chromosome fragility is measured by integrating "breaks/cell" and "%aberrant cells" in a "spontaneous chromosome fragility index" (SCFI), which results from multiplying "% of aberrant cells" and "breaks/cell".

Finally, to further understand the mechanism that produces spontaneous chromosome fragility in FA patients, the correlation between spontaneous and DEB-induced chromosome fragility was evaluated. As shown in Figure 4, a highly significant correlation between spontaneous and DEB-induced chromosome fragility was detected among FA patients (excluding mosaics) when using "% of aberrant cells" (Figure 4a) or "breaks/cell" (Figure 4b).

Cell viability to MMC

To further support the diagnosis by chromosome fragility assay, cell viability upon MMC treatment was also evaluated in non-FA and FA individuals during the genetic subtyping by retroviral-mediated complementation. [START_REF] Casado | A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network[END_REF] As shown in Figure 5a, the non-FA population were resistant to MMC while FA non-mosaics were highly sensitive, as expected. However, a wide range of MMC sensitivity is observed among FA mosaic patients. Cell viability to MMC was seen to correlate with the proportion of aberrant cells detected with the DEB-induced chromosome fragility assay among FA mosaic patients (Figure 5b). While FA-mosaic patients with 20 to 40% of aberrant cells can be either sensitive or resistant to MMC, patients with ≤20% aberrant cells invariably show resistance to MMC (Figure 5b). Finally, a very good correlation between cell sensitivity to MMC and DEB-induced chromosome fragility was also observed in the FA nonmosaic patient group (Figure 5c), indicating that the excess cell mortality in FA is mainly attributable to the death of cells bearing chromosome breaks.

Correlation between cell sensitivity and severity of patient's clinical phenotype

We finally tried to identify whether ICL sensitivity could be used as a clinical marker to predict the severity of the disease and, therefore, as a prognostic variable at the time of diagnosis. To evaluate the clinical severity, two different clinical markers were used: the number of congenital malformations and the age of onset of hematological disease.

Mean age of onset of hematological disease for this population is 6.75 years, similar to that of other previously published cohorts. Correlations with spontaneous chromosome fragility and cell sensitivities to DEB and MMC are presented in Table 2. We could not detect any correlation between spontaneous chromosome fragility and clinical markers. However, a good correlation was found between number of congenital malformations and DEB-induced chromosome fragility or cell sensitivity to MMC. As shown in Figure 6a and 6b, cells from FA patients with a higher number of malformations statistically have more DEB-induced chromosome breaks (P<.001) and more sensitivity to MMC.

To rule out a possible effect of subtype as a confounding factor in this analysis, the same correlation was assessed within the group of FA-A patients with available clinical data (n=46), and the same highly significant correlation was detected between the number of malformations and DEB-induced fragility (P<.001). On the contrary, cell sensitivity to ICLs did not correlate with the age of onset of hematological disease and, therefore, this variable has no prognostic value in FA.

Discussion

The chromosome fragility assay upon treatment with ICL-inducing agents is the most widely used test for the diagnosis of FA, although it is very laborious and requires specialized personnel. Among the different ICL inducers, DEB is commonly chosen, due to high compound stability and high specificity, as no other group of individuals with sensitivity to DEB comparable to FA patients has been described. To further improve our understanding of chromosome fragility in FA and find rational criteria to correctly and unambiguously diagnose FA patients, including mosaics with a high percentage of reverted cells in their blood, we have performed a comprehensive study to quantify the variability in spontaneous and DEB-induced chromosome fragility among FA patients and the non-FA population. The results on the chromosome fragility test presented here have been obtained in an ethnically homogenous population of Spanish Caucasian individuals, and the cytogenetic analysis has been performed systematically in the same laboratory and under controlled conditions over a period of 11 years (1999-2009).

Typically reported indexes to measure chromosome fragility ("breaks/cell" or "% aberrant cells") allow clear discrimination between non-FA population and non-mosaic FA patients. [START_REF] Auerbach | Diagnosis of Fanconi Anemia by Diepoxybutane Analysis[END_REF] However, mosaic patients present intermediate values and can be easily misdiagnosed. Similarly, non-FA individuals with higher sensitivity to ICL due to genetic background or other unknown factors can have a spontaneous or DEB-induced frequency of cells with breaks of up to 16% or 22 %, respectively, after DEB. These levels of chromosome damage, together with the presence of multiradial figures in a few cases (we detected at least 3 non-FA patients with one multiradial figure) can lead to a false positive diagnosis of mosaicism in a non-specialized laboratory. However, cells with breaks in FA mosaics are usually multiaberrant. This fact led us to develop a novel index (CFI) that integrates "% of aberrant cells" and "breaks/multiaberrant cell" to unambiguously discriminate mosaic patients from non-FA population, as it takes into account the parameters that are more significant for a correct diagnosis of FA. In our hands a CFI=40 can be used as a cut-off to separate FA patients (including mosaics) from non-FA population. While it is possible that this cut-off level can vary between laboratories, we believe that the implementation of the CFI in diagnostic laboratories is highly recommended as it adds to the integrated analysis of the cell distribution of chromosome fragility, for a better and more reliable diagnosis of FA.

FA mosaic patients are usually discriminated from FA non-mosaics depending on % of aberrant cells. However, the mosaicism phenomenon has a progressive nature and, therefore, the differentiation between FA mosaic and FA non-mosaic (full FA) is not always possible. Using restrictive criteria, when only FA patients with 40% or less of aberrant cells are considered as mosaics, 18% of Spanish FA patients are mosaic. With a less conservative upper limit of 50% of aberrant cells, the incidence of mosaicism would increase to more than 20%. Similar criteria for the detection of mosaicism have been proposed for other specialized laboratories [START_REF] Auerbach | Fanconi anemia and its diagnosis[END_REF] and our frequency of mosaicism is also within the range previously reported in other populations (15-25%). [START_REF] Soulier | Detection of somatic mosaicism and classification of Fanconi anemia patients by analysis of the FA/BRCA pathway[END_REF][START_REF] Lo Ten Foe | Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance[END_REF] Cell viability after MMC treatment can be useful in the discrimination of mosaic patients, as the majority of them are resistant to MMC, given the high proportion of wild-type T-cells in their blood. However, there are mosaic patients with high sensitivity to MMC while having a high percentage of cells without breaks in their peripheral blood. The reason for this apparent contradiction is not known. It is also important to mention that the mosaicism observed in the T-cell lineage does not always imply normal patient blood counts, and therefore, mosaicism may not necessarily have clinical implications. Likewise we have detected a patient with improved blood counts over time since first being diagnosed (platelets, hemoglobin and neutrophils) but with a stable percentage of aberrant T-cells over a period of at least 7 years: DEB tests performed in 2002, 2008 and 2009 with % of aberrant cells of 64%, 59% and 69%, respectively. This observation suggests a lack of mosaicism in the lymphoid lineage of the hematopoiesis, but somatic reversion in the myeloid lineage.

FA has long been considered a spontaneous chromosome fragility syndrome since the pioneering work of Professor Traute Schroeder in 1964. [START_REF] Schroeder | Cytogenetic and cytologic findings in enzymopenic panmyelopathies and pancytopenias. Familial myelopathy of Fanconi, glutathione-reductase deficiency anemia and megaloblastic B12 deficiency anemia[END_REF] Results obtained in this study show that, while on average the FA population has an increased level of spontaneous DNA damage, 54% of FA patients (excluding mosaics) have a spontaneous chromosome fragility level within the normal range of non-FA patients. Therefore, spontaneous chromosome fragility, although helpful when positive, cannot be used as a diagnostic tool for FA.

It is well known that FA patients are highly sensitive to ICLs, although it has been proposed that other types of DNA damage, like oxidative stress, can be responsible for spontaneous chromosome fragility. [START_REF] Joenje | Oxygendependence of chromosomal aberrations in Fanconi's anaemia[END_REF][START_REF] Pagano | Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotype[END_REF][START_REF] Nordenson | Effect of superoxide dismutase and catalase on spontaneously occurring chromosome breaks in patients with Fanconi's anemia[END_REF] In this study we show that spontaneous and DEB-induced fragility, as well as cellular sensitivity to MMC are all highly variable among FA patients, but a good correlation between sensitivity to ICL-inducing agents and spontaneous chromosome fragility has been observed. This result indicates that both types of DNA damage are modulated by the same factors, and therefore, that spontaneous chromosome fragility is also a consequence of cellular inability to repair stalled replication forks probably induced by replication errors or endogenously produced ICL agents. How ICLs are generated endogenously is not yet clear, although products of lipid peroxidation seem to be able to cause this type of DNA damage. [START_REF] Pang | Fanconi anemia proteins and endogenous stresses[END_REF] Whether the clinical phenotype is directly caused by the deficiency in the repair of stalled replication forks is also an important question yet to be solved. ICLs toxicity can explain cell proliferation deficiencies and why FA cells are prone to apoptosis. However, oxidative damage, telomeric dysfunctions, inflammatory response or replication stress can also explain part of the clinical phenotype. [START_REF] Bogliolo | The Fanconi anaemia genome stability and tumour suppressor network[END_REF] Interestingly, good correlation between cell sensitivity to DEB or MMC and the number of congenital malformations has been detected in this study. This result supports the hypothesis that cell death during embryonic development as a consequence of the cell inability to repair stalled replication forks, may be responsible for congenital malformations of FA patients. DNA repair genes seem to play an important role during embryonic development, as congenital malformations are frequently seen in chromosome instability disorders including Bloom, Nijmegen breakage syndrome or Seckel syndrome.

[32] The fact that this correlation was also significant when the analysis was restricted to FA-A patients was indeed expected as the vast majority of patients included in this study belong to this complementation group. However, it cannot be extrapolated to other genetic subtypes as there is increasing evidence that core complex and downstream FA proteins do not always function in a single unit or pathway. [START_REF] Kachnic | Fanconi anemia pathway heterogeneity revealed by cisplatin and oxaliplatin treatments[END_REF][START_REF] Pulliam-Leath | Genetic disruption of both Fancc and Fancg in mice recapitulates the hematopoietic manifestations of Fanconi anemia[END_REF][START_REF] Wilson | FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3[END_REF] On the other hand, no relationship between cell sensitivity to ICLs sensitivity and the age of onset of hematological disease has been detected. Hematopoietic cell progenitors are highly sensitive to pro-apoptotic cytokines that are expressed in bone marrow under stress conditions. Therefore, our data supports the notion that development of bone marrow failure in FA patients could be determined by factors other than DNA repair deficiency alone. Consistently, we have FA patients with late onset of the blood disease but high sensitivity to ICLs and vice versa. Thus, this study suggests a model where genome instability is necessary, but not sufficient, to induce early bone marrow failure in FA patients. Uncovering the factors that modulate the age of onset and evolution of the hematological disease is a promising line of research aimed to ameliorate the hematological complications of FA. Reference line on X-axis indicates the upper level observed in non-FA population.

Reference line on Y-axis indicates proportion of FA patients with SCFI below this limit.

Figure 1 .

 1 Figure 1. DEB-induced chromosome fragility in FA and non-FA groups expressed by

Figure 2 .

 2 Figure 2. DEB-induced chromosome fragility in no-FA, FA mosaic and FA non-mosaic

Figure 3 .

 3 Figure 3. Spontaneous chromosome fragility in no-FA, FA mosaic and FA non-mosaic

Figure 4 .

 4 Figure 4. Correlation between spontaneous chromosome fragility and DEB-induced

Figure 5 .

 5 Figure 5. Cell viability to 33nM of MMC in no-FA, FA mosaic and FA non-mosaic

Figure 6 .

 6 Figure 6. Correlations between number of congenital malformations and cell sensitivity

Table 1 .

 1 Spontaneous and DEB-induced chromosome fragility test: Summary of results for FA and non-FA groups. FA patients are subdivided in 3 categories: FA non-mosaic, FA possible mosaic and FA mosaic.

	Group	% aberrant cells n Mean Interval	% multiaberrant cells n Mean Interval	Breaks / cell n Mean Interval	Breaks / multiaberrant cell n Mean Interval
	Spontaneous chromosome fragility
	No FA	105 3.21	0 -16	105 0.51	0 -12	105 0.03	0 -0. 24	9	2	2.00 -2.00
	FA	93 15.24 0 -64	93	4.57	0 -56	93 0.21	0 -1.6	51	2.26	2.00 -4.00
	FA non-mosaic	68 17.56 0 -64	68	5.34	0 -56	68 0.25	0 -1.60	40	2.27	2.00 -4.00
	FA possible mosaic	8 12.50 0 -20	8	3.00	0 -8	8	0.17	0 -0.28	4	2.50	2.00 -3.00
	FA mosaic	17	7.23	0 -24	17	2.23	0 -12	17 0.09	0 -0.28	7	2	2.00 -2.00
	DEB-induced chromosome fragility
	No FA	105 5.83	0 -22	105 0.84	0 -6	105 0.06	0 -0.26	38	2.06	2.00 -2.80
	FA	91 68.10 10 -100	91 54.81 6 -100	91 3.44 0.31 -10.00	91	5.65	2.40 -11.80
	FA non-mosaic	66 81.15 60 -100	66 67.71 36 -100	66 4.34 1.38 -10.00	66	5.94	2.80 -11.80
	FA possible mosaic	8 50.75 46 -58	8	32.00 28 -38	8	1.38 1.06 -1.78	8	3.76	3.16 -4.76
	FA mosaic	17 25.62 10 -38	17 15.44	6 -30	17 0.92 0.31 -1.52	17	5.43	2.40 -9.37

Acknowledgments

We would like to thank all Spanish Fanconi anemia families and their clinicians for their continuous support and for providing samples, Aurora de la Cal and Ana Molina for sampling coordination, and Glòria Umbert for technical support. This work was funded by the Generalitat de Catalunya (SGR0489-2009), the La Caixa Fundation Oncology Program (BM05-67-0), Fundación Genoma España, the Spanish Ministry of Science and Innovation (projects FIS PI06-1099, CB06/07/0023, SAF2006-3440, SAF2009-11936, SAF2009-07164 and PLE 2009-0100), the Commission of the European Union (project RISC-RAD FI6R-CT-2003-508842 and VII FWP PERSIST 222878), and the European Regional Development Funds. CIBERER is an initiative of the Instituto de Salud Carlos III. Authors contribution: MC performed research, analyzed data, and wrote the paper; RP, EC, MJR, JAC, MT, TF performed research; AM, JS, LM, EC, CB, CDdH, TO, JSdT, IB, JE, AD, AR-V, PG, MT, AM, and AF analyzed data and contributed vital analytical tools and materials; JAB analyzed data and JS coordinated the study, performed research, designed the research, analyzed data and wrote the paper.

Conflict of interest disclosure: the authors declare that there are no financial conflicts of interest in the publication of this article.

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Jennifer R Hiscock

Philip A Gale
email: philip.gale@soton.ac.uk

Claudia Caltagirone

Michael B Hursthouse

Mark E Light

N H Hn

Fluorescent carbazolylurea and carbazolylthiourea based anion receptors and sensors

Keywords: Anion binding, indole, hydrogen bonding, crystallography Anion binding, indole, hydrogen bonding, crystallography

A series of carbazolylurea and carbazolylthiourea-based receptors have been synthesized and their anion complexation and fluorescence properties studied. The urea compounds show selectivity for oxoanion complexation over chloride and the fluorescence of compound 1 is selectively quenched by benzoate anions in DMSO/0.5% water. However the thiourea compounds show reduced anion affinities compared to the urea analogues.

Introduction

Interest in new hydrogen bond donor receptors for anionic species has led our group and others to begin to explore indole, carbazole, indolocarbazoles and biindoles as components of new anion complexation agents. [START_REF] Sessler | reviews on anion complexation see[END_REF] We have recently reported that indole appended isophthalamides and pyridine-2,6-dicarboxamides function as selective fluoride binding agents, 2 and our interest in urea 3 as a receptor for oxo- were purchased from chemical sources where available. NMR titrations were performed by adding aliquots of the putative anionic guest (as the TBA/TEA) salt (0.15 M) in a solution of the receptor (0.01 M) in DMSO-d 6 to a solution of the receptor (0.01 M). Fluorescence and UV-Vis titrations were performed by adding aliquots of the putative anionic guest (as the TBA salt or TEA salt in the case of bicarbonate) (1 × 10 -3 M) in a solution of the receptor (1 × 10 -5 M) in DMSO/water 0.5%. All fluorescence and UV-Vis spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer and Thermo Nicolet Evolution 300 spectrophotometer, respectively. Luminescence quantum yields were determined using quinine sulfate in a 1 M H 2 SO 4 aqueous solution (Φ = 0.546) as a reference.

Compounds 1 -3 were synthesised by our previously published procedures. 7 1-Aminocarbazole 1-Nitrocarbazole was synthesised according to a literature procedure 8 modified by separation of the 1-and 3-nitrocarbazoles via chloroform flash chromatography (see ESI for details). Characterisation data agreed with previously published characterisation data. 9

1-Isothiocyanato-9H-carbazole was prepared by reaction of 1-aminocarbazole with thiophosgene in a mixture of dichloromethane and a saturated aqueous solution of NaHCO 3 (see ESI for details).

1,3-di(9H-Carbazol-1-yl)thiourea (4)

A solution of the isothiocyanate (0.23 g, 1.04 mM) in chloroform (20 ml) was then added dropwise to a solution of 1-aminocarbazole (0.19 g, 1.04 mM) in chloroform (25 ml). The solution was then heated at reflux overnight with triethylamine (2 mL).

The solution was then taken to dryness and the product isolated by filtration by recrystallisation from an ether/methanol slow evaporation to give a light green solid.

Results and Discussion

The synthesis of compounds 1-3 has been communicated previously. 7 Stability constants for the compounds with a range of anionic guests were determined using 1 H NMR titration techniques with stability constants calculated using the EQNMR computer program (Table 1). 10 The results show that all the compounds bind acetate and bicarbonate strongly. NMR titrations with H 2 PO 4 -show titration profiles usually indicative of strong binding but the titration curves for compounds 1

and 2 show features at 1 and 2 equivalents of anion that cannot be fitted adequately to a binding model. 7 It is possible that an anion deprotonation process is occurring in these cases. 6 Titrations with F -resulted in broadening of the NH resonances in all cases thus it was not possible to determine an accurate stability constant. Shifts of the NH resonances suggest weak complex formation (i.e. a continuous downfield shiftsee ESI for compounds 2 and 3).

Table 1 The stability constants (K a /M -1 ) of compounds 1 , 2 and 3 with a variety of anionic guests (added as tetrabutylammonium salts except bicarbonate which was added as a tetraethylammonium salt) at 298K in DMSO-d 6 /0.5% water as determined by following urea NH resonance adjacent to the carbazole. In all cases 1:1 receptor: a NMR titration curve could not be fitted to a 1:1 or 1:2 receptor:anion binding stoichiometry but indicates strong binding.

Compounds 4-6 displayed reduced affinties for anions as compared to the urea analogues 1-3 (Table 2). This is unusual as thioureas are more acidic than ureas and hence would be expected to form stronger hydrogen bonding interactions.

Interestingly though, the reduction in affinity in thiourea analogues of urea based receptors was also observed with diindolylurea based receptors which was attributed either to size or conformational interconversion effects. 5 The work presented here further highlights that thiourea based receptors do not always possess higher affinities for anions than urea based systems. a Peak broadening prevented a stability constant from being determined in these cases.

Figure 1 shows a comparison of the 1 H NMR titration curves for compounds 1 and 4

with tetrabutylammonium benzoate in DMSO-d 6 /0.5% water solution. Upon addition of excess benzoate salt to compound 1 (the dicarbazolylurea), the urea NH shifts downfield by 2.3 ppm whilst the indole NH shifts downfield by approximately 1 ppm.

Compound 4 is the thiourea analogue of compound 1. In this case whilst the thiourea NH groups shift downfield by 2.8 ppm, the indole NH shifts downfield by only 0.4

ppm. The lower affinity of the thiourea analogues of the original diindolylurea compounds was attributed to a conformation effect that caused the thiourea receptors to adopt a twisted conformation due to the large size of the sulfur atom present in these compounds. 5 The NMR evidence presented here is consistent this theory in that if the compound is adopting a twisted conformation we would expect a less linear relationship between the carbazole NH hydrogen bond donors and the benzoate oxygen atoms resulting in a weaker interaction. and HCO 3 -(I res = 73%). Chloride did not effect the emission of the system. The trend in fluorescence quenching is similar to that found for the stability constants by 1 H-NMR titrations with the exception for benzoate. This may be due to a π-π interaction in the excited state between the aromatic groups of the receptor and guest. values agree with previously published characterisation data. 9

1-Isothiocyanato-9H-carbazole 1-Aminocarbazole (0.43 g, 2.36 mM) was dissolved in a mixture of dichloromethane (20 mL) and a saturated aqueous solution of NaHCO 3 (40 mL).

Thiophosgene (0.11 mL, 2.36 mM) was added dropwise in dichloromethane (20 mL) and the reaction mixture stirred under nitrogen atmosphere at room temperature overnight. The organic layer was washed with water, dried over MgSO 4 , filtered and reduced in vacuo. The pure product was obtained by recrystallisation from hot MeOH. The isothiocyanate was isolated as a white solid in quantitative yield.

Figure S23

Changes in the fluorescence emission and absorption of 5 upon addition of TBAF in dmso.

Figure S24

Changes in the fluorescence emission and absorption of 5 upon addition of TBAOAc in dmso.

Figure

Figure S27

Changes in the fluorescence emission and absorption of 5 upon addition of TBACl in dmso.

Figure S28

Changes in the fluorescence emission and absorption of 6 upon addition of TBAF in dmso.

Figure S29

Changes in the fluorescence emission and absorption of 6 upon addition of TBAOAc in dmso.

  Crystal data for compound 1.tetrabutylammonium benzoate: C 48 H 59 N 5 O 3 , Mr = 754.00, T = 120(2) K, triclinic, space group P1 a =10.980(5), b = 13.774(5), c = 15.173(5) Å, α =68.760(5)°, β =85.232(5)°, γ =81.541(5)°, V =2114.5(14) Å 3 , ρ calc = 1.184 g cm -3 , µ = 0.074 mm -1 , Z = 2, reflections collected: 44837, independent reflections: 9728 (R int = 0.1052), final R indices [I > 2σI]: R1 = 0.0661, wR2 = 0.1368, R indices (all data): R1 = 0.1190. wR2 = 0.1589.7

Figure 1 7 Figure 2

 172 Figure 1 Proton NMR titration curves for compounds 1 and 4 with tetrabutylammonium benzoate in DMSO-d 6 /0.5% water showing shifts of indole and urea NH groups.

Figure 3 .

 3 Figure 3. Fluorescence quenching of receptor 1 in DMSO/0.5% water upon addition of increasing amounts of tetrabutylammonium benzoate (for benzoate concentrations for each fluorescence spectrum see Figure 4).

Figure 4 .

 4 Figure 4. Effect of increasing anion concentration upon the relative fluorescence emission of receptor 1 in DMSO/0.5% water.

Figure S1 1 H

 1 Figure S1 1 H NMR spectrum of compound 4 in DMSO-d 6 .

FigureFigure S3 1 H

 1 Figure S2 13 C NMR spectrum of compound 4 in DMSO-d 6 .

FigureFigure S5 1 H

 1 Figure S4 13 C NMR spectrum of compound 5 in DMSO-d 6 .

FigureFigure S18 Figure S19 Figure S20 Figure S21 Figure S22

 S18S19S20S21S22 Figure S6 13 C NMR spectrum of compound 6 in DMSO-d 6 .

Figure S26

 S26 Figure S25Changes in the fluorescence emission and absorption of 5 upon addition of TBAH 2 PO 4 in dmso.Page 23 of 32

Figure S30

 S30 Figure S30Changes in the fluorescence emission of 6 and absorption upon addition of TBAH 2 PO 4 in dmso.

Figure S31

 S31 Figure S31Changes in the fluorescence emission and absorption of 6 upon addition of TBAOBz in dmso.

Figure S32

 S32 Figure S32Changes in the fluorescence emission and absorption of 6 upon addition of TBACl in dmso.Page 25 of 32

Table 2

 2 The stability constants (K

	Anion	Compound	Compound	Compound
		4	5	6
	acetate	2230	1800	1780
	benzoate	658	675	870
	dihydrogen	687	1340	a
	phosphate
	chloride	15	17	23
	bicarbonate a	a	a

a /M -1 ) of compounds 4 , 5 and 6 with a variety of anionic guests (added as tetrabutylammonium salts except bicarbonate which was added as a tetraethylammonium salt) at 298K in DMSO-d 6 /0.5% water as determined by following urea NH resonance adjacent to the carbazole. In all cases 1:1 receptor: anion stoichiometry was observed. Errors estimated to be no more than ±15%

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Supramolecular Chemistry

Acknowledgements

We thank the EPSRC for studentship funding (JRH) and for access to the crystallographic facilities at the University of Southampton. CC would like to thank thank the Italian Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (MIUR) (Project PRIN-2007C8RW53) and Università degli Studi di Cagliari (fondo 5%) for financial support.


Maria Põllupüü
email: maria.pollupuu@ut.ee

Mart Simm

Henn Ojaveer

Life history and population dynamics of the marine cladoceran Pleopis polyphemoides (Leuckart) (Cladocera, Crustacea) in a shallow temperate Pärnu Bay (Baltic Sea)

Keywords: Baltic Sea, fecundity, Pleopis polyphemoides, reproduction, seasonal dynamics Baltic Sea, fecundity, Pleopis polyphemoides, reproduction, seasonal dynamics

Life history and population dynamics of the marine cladoceran Pleopis polyphemoides (Leuckart) (Cladocera, Crustacea) in a shallow temperate Pärnu

Bay (Baltic Sea)

Introduction

Altogether, there are eight truly marine cladoceran species belonging to the families Podonidae (containing also Pleopis polyphemoides (Leuckart)) and Sididae, characterised by a very broad distribution range from tropical to subarctic waters (Gieskes, 1971a(Gieskes, , 1971b(Gieskes, , 1971c;;Della Croce, 1974;[START_REF] Mordukhai-Boltovskoi | Predatory Cladocerans Podonidae, Polyphemidae, Cercopagidae, and Leptodoridae of the World Fauna[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]. Phylogenetic analysis suggests the initial radiation of marine Podonidae during the Miocene or early Pliocene and start of their oceanic expansion via the Mediterranean at times when the Ponto-Caspian basin was connected with the world's oceans [START_REF] Cristescu | Phylogeny and adaptative radiation in the Onychopoda (Crustacea, Cladocera): evidence from multiple gene sequences[END_REF][START_REF] Durbin | Comparative phylogeography of marine cladocerans[END_REF]. However, recent expansion (incl. transoceanic) of several species of marine and also brackish cladocerans might be a result of human intervention [START_REF] Cristescu | An invasion history for Cercopagis pengoi based on mitochondrial gene sequences[END_REF][START_REF] Durbin | Comparative phylogeography of marine cladocerans[END_REF][START_REF] Põllupüü | Successful establishment of the Ponto-Caspian alien cladoceran Evadne anonyx G.O.Sars 1897 in low-salinity environment in the Baltic Sea[END_REF] as has been suggested for the freshwater predatory cladoceran Bythotrephes cederstroemi [START_REF] Sprules | Dynamics of the Bythotrephes invasion of the St. Lawrence Great Lakes[END_REF].

Cladocerans have high seasonal variability due to the alternation of parthenogenetic and gamogenetic reproduction modes [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]. A rapid increase in densities attained as a result of high rates of embryonic and postembryonic growth is characteristic of marine cladocerans, including P. polyphemoides. The density increase is accelarated by paedogenesis, namely, the development of parthenogenetic eggs beginning in the brood chambers of embryos before they are released from their parent, a phenomenon described commonly in marine Podonidae [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]. The exponential growth of the parthenogenetically multiplying population is probably additionally accelerated by the development of new parthenogenetic eggs in the parent brood chamber before the previous embryos are fully developed and released.

Although cladocerans are often viewed as a 'secondary' constituent of marine zooplankton communities, they are widely distributed in marine ecosystems and may form a significant fraction in zooplankton communities in the Baltic Sea [START_REF] Hessle | Undersökningar över plankton och dess växlinger i Östersjön under åren 1925-1927. Svenska Hydrogr[END_REF][START_REF] Ackefors | Seasonal and vertical distribution of the zooplankton in the Askö area (Northern Baltic proper) in relation to hydrographical conditions[END_REF][START_REF] Sandström | the Gulf of Riga) in the spring-summer period[END_REF][START_REF] Kankaala | Structure, dynamics and production of mesozooplankton community in the Bothnian Bay, related to environmental factors[END_REF][START_REF] Lumberg | On the environment and zooplankton dynamics in the Gulf of Finland in 1961-1990[END_REF][START_REF] Line | Zooplankton of the Gulf of Riga[END_REF][START_REF] Viitasalo | Mesozooplankton dynamics in the northern Baltic Sea: implications of variations in hydrography and climate[END_REF][START_REF] Möllmann | Climate, zooplankton, and pelagic fish growth in the central Baltic Sea[END_REF][START_REF] Ikauniece | Changes in the cladoceran community as an indicator of climatic variability in the Gulf of Riga, NE Baltic Sea[END_REF]. Cladocerans possess an ability to increase their abundance by using parthenogenetic reproduction if the environment is favourable. This ability makes them especially adapted to opportunistic utilisation of seasonally changing resources [START_REF] Brandl | Methodology and general ecology[END_REF], enabling them to achieve seasonally very high abundances and play significant roles in the diet of various planktivorous fish and invertebrate predators at high densities [START_REF] Hansson | Selective predation by herring and mysids, and zooplankton community structure in a Baltic Sea coastal area[END_REF][START_REF] Rudstam | Dynamics of planktivory in a coastal area of the northern Baltic Sea[END_REF][START_REF] Flinkman | Planktivorous Baltic herring (Clupea harengus) prey selectively on reproducing copepods and cladocerans[END_REF][START_REF] Flinkman | Changes in northern Baltic zooplankton and herring nutrition from 1980s to 1990s: top-down and bottom-up processes at work[END_REF][START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]Lankov et al., unpublished data). It has been also suggested that cladoceran populations (incl. Podonidae) are, in addition to key abiotic parameters and diet availability, regulated by top-down processes through invertebrates and fish (e.g., Horsted et al., 1988;[START_REF] Põllumäe | Factors describing the distribution of the zooplankton community in the Gulf of Finland in the context of interactions between native and introduced predatory cladocerans[END_REF].

The cladoceran community of the Baltic Sea is dominated by five taxa: Bosmina spp., Podon intermedius, Podon leuckarti, Pleopis polyphemoides, Evadne nordmanni and the alien Cercopagis pengoi. Pleopis polyphemoides is a eurythermic species and prefers brackish and fresher parts of river esturies. Unfortunately, in the quantitative analysis of most Baltic zooplankton studies which has included cladocerans, P. polyphemoides has not been distinguished from the morphologically relatively similar other podonids Podon leuckarti and Podon intermedius (e.g., [START_REF] Dippner | Long-term variability of mesozooplankton in the Central Baltic Sea[END_REF][START_REF] Uitto | Diurnal and vertical grazing activity of mesozooplankton during summer on the SW coast of Finland[END_REF][START_REF] Möllmann | Long-term trends in abundance of cladocerans in the Central Baltic Sea[END_REF][START_REF] Aleksandrov | Long-term dynamics of zooplankton in the Southeastern Baltic Sea[END_REF] and there is no information about the reproduction of P. polyphemoides in this region. However, there are a number of relatively detailed investigations with high spatio-temporal resolution of annual density development, population structure, fecundity and production of P. polyphemoides elsewhere in the world, such as Chesapeake Bay (Bosch andTaylor, 1973a, 1973b), the North Atlantic and the North Sea (Gieskes, 1971a(Gieskes, , 1971b(Gieskes, , 1971c)), the Inland Sea of Japan [START_REF] Onbé | Preliminary notes on the biology of the resting eggs of marine cladocerans[END_REF][START_REF] Onbé | Seasonal fluctuations in the abundance of populations of marine cladocerans and their resting eggs in the Inland Sea of Japan[END_REF]Onbe et al., 1977), Narragansett Bay [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF], Guanabara Bay in south-eastern Brazil (Marazzo and Valentin, 2003c;[START_REF] Valentin | The diel vertical migration pattern of the cladoceran Pleopis polyphemoides (Leuckart, 1859) in a stratified tropical bay[END_REF] and a semi-enclosed bay in Hong Kong [START_REF] Wong | Diel cycle in the percentage abundance of parthenogenetic females with embryos of different developmental stages in four species of marine cladocerans[END_REF].

The the lack of knowledge for the Baltic and the importance of cladocerans in the Baltic zooplankton communities as well as energy transfer to higher trophic levels led us to carry out detailed seasonal investigations on P. polyphemoides. The objective of the current study was to investigate the species in a shallow eutrophic Pärnu Bay during the ice-free season with respect to density dynamics, reproduction modes, individual fecundity and body length in relation to selected key environmental background parameters such as water temperature, salinity and chlorophyll a concentration.

Method

Study area

Pärnu Bay, located in the northeastern part of the Gulf of Riga (Fig. 1), is a relatively enclosed and very shallow (maximum depth around 10 m) sea area covering approximately 700 km 2 with a volume of 2 km 3 . In most years Pärnu Bay is covered by ice from December/January to March/April. In summer, the average surface water temperature has reached 22-23ºC during July-August. The salinity of the bay fluctuates from almost freshwater to 7.5 psu. The hydrographic 5 conditions are formed under the complex influence of ice conditions, freshwater inputs from the Pärnu River and the water exchange with the Gulf of Riga. The bay suffers from extensive human pressures (recreation, eutrophication, fishing) (e.g., [START_REF] Kotta | Gulf of Riga and Pärnu Bay[END_REF]. Based on the coastal morphology, associated hydrographic features and composition and seasonal dynamics of biota, Pärnu Bay falls into two parts, the inner and the outer basin.

Sampling

Zooplankton samples were collected during daytime at two monitoring stations located in the inner and outer basins of Pärnu Bay during the ice-free period (April-November) in 2006. The sampling frequency was three times a month throughout the study period; the results obtained are considered as being representative of the 10-day periods per month. The inner bay sampling station (called herafter the coastal station) is located in the vicinity of the Pärnu River estuary at 6-7 m depth and is directly and strongly under riverine freshwater inputs. The outer bay station (hereafter the open sea station) is located at a depth of 10 m. The conditions there are influenced by hydrological processes in the Gulf of Riga.

Sampling was performed using a Juday net (mouth opening 0.1 m 2 ; mesh size 100 µm) integrating the whole water-column vertically. The samples were preserved immediately after sampling with formalin; final concentration ca 4%. Measurements of temperature, salinity and chlorophyll a (Chl a) were made by means of a CTD (model SAIV SD204). Zooplankton sampling and analysis followed the guidelines developed for the HELCOM COMBINE programme [START_REF] Helcom | Manual for Marine Monitoring in the COMBINE Programme of HELCOM[END_REF].

Sample analysis

Body length of P. polyphemoides was measured from the top of the head to the end of the caudal outgrowths. Total length was determined from the top of the head to the end of the brood pouch (detailed information on conventional measurements of Pleopis is provided in [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF].

All P. polyphemoides individuals were counted in the following categories: parthenogenetic females, gamogenetic females and males. Additionally, fecundity of parthenogenetic (number of embryos) and gamogenetic females (number of resting eggs) was determined. Embryonic development in P. polyphemoides was divided into four stages (I to IV), based on easily distinguishable external characteristics found in Podonidae [START_REF] Wong | Diel cycle in the percentage abundance of parthenogenetic females with embryos of different developmental stages in four species of marine cladocerans[END_REF][START_REF] Atienza | Life history and population dynamics of the marine cladoceran Penilia avirostris (Branchiopoda: Cladocera) in the Catalan Sea (NW Mediterranean)[END_REF]. The body length of parthenogenetic embryos was measured by different developmental stages separately. Altogether, 8694 P. polyphemoides individuals were analysed. Data analyses were performed by the statistical software "Statistica" version 8.0 and R. The R package relaimpo (relative importance of regressors in the linear models) method LMG was applied for tests between the total abundance (in 10-day periods), body size, fecundity of P. polyphemoides and different continuous abiotic variables. The relationship between the body size and fecundity of parthenogenetic females was estimated by a Spearman rank correlation analysis. The significance of differences in the body size of parthenogenetic females within and between the coastal and the open sea station was estimated by the post hoc Bonferroni test in repeated measures analysis of variance (ANOVA) where 10-day period was used as a time factor. The significance of differences in the body size of gamogenetic females and males between the coastal and the open sea station was estimated by one-way ANOVA. The difference between the body size of gamogenetic females and parthenogenetic females within and between stations was estimated by one-way ANOVA. The differences were considered significant at p < 0.05.

Results

Background conditions

In 2006, the retreat of the ice cover from Pärnu Bay was recorded at the end of April. At that time the water temperature exceeded 5°C at both the coastal and the open sea station. The seasonal temperature development was very similar at both stations with a sharp increase during the beginning of June and a peak in mid-July (25.5°C in the coastal and 23.1°C in the open sea station).

A progressive decrease in water temperature was recorded for the rest of the season (Fig. 2A).

There was a notable difference in salinity between the coastal and the open sea station, especially in spring with very low values at the coastal station. Salinity varied from 1.3 to 5.9 psu (mean 4.4 psu) at the coastal and from 3.6 to 5.9 psu (mean 5.0 psu) at the open sea station. In general, salinity values increased progressively during summer in both areas of the bay (Fig. 2B). The highest Chl a concentrations were recorded during the spring bloom: at the coastal station at the end of May (40.4 mg m -3 ) and at the open sea station at the beginning of May (26.9 mg m -3 ). After the spring bloom, Chl a concentrations dropped sharply, the values were mostly below 4 mg m -3 during June-July. A moderate continuous increase in Chl a concentration (from 0.8 to 8.4 mg m -3 ) was observed at both stations from the end of July until the end of the season (Fig. 2C). The first individuals of P. polyphemoides appeared in Pärnu Bay at the beginning of May. At the coastal station, the densities reached the level of 1000 ind. m -3 already by the end of May while at the open sea station, the density was substantially lower, around 600 ind. m -3 . A rapid increase in the abundance of P. polyphemoides was observed from the beginning of June onward. The maximum abundance at the coastal station was registered in mid-June (6729 ind. m -3 ) while at the open sea station the maximum (7350 ind. m -3 ) was recorded 10 days later. The seasonal abundance peak was followed by a rapid decrease until mid-July (Fig. 3). Compared to the open sea station, the duration of the occurrence of P. polyphemoides was substantially more restricted at the coastal station: at the former, the species disappeared from the plankton community about two weeks later than at the latter. However, a few individuals were found at both stations as late as in December.

Seasonal abundance dynamics

A strong positive relationship between the abundance of P. polyphemoides and water temperature was evident for parthenogenetic individuals (R 2 = 0.82; relaimpo) as well as for gamogenetic females and males (R 2 = 0.9; relaimpo). The relationship was weak and negative between abundance and Chl a concentration. Salinity and Chl a seemed to have no impact on P. polyphemoides abundance.

Reproduction mode

Parthenogenetic females of P. polyphemoides dominated in the population at both stations (Fig. 4).

In May, only parthenogenetic females were found. Gamogenetic females and males appeared at the coastal station about 10 days earlier than at the open sea station (accounting for 2% and 11% of the total population abundance at the coastal and the open sea station, respectively). Gamogenetic reproduction was the most intense during or immediately after the P. polyphemoides population abundance peak (from the middle to the end of June) when the proportion of parthenogenetic females was already in a sharply decreasing phase (constituting 36% of the gamogenetic females and males at the coastal and 17% at the open sea station). In both areas, the abundance of gamogenetic individuals of P. polyphemoides rapidly decreased after their abundance peak. Males of P. polyphemoides disappeared from the plankton community in early July at the coastal station and about a month later at the open sea station. However, gamogenetic females were sporadically found also in autumn (in October) as was the case for parthenogenetic females.

Body size

The body length of parthenogenetic females of P. polyphemoides significantly differed between the coastal and open sea stations (p < 0.01; post hoc Bonferroni test, repeated measures ANOVA) with The temporal dynamics of the total length of parthenogenetic females was similar at both stations with the largest values recorded prior to the population abundance peak (Table I). In general, parthenogenetic females were found to be the smallest in terms of the total length during or after the population peak. However, no clear patterns could be established for the body length of P. polyphemoides in terms of the population peak (Table I). A few large parthenogenetic females were found as late as October with total and body lengths of 0.42 ± 0.03 and 0.39 ± 0.02 mm, respectively. The highest total length/body length ratio was observed when the species first appeared in the plankton community. At that time, only parthenogenetic females were present.

Subsequently, when intensive parthenogenetic reproduction began and in addition to large parthenogenetic females young individuals were also present, a lower total length/body length ratio was observed.

The total length of parthenogenetic females was inversely correlated with water temperature (R 2 = 0.72; relaimpo) (Fig. 5). However, the body length was positively related to Chl a (R 2 = 0.82; relaimpo). The total length and the body length of gamogenetic females, on the contrary, were both affected by water temperature, R 2 = 0.58 and R 2 = 0.53, respectively (Table II). No relationship was found between the body size of males and abiotic environmental parameters, and we were not able to find any significant influence of salinity on the body length and total length of P. polyphemoides either.

In gamogenetic females of P. polyphemoides no significant difference was found in the total length or body length between the coastal and the open sea station. The total length and body length were greatest in June, when gamogenetic females first appeared to the plankton community. Although they generally disappeared in August, a few individuals were still observed in October with a total length of 0.53 ± 0.01 mm and body length of 0.43 ± 0.00 mm. The total length and body length of gamogenetic females significantly differed from that of parthenogenetic females at the coastal and open station (p < 0.01; one-way ANOVA). Similarly to gamogenetic females, males with the maximum total length and body length occurred at the beginning of June, when they first appeared in the zooplankton community. Later in the season, males were significantly smaller (Table I). The body size of males did not differ between the coastal and the open sea station. On some occasions, the body length of males exceeded their total length. The fecundity of parthenogenetic females of P. polyphemoides was 5.4 ± 0.3 at the coastal station and 2.9 ± 0.1 embryos female -1 at the open sea station. In both areas, the highest fecundity was associated with the first appearance of the species in the zooplankton community, which occurred at the beginning of May at the coastal station and about a month later at the open sea station (Table III). The maximum number of embryos in a brood pouch (10 embryos female -1 ) was observed also during the initial phase of population growth. The number of embryos per female correlated significantly with the total length at both the coastal (r = 0.77; p < 0.01) and the open sea station (r = 0.93; p < 0.01) (Fig. 6). However, there was no correlation between fecundity and body length.

Fecundity

Parthenogenetic fecundity of P. polyphemoides was found to be higher at lower water temperatures (R 2 = 0.69; relaimpo). No effect of Chl a concentration or salinity on fecundity was found.

During daytime most P. polyphemoides parthenogenetic embryos were at stage II (42%) or III (34%). Only 8% of the embryos were at early developmental stage I and 16% were determined as fully developed (stage IV). At both stations, the size of embryos increased with advancing developmental stage (Table IV). The maximum embryo size before its release from the brood pouch was 0.32 mm. Gamogenetic females of P. polyphemoides carried only one resting egg in the brood pouch. The diameter of the resting egg was 0.12 ± 0.01 mm on average both in the coastal and the open sea station. The gamogenetic female with the largest resting egg (0.2 mm) was recorded at the open sea station in July.

Discussion

In the Baltic Sea, the abundance dynamics of cladocerans (incl. P. polyphemoides) is characterised as extremely seasonal: they appear in spring, reach maximum densities in summer and disappear in autumn [START_REF] Ackefors | Seasonal and vertical distribution of the zooplankton in the Askö area (Northern Baltic proper) in relation to hydrographical conditions[END_REF][START_REF] Vuorinen | Dynamics of marine meso-zooplankton at Seili, Northern Baltic Sea, in 1967-1975[END_REF][START_REF] Viitasalo | Mesozooplankton dynamics in the northern Baltic Sea: implications of variations in hydrography and climate[END_REF][START_REF] Möllmann | Long-term trends in abundance of cladocerans in the Central Baltic Sea[END_REF]. This was also confirmed by the current study. A similar seasonal distribution pattern of P. polyphemoides has been observed in several other areas of the world (Gieskes, 1971a(Gieskes, , 1971b;;[START_REF] Platt | Population ecology of marine cladocera in St. Margaret's Bay, Nova Scotia[END_REF][START_REF] Uye | Length-weight relationships of important zooplankton from the Inland Sea of Japan[END_REF][START_REF] Turner | Zooplankton feeding ecology: grazing during enclosure studies of phytoplankton blooms from the west coast of Sweden[END_REF]Marazzo and Valentin, 2003c). However, there is evidence from the Chesapeake Bay (Bosch and Taylor, 1973b), Narragansett Bay [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF] and the Marmara Sea [START_REF] Isinibilir | Annual cycle of zooplankton abundance and species composition in Izmit Bay (the northeastern Marmara Sea)[END_REF] that this species may have a two-peak seasonal distribution pattern. Further, P. polyphemoides is usually a spring to early summer species in the Inland Sea of Japan [START_REF] Onbé | Seasonal fluctuations in the abundance of populations of marine cladocerans and their resting eggs in the Inland Sea of Japan[END_REF] and a winter to spring species in St. Andrew's Bay, Florida [START_REF] Hopkins | feeding bivalves and fish in estuarine enclosures[END_REF]. Thus, there is evidence to suggest that regional variability occurs in respect to seasonal development and duration of occurrence of P. polyphemoides in different seas of the world.

The densities of P. polyphemoides have been found to substantially differ in different marine areas.

Very high densities (over 30 000 ind. m -3 ) of P. polyphemoides have been recorded in Chesapeake Bay (Bosch and Taylor, 1973a), in the Inland Sea of Japan [START_REF] Onbé | Seasonal fluctuations in the abundance of populations of marine cladocerans and their resting eggs in the Inland Sea of Japan[END_REF] and in Narragansett Bay [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]. In the Dardanelles, P. polyphemoides is one of the most abundant cladoceran species (maximum over 10 000 ind. m -3 ) after Penilia avirostris (Buyukates and Đnanmaz, 2007).

Considerably lower densities (ca. 150 ind. m -3 ) have been observed in the Marmara Sea [START_REF] Isinibilir | Annual cycle of zooplankton abundance and species composition in Izmit Bay (the northeastern Marmara Sea)[END_REF] and ca. 1 500-3 500 in Guanabara Bay, south-eastern Brazil, (Marazzo and Valentin, 2003c) and the west coast of Sweden [START_REF] Eriksson | The occurrence of marine Cladocera on the West Coast of Sweden[END_REF], while the species was almost absent in the North Sea (Gieskes, 1971a). The densities of P. polyphemoides recorded within the current study were similar to those reported from the other areas of the Baltic Sea (below 10 000 ind. m -3 ), e.g., the northern Baltic Sea [START_REF] Vuorinen | Dynamics of marine meso-zooplankton at Seili, Northern Baltic Sea, in 1967-1975[END_REF], Gulf of Finland [START_REF] Põllumäe | Factors describing the distribution of the zooplankton community in the Gulf of Finland in the context of interactions between native and introduced predatory cladocerans[END_REF] and Pomeranian Bay [START_REF] Chojnacki | Seasonal changes in the neritic zone mesozooplankton of Pomeranian Bay in 2000[END_REF].

The obvious reason behind the abrupt increase of cladoceran populations in spring/early summer is rapid and intense parthenogenetic reproduction, associated with rising water temperature [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]Marazzo and Valentin, 2003a, 2003b, 2003c). The population of P. polyphemoides in Pärnu Bay in spring and early summer, i.e., before the abundance peak when the water temperature increased rapidly, was characterised by the presence of relatively large parthenogenetic individuals, which were found to be generally more fecund than smaller individuals. This was shown not only by a significant positive correlation between parthenogenetic fecundity and total female length, but also by a negative correlation between parthenogenetic fecundity and water temperature. A positive relationship between total length and fecundity was expected, as total length is mainly determined by the reproductive phase and number of embryos in a brood pouch (Marazzo and Valentin, 2003c). However, body length, was not related to fecundity characteristics. Remarkably, some very large (and highly fecund: 10 embryos female -1 ) parthenogenetic individuals, probably hatched from resting eggs, were found during the very initial stages of population development. Overall, the parthenogenetic females exhibited significant seasonal variability in their total length, achieving the largest values just prior to the population abundance peak and having usually smaller values afterwards with lowered individual fecundity. In general, the fecundity of marine cladocerans typically appears to be higher during the initial phases of population growth, decreasing rapidly as the population increases [START_REF] Platt | Prenatal mortality in a marine cladoceran, Evadne nordmanni[END_REF]; 11 between parthenogenetic fecundity and P. polyphemoides density. The observed parthenogenetic fecundity of P. polyphemoides in Pärnu Bay seems to be comparable to that of this species in other areas [START_REF] Murakami | Notes on the marine cladocerans observed at a definite station near Tomo Marine Biological Station[END_REF][START_REF] Onbé | Studies on the ecology of marine cladocera[END_REF]Marazzo and Valentin, 2003c).

The eyes of the embryos of marine cladocerans become pigmented at the final stage when the embryos are fully developed [START_REF] Platt | Prenatal mortality in a marine cladoceran, Evadne nordmanni[END_REF]. Because planktivorous fish select for the pigmented structures [START_REF] Zaret | Fish predation on Bosmina longirostris: body-size selection versus visibility selection[END_REF][START_REF] Flinkman | Planktivorous Baltic herring (Clupea harengus) prey selectively on reproducing copepods and cladocerans[END_REF], it has been suggested that the reason for the release of neonates by cladocerans at night is the decrease in fish predation [START_REF] Wong | Diel cycle in the percentage abundance of parthenogenetic females with embryos of different developmental stages in four species of marine cladocerans[END_REF]. Our observations showed that during the daytime, most of the embryos in the female brood pouch were in stages II and III while the share of fully developed embryos (i.e., stage IV) was very low. However, whether this was due to nocturnal release of neonates and predation remains unclear and should be investigated in the future. Marine cladocerans attain high population abundances very rapidly, but these are generally sustained only for a very short time. Reasons for a sudden decrease of cladoceran populations are relatively poorly understood. However, this decrease is typically accompanied by an intensive gamogenetic reproduction [START_REF] Onbé | The life cycle of marine cladocerans[END_REF] resulting in the production of diapausing (resting) eggs at the end of the reproductive season. This has been suggested to be triggered by changes in environmental conditions and aimed at ensuring survival of the species over the winter [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]. In contrast to this, in the current study no strong and significant relationships were found between the appearance of gamogenetic females carrying resting eggs and the abiotic variables measured. This is probably because no clear shift between the two reproduction modes was detected and the species was found to reproduce both parthenogenetically and gamogenetically almost during the whole reproductive season, with relatively intense gamogenetic reproduction lasting for about a month. It has been suggested earlier that the onset of sexual reproduction in podonids generally follows a sharp reduction in parthenogenetic fecundity [START_REF] Bainbridge | Some observations on Evadne nordmanni Loven[END_REF][START_REF] Onbé | Studies on the ecology of marine cladocera[END_REF][START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]. Although no clear shift in reproduction modes was detected in this study, we did observe substantially intensified gamogenetic reproduction at the population abundance maximum or soon after it, which was accompanied by decreasing fecundity of parthenogenetic females. After intense production of resting eggs begins, the population of P. polyphemoides may almost disappear but parthenogenetic reproduction may still continue for several months [START_REF] Della Croce | Penilia avirostris Dana in the Indian Ocean (Cladocera)[END_REF][START_REF] Rivier | The Predatory Cladocera (Onychopoda: Podonidae, Polyphemidae, Cercopagidae) and Leptodorida of the World[END_REF]. This pattern was also observed in our study.

The sexual reproduction process may be terminated by water temperature. It has been found that gamogenetic females are not present in cladoceran communities above certain water temperatures. For instance, in the Chesapeake Bay, gamogenetic females disappeared at water temperatures exceeding 23°C (Bosch and Taylor, 1973b) and in Narragansett Bay at 22.5°C [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF].

Temperature control may serve as a possible explanation for the disappearance of gamogenetic females of P. polyphemoides from Pärnu Bay at water temperatures exceeding 22°C.

It was found that gamogenetic females of P. polyphemoides carried one resting egg in Pärnu Bay. In other studies gamogenetic females of P. polyphemoides with more than one resting egg have not been seen either [START_REF] Onbé | Studies on the ecology of marine cladocera[END_REF][START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]. The maximum recorded percentages of gamogenetic individuals in a population range from less than 10% for P. polyphemoides in the Inland Sea of Japan [START_REF] Onbé | The life cycle of marine cladocerans[END_REF] and in the Chesapeake Bay (Bosch and Taylor, 1973b) to 46% in Narragansett Bay [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]. In a tropical Guanabara Bay, characterised by substantially lower densities of P. polyphemoides, the share of gamogenetic indviduals was remarkably more stable (20-50%) (Marazzo and Valentin, 2003c), indicating that a smaller population continuously sustains higher production of resting eggs throughout the reproductive season. In contrast, the proportion of gamogentic females of P. polyphemoides in Pärnu Bay varied over a very wide range.

It has been suggested that low incidences of gamogenetic reproduction may result from the failure of parthenogenetic females to be trasformed to gamogenetic females, as was the case for P. polyphemoides in Narragansett Bay [START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF].

Several investigators have argued that the environment plays a key role in declines of cladoceran populations. Amongst others, temperature, salinity, food availability, water transparency and predation have been indicated as regulating factors [START_REF] Calbet | Annual zooplankton succession in coastal NW Mediterranean waters: the importance of the smaleer size fractions[END_REF][START_REF] Marques | Zooplankton and ichthyoplankton communities in a temperate estuary: spatial and temporal patterns[END_REF][START_REF] Atienza | Life history and population dynamics of the marine cladoceran Penilia avirostris (Branchiopoda: Cladocera) in the Catalan Sea (NW Mediterranean)[END_REF]. Further, it is possible that instead of any single factors, interaction of several ones (such as stratification of the water column, higher water temperatures and associated prey availability) are important [START_REF] Atienza | Life history and population dynamics of the marine cladoceran Penilia avirostris (Branchiopoda: Cladocera) in the Catalan Sea (NW Mediterranean)[END_REF]. In the Baltic Sea, temperature, salinity and predation by planktivorous fish as well as predatory invertebrates have been indicated to influence the abundance dynamics of small-sized cladocerans at various spatio-temporal scales [START_REF] Ikauniece | Long-term abundance dynamics of coastal zooplankton in the Gulf of Riga[END_REF][START_REF] Möllmann | Long-term trends in abundance of cladocerans in the Central Baltic Sea[END_REF][START_REF] Kotta | Factors controlling long-term changes of the eutrophicated ecosystem of Pärnu Bay, Gulf of Riga[END_REF][START_REF] Ojaveer | Population dynamics and ecological impact of the non-indigenous Cercopagis pengoi in the Gulf of Riga (Baltic Sea)[END_REF][START_REF] Põllumäe | Factors describing the distribution of the zooplankton community in the Gulf of Finland in the context of interactions between native and introduced predatory cladocerans[END_REF][START_REF] Casini | Trophic cascades promote threshold-like shifts in pelagic marine ecosystems[END_REF][START_REF] Kotta | Separate and interactive effects of eutrophication and climate variables on the ecosystem elements of the Gulf of Riga[END_REF]. In the current study, it was found to be unlikely that temperature triggered the observed sharp density drop because the P. polyphemoides population started to decline at the time when water temperature was at its maximum. Neither was there any relationship between salinity and population abundance. Most investigators assume that podonids primarily feed on dinoflagellates because of the observed high abundance of podonids during dinoflagellate blooms [START_REF] Bainbridge | Some observations on Evadne nordmanni Loven[END_REF][START_REF] Egloff | Reproductive biology of marine cladocerans[END_REF]. This, however, could not be the case in Pärnu Bay, because in this bay dinoflagellates form an insignificant fraction of the phytoplankton 13 community during summer (A. Jaanus, Tallinn, personal communication) when P. polyphemoides is present. Besides, in the current study, population abundance correlated negatively with Chl a concentration. We suggest that it was rather predator-prey interactions that were responsible for the observed decline as the predation pressure on zooplankton should be high in the study area, which is biologically a very productive region and acts as an important spawning and nursery ground for several fish species (Simm and Ojaveer, 2000;[START_REF] Lankov | Perch and herring -different feeding strategies in early life history?[END_REF]. As there is evidence on the predation-induced mortality of small-sized cladocerans in the Baltic Sea [START_REF] Kotta | Factors controlling long-term changes of the eutrophicated ecosystem of Pärnu Bay, Gulf of Riga[END_REF][START_REF] Ojaveer | Population dynamics and ecological impact of the non-indigenous Cercopagis pengoi in the Gulf of Riga (Baltic Sea)[END_REF][START_REF] Põllumäe | Factors describing the distribution of the zooplankton community in the Gulf of Finland in the context of interactions between native and introduced predatory cladocerans[END_REF] as well as for other regions [START_REF] Gliwicz | Species-specific population-density thresholds in cladocerans?[END_REF]Marazzo and Valentin, 2003c), we hypothesize that predation should be an important factor behind the sharp drop of the abundance of P. polyphemoides during summer. This hypothesis is backed by the fact that the size of parthenogenetic females was significantly lower at and/or soon after the seasonal population peak, potentially indicating predation mortality of larger individuals.

Essentially because of a very short generation time, cladoceran abundance is highly variable at the fine-scale level, stressing thus the importance of spatio-temporal scale considerations when designing studies on cladocerans. The current investigation provides additional information on the spatio-temporal dynamics of vital rates of one of the most abundant cladoceran species in the Baltic Sea, P. polyphemoides, which is, amongst others, one of the most important diet items for several pelagic fish species in the Gulf of Riga (Lankov et al., unpublished data.). Also, it was suggested [START_REF] Põllumäe | Factors describing the distribution of the zooplankton community in the Gulf of Finland in the context of interactions between native and introduced predatory cladocerans[END_REF]

  at the coastal station. Such a difference was not evident for total length.

  that in the Gulf of Finland the non-indigenous C. pengoi may suppress P. polyphemoides abundance and significantly modify its density dynamics. While only a few measured parameters (e.g., abundance, parthenogenetic and gamogenetic fecundity, body size of gamogenetic individuals) were relatively stable across the sampled stations in Pärnu Bay, several key characteristics (such as timing, duration as well as the body size of P. polyphemoides) exhibited differences between the coastal and the open sea station. It should be noted that both stations are in fact relatively coastal, shallow and sheltered from strong winds. Further studies should resolve some of the important issues discussed in the study, like causes of the abundance drop in summer, mechanisms behind the dissimilar performance of P. polyphemoides in the inner and the outer bay as well as annual-scale variability in the seasonal dynamics of P. polyphemoides. Finally, results of the current study provide additional considerations for limitations of interpretation of analysis of long-time datasets based on a relatively coarse time-scale sampling design.

  Fig. 1. Location of the sampling sites, coastal station in the inner bay (empty circle) and open sea station in the outer bay (filled circle) in Pärnu Bay (Baltic Sea).

Fig. 2 .

 2 Fig. 2. Dynamics of sea surface temperature (A), salinity (B) and chlorophyll a concentration (C) in the coastal (solid line) and the open sea station (bold line) in Pärnu Bay in 2006.

Fig. 3 .

 3 Fig. 3. Abundance dynamics of Pleopis polyphemoides in the coastal (solid line) and the open sea station (bold line) in Pärnu Bay in 2006.

Fig. 4 .

 4 Fig. 4. Abundance dynamics of parthenogenetic females (solid line, left scale) and gamogenetic females (black columns, right scale) and males (white columns, right scale) of Pleopis polyphemoides in the coastal (A) and the open sea station (B) in Pärnu Bay in 2006.

Fig. 5 .

 5 Fig. 5. Seasonal dynamics of the total length of parthenogenetic females of Pleopis polyphemoides and surface water temperature (dotted line) in Pärnu Bay in 2006.

Fig. 6 .

 6 Fig. 6. Scatterplot of total length and fecundity (number of embryos female -1 ) of Pleopis polyphemoides in the coastal (empty circles) and the open sea (filled circles) station in Pärnu Bay.

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .

 123456 Fig. 1. Location of the sampling sites, coastal station in the inner bay (empty circle) and open sea station in the outer bay (filled circle) in Pärnu Bay (Baltic Sea). 79x99mm (600 x 600 DPI)

Table I .

 I Dynamics of the body length (BL) and total length (TL) (mm; mean ± SE) of Pleopis polyphemoides in the coastal and the open sea station in Pärnu Bay by 10-day periods (D) during

	May-August in 2006
	Parthenogenetic females		Gamogenetic females		Males
	Month D	n	BL	TL	n	BL	TL	n	BL	TL
			F o
				r
				P
				e e r
						R e v i e w

Table II .

 II Relative importances (R 2 ) of different environmental variables for the total length (TL) and the body length (BL) of Pleopis polyphemoides

		Parthenogenetic females	Gamogenetic females
		n	TL	BL	TL	BL
	Chl a concentration	15	0.13	0.82	0.08	0.09
	Temperature	15	0.72	0.12	0.58	0.53
	Salinity	15	0.14	0.05	0.34	0.39
	F o
	r
	P
		e e r
			R e v i e w

Table II .

 II Fecundity (embryos female -1 ; mean ± SE) of parthenogenetic females of Pleopis polyphemoides in the coastal and the open sea station in Pärnu Bay by 10-day periods (D)

	Journal of Plankton Research	Page 30 of 31
	Month D	n	Embryos female -1
		Coastal station
	May	2 23	7.7 ± 0.4
		3	7	5.9 ± 0.3
	June	1 21	5.6 ± 0.3
		2 19	2.3 ± 0.2
		Open sea station
	June	1 13	4.0 ± 1.0
		2 105	3.2 ± 0.1
	July	1 16	1.6 ± 0.2
	2 12 3 27 August 1 17 o F	1.5 ± 0.2 3.0 ± 0.3 1.2 ± 0.1
	r
	P
	e e r
			R e v i e w

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Table III .

 III Length (mm; mean ± SE) of parthenogenetic embryos of Pleopis polyphemoides by different developmental stages in the coastal and the open sea station in Pärnu Bay

	Page 31 of 31
	Embryo stage n	avg ± SE	min -max
		Coastal station
	I	8 0.13 ± 0.01 0.08 -0.17
	II	22 0.18 ± 0.01 0.11 -0.24
	III	17 0.21 ± 0.01 0.14 -0.27
	IV	24 0.26 ± 0.00 0.19 -0.31
		Open sea station
	I	12 0.11 ± 0.01 0.05 -0.17
	II	87 0.16 ± 0.00 0.08 -0.24
	III	73 0.21 ± 0.00 0.14 -0.27
	IV	19 0.23 ± 0.01 0.15 -0.32

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The authors thank all the colleagues involved in the fieldwork and Jonne Kotta for help with statistical analysis. We are also grateful to three anonymous referees whose comments improved the quality of the manuscript and thank Tiia Kaare for linguistic corrections.

Acknowledgements

Funding

This work was financed by the Estonian Ministry of Education and Research (grants nos SF01800135s08 and SF0180005s10) and by the Estonian Science Foundation (grant no. 6751).