Towe Holmborn

Erica Goetze

Maria Põllupüü

Arno Põllumäe

Genetic species identification and low genetic diversity in Pseudocalanus acuspes of the Baltic Sea

Keywords: Pseudocalanus acuspes, Pseudocalanus elongatus, Pseudocalanus minutus, Pseudocalanus minutus elongatus, taxonomy, mtCOI, haplotype diversity Pseudocalanus acuspes, Pseudocalanus elongatus, Pseudocalanus minutus, Pseudocalanus minutus elongatus, taxonomy, mtCOI, haplotype diversity Pseudocalanus acuspes, Pseudocalanus elongatus, Pseudocalanus minutus, Pseudocalanus minutus elongatus, taxonomy, mtCOI, haplotype diversity

Genetic species identification and low genetic diversity in Pseudocalanus acuspes of the Baltic Sea

Towe Holmborn, Erica Goetze, Maria Põllupüü, Arno Põllumäe

Introduction

The calanoid copepod genus Pseudocalanus is comprised of temperate-boreal, subarctic and arctic marine species that are often abundant in near-coastal waters. In the Baltic Sea, marine Pseudocalanus spp. live on the edge of their distribution with respect to salinity [START_REF] Hessle | Undersökningar över plankton och dess växlingar i Östersjön under åren 1925-1927. Svenska Hydrografisk-biologiska kommisionens skrifter[END_REF][START_REF] Ackefors | Zooplankton[END_REF]. Pseudocalanus is abundant throughout the Baltic proper, but is mostly found in offshore areas rather than in coastal zones, and is rarely present above the thermocline during the warm summer period [START_REF] Hessle | Undersökningar över plankton och dess växlingar i Östersjön under åren 1925-1927. Svenska Hydrografisk-biologiska kommisionens skrifter[END_REF][START_REF] Ackefors | Zooplankton[END_REF]. In offshore deep areas, this genus can constitute 70 -100% of all copepods in the water column [START_REF] Ackefors | Djurplankton I Östersjöområdet[END_REF]. In the Baltic Sea food web, Pseudocalanus is recognized as an important link between lower trophic levels and zooplanktivorous fish [START_REF] Peters | Trophodynamics and seasonal cycle of the copepod Pseudocalanus acuspes in the Central Baltic Sea (Bornholm Basin): evidence from lipid composition[END_REF], such as commercially-harvested cod, herring, and sprat [START_REF] Hinrichsen | Biophysical modeling of larval Baltic cod (Gadus morhua) growth and survival[END_REF]Möllman et al., 2003;[START_REF] Casini | Feeding preferences of herring (Clupea harengus) and sprat (Sprattus sprattus) in the southern Baltic Sea[END_REF]. In fact, the unsuccessful recruitment of cod seen in the Baltic during the 1980s has been suggested to be, at least partially, a result of decreased abundances of Pseudocalanus spp. available for cod larvae [START_REF] Voss | Comparing the feeding habitats of co-occurring sprat (Sprattus sprattus) and cod (Gadus morhua) larvae in the Bornholm Basin, Baltic Sea[END_REF]. Degraded weight-at-age and stock size in herring and degraded weight-at-age in sprat during the late 1980s and early 1990s have also been associated with a shift from a Pseudocalanus-rich diet to a diet consisting of other taxa (e.g. [START_REF] Raid | Recent changes in the growth and feeding of Baltic herring and sprat in the northeastern Baltic Sea[END_REF][START_REF] Möllmann | Population dynamics of calanoid copepods and the implications of their predation by clupeid fish in the Central Baltic Sea[END_REF][START_REF] Rönkkönen | Long-term changes in Baltic herring (Clupea harengus membra) growth in the Gulf of Finland[END_REF]. The reason behind the observed diet shifts is unclear but has been suggested to be a result of both increased competition between zooplanktivorous fish and a salinity-related decline in the Pseudocalanus stock [START_REF] Möllmann | The marine copepod, Pseudocalanus elongatus, as a mediator between climate variability and fisheries in the Central Baltic Sea[END_REF][START_REF] Casini | Inter-annual variation in herring, Clupea harengus, and sprat, Sprattus sprattus, condition in the central Baltic Sea: what gives the tune?[END_REF].

Regardless of the mechanism, these events highlight the importance of Pseudocalanus as prey in the Baltic Sea pelagic ecosystem.

Although this genus is recognized to play a key role in the trophodynamics of the Baltic Sea [START_REF] Rönkkönen | Long-term changes in Baltic herring (Clupea harengus membra) growth in the Gulf of Finland[END_REF][START_REF] Möllmann | Food consumption by clupeids in the central Central Baltic: evidence for top-down control?[END_REF], there are uncertainties regarding the species identity of Pseudocalanus in this area. This uncertainty is particularly evident in the variable use of different species names in publications from the Baltic Sea, including in literature from the same geographic area. The species name P. minutus elongatus has been used frequently in studies in the Gulf of Finland [START_REF] Rönkkönen | Long-term changes in Baltic herring (Clupea harengus membra) growth in the Gulf of Finland[END_REF][START_REF] Ojaveer | Highlights of zooplankton dynamics in Estonian waters (Baltic Sea)[END_REF] and northwestern Baltic proper [START_REF] Ackefors | On the zooplankton fauna at Askö (The Baltic -Sweden)[END_REF][START_REF] Hansson | Selective predation by herring and mysids, and zooplankton community structure in the Baltic Sea coastal area[END_REF], as well as in work from the Gdańsk Deep (Southern Baltic Sea;[START_REF] Dzierzbicka-Glowacka | Seasonal dynamics of Pseudocalanus minutus elongatus and Acartia spp. in the southern Baltic Sea (Gdańsk Deep)numerical simulations[END_REF]. In studies of the region stretching from the Latvian coast to the eastern slope of Gotland Deep, [START_REF] Dippner | Long-term variability of mesozooplankton in the Central Baltic Sea[END_REF] and [START_REF] Möllmann | The marine copepod, Pseudocalanus elongatus, as a mediator between climate variability and fisheries in the Central Baltic Sea[END_REF] used the name P. elongatus. At least two different species names can be found in the recent literature of the Bornholm Basin; P. elongatus [START_REF] Hinrichsen | Biophysical modeling of larval Baltic cod (Gadus morhua) growth and survival[END_REF][START_REF] Möllmann | The marine copepod, Pseudocalanus elongatus, as a mediator between climate variability and fisheries in the Central Baltic Sea[END_REF] and P. acuspes [START_REF] Schulz | Vertical zonation of the zooplankton community in the Central Baltic Sea in relation to hydrographic stratification as revealed by multivariate discriminant function and canonical analysis[END_REF][START_REF] Renz | Life cycle of Pseudocalanus acuspes Giesbrecht (Copepoda, Calanoida) in the central Baltic Sea: II. Reproduction, growth and secondary production[END_REF][START_REF] Peters | Trophodynamics and seasonal cycle of the copepod Pseudocalanus acuspes in the Central Baltic Sea (Bornholm Basin): evidence from lipid composition[END_REF]. Finally, the inclusive term, ), has been used in studies from the Bornholm Basin and Kiel Bay [START_REF] Hansen | Spatio-temporal distribution and production of calanoid copepods in the central Baltic Sea[END_REF][START_REF] Isla | Physiological response of the calanoid copepod Pseudocalanus sp. in the Baltic Sea at different thermal scenarios[END_REF].

The taxonomic confusion seen in the Baltic Sea nomenclature of Pseudocalanus is not unique to this region. This genus has had, as stated by [START_REF] Corkett | The biology of Pseudocalanus[END_REF], "problems with nomenclature and of delimitations of named species pervading in the literature", ever since the first description of a species within this genus (P. minutus, originally named Calanus minutus). The confusion stems, in part, from the difficulty in distinguishing Pseudocalanus species morphologically, due to conservatism in traits commonly used for species identification. In an effort to resolve the confusing nomenclature and taxonomy of Pseudocalanus, [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] revised the entire taxonomy of this genus. [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] described seven species worldwide, of which three were new: P. minutus (Krøyer, 1845); P. acuspes (Giesbrecht, 1881); P. elongatus (Boeck, 1865); P. major (Sars, 1900); P. moultoni n.sp.; P. newmani n.sp; and P. mimus n.sp. Three species were described from the north-east Atlantic Ocean and adjacent waters. Pseudocalanus minutus and P. acuspes were reported from the Norwegian Sea region, and P. acuspes and P. elongatus were sampled in the North Sea and in coastal waters around the British Isles. In his samples from the Baltic Sea, based on material sampled in March and June in the Gotland basin and from all seasons in Kiel Bay, only P. acuspes was identified. However, in a subsequent study [START_REF] Bucklin | Molecular systematics and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae[END_REF] acknowledged the possibility that other Pseudocalanus species may co-occur with P. acuspes in the Baltic Sea. The taxonomic revisions of [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] were confirmed with molecular data from allozymes of the GPI (Glucose Phosphate Isomerase) locus [START_REF] Sévigny | Discrimination among and variation within species of Pseudocalanus based on the GPI locus[END_REF], and [START_REF] Bucklin | Molecular systematics and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae[END_REF] also found species differences in DNA sequence variation in the mitochondrial locus cytochrome oxidase I (mtCOI) for six Pseudocalanus species (no data reported for P. major).

Currently, the only confirmed Pseudocalanus species found in the Baltic Sea is P. acuspes.

However, all efforts to confirm this were carried out in the southern parts of the Baltic Sea. The Baltic Sea has a dramatic south -north salinity gradient (~20-0 psu; e.g, [START_REF] Fonselius | One hundred years of hydrographic measurements in the Baltic Sea[END_REF], and different zooplankton species are known to dominate in distinct areas along this salinity gradient [START_REF] Ackefors | Djurplankton I Östersjöområdet[END_REF]. Therefore, it is not certain that the Pseudocalanus species found in the northern Baltic proper and Gulf of Finland is the same as that confirmed in the south, although this may well be the case. There may also be seasonal dependency in the species of Pseudocalanus present, as temperature is known to affect the salinity tolerance of this marine genus [START_REF] Ackefors | Zooplankton[END_REF].

In this study, we investigated the species composition, in time and space, of the genus Pseudocalanus in the northern, central, and southern parts of the Baltic proper and in the Gulf of Finland using a Restriction Fragment Length Polymorphism (RFLP) approach. Our goal was to formally confirm the Pseudocalanus species present in these areas and thereby resolve the taxonomic confusion found in the ecological literature of the Baltic Sea.

Method

Zooplankton sampling and preservation -Copepods were sampled at 13 different stations on a total of 22 different occasions (Table 1; Fig 1), stretching from November 2006 until July 2008, in the Baltic proper andGulf of Finland (Table 1, Fig. 1). Copepods were collected from the upper 100 m by vertical hauls. The content of the tows was rinsed into jars with 96 % ethanol (non-denatured) and stored at -20 °C. After 12 -24 h the alcohol was replaced by fresh ethanol and samples were stored at -20 °C until sorting. Adult females and copepodite stage five females of Pseudocalanus spp. were sorted from bulk plankton samples for analysis. In total, 888 individuals were screened using our RFLP protocol (detailed description below) to establish species identity (Table 1).

DNA extraction -DNA was extracted from individual copepods following a lysis buffer protocol [START_REF] Goetze | Cryptic speciation on the high seas: global phylogenetics of the copepod family Eucalanidae[END_REF][START_REF] Lee | Morphological stasis in the Eurytemora affinis species complex (Copepoda: Temoridae)[END_REF]. Briefly, individual specimens were removed from ethanol, blotted dry, and transferred to a 1.5 mL microcentrifuge tube with 40 µl of lysis buffer [10mM Tris pH 8.3, 50 mM KCl, 0.5% Tween 20, 200 ug/ml proteinase K]. The specimen was ground with a pestle, incubated at 65 °C for 1 hour, followed by a 15 minute incubation at 95 °C for denaturation.

DNA extracts were stored at -80 °C when not in use.

Restriction Fragment Length Polymorphism (RFLP) protocol development -We designed an RFLP

protocol to discriminate P. acuspes, P. elongatus, and P. minutus, the three species reported by [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] to occur in the Northeastern Atlantic and adjacent waters. The mitochondrial gene cytochrome c oxidase subunit I (mtCOI) was selected as the basis for the RFLP protocol, due to the prior availability of mtCOI DNA sequence data for Pseudocalanus species and because mtCOI is known to have sufficient variation for species discrimination in many calanoid copepods (e.g., [START_REF] Bucklin | Distribution and relative abundance of Pseudocalanus moultoni and P. newmani (Copepoda: Calanoida) on Georges Bank using molecular identification of sibling species[END_REF][START_REF] Bucklin | Molecular systematics and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae[END_REF][START_REF] Blanco-Bercial | RFLP procedure to discriminate between Clausocalanus Giesbrecht, 1888 (Copepoda, Calanoida) species in the Central Cantabrian Sea[END_REF].

Pseudocalanus mtCOI DNA sequences were retrieved from GenBank for initial RFLP design and testing (Accession numbers: P. acuspes -AF462317, P minutus -AF332794, P. elongatus -AF259669, AY144467, AY604523; [START_REF] Bucklin | Molecular systematics and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae[END_REF][START_REF] Unal | Phylogeography of Calanus helgolandicus and the Black Sea copepod Calanus euxinus, with notes on Pseudocalanus elongatus (Copepoda, Calanoida)[END_REF]Felgueroso and Braig, direct submission). Sequences were aligned and examined in Vector NTI software (Invitrogen) to identify recognition sites for commonly-used restriction enzymes. The enzyme HhaI was found to have distinct number and location of recognition sites between P. elongatus, P. minutus, and P. acuspes. Within the Folmer region [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], there were no HhaI recognition sites in P. 5 minutus, and one recognition site each in P. elongatus and P. acuspes (> 184 bp apart). Using this preliminary information, we developed an initial RFLP protocol using universal primers for polymerase chain reaction (PCR) amplification of mtCOI followed by a restriction digest using HhaI. A 702-bp fragment of mtCOI was amplified using primers COI_VH [5' -TAA ACT TCA GGG TGA CCA AAA AAT CA -3'] and L1384 [5' -GGT CAT GTA ATC ATA AAG ATA TTG G -3'] [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF][START_REF] Machida | Large-scale gene rearrangements in the mitochondrial genomes of two calanoid copepods Eucalanus bungii and Neocalanus cristatus (Crustacea), with notes on new versatile primers for the crRNA and COI genes[END_REF]. PCR conditions included a 1 min 95 °C initial denaturation step, followed by 40 cycles of 95 °C for 30 sec (denaturation), 42 °C for 30 sec annealing), and 72 °C for 1 min (extension), with a final extension step of 72 °C for 4 min.

Digestion of this 702-bp mtCOI fragment with HhaI resulted in distinct RFLP profiles for our three target Pseudocalanus species; Pseudocalanus acuspes with two fragments (123 and 518 bp), P. elongatus with two fragments (303 and 307 bp), and because P. minutus has no HhaI recognition site, a single band at the initial 702-bp fragment length remained visible on the agarose gel. This initial RFLP protocol was used to screen DNA extracts of Pseudocalanus specimens from the Norwegian, North, and Baltic Seas in order to find material of species P. minutus, P. elongatus, and P. acuspes from our collections. Our goal was to obtain new DNA sequence data for all three species from the region of interest, in order to verify that our RFLP protocol would capture the genetic variation present in Pseudoclanus populations in the regions adjacent to and within the Baltic Sea. A total of 110 individuals were screened from 4 samples, including material from 65.461N/1.030 W (Norwegian Sea), 63.086N/4.585E (Norwegian Sea), 57.42N/7.93E (North Sea), 55.292 N/14.750E (Baltic Sea), and we sequenced 680-690 bp of mtCOI from 20 of these specimens (DNA sequencing methods described below; Genbank accession numbers HM77004 -HM770078). Despite screening 78 specimens from the North and Norwegian Seas, we did not find any P. acuspes outside the Baltic Sea. The North Sea material contained exclusively P. elongatus, and the Norwegian Sea samples contained both P. elongatus and P. minutus. Although this initial RFLP protocol was effective at species identification, it had the disadvantage of requiring low annealing temperatures in the PCR due to the use of 'universal' PCR primers. Therefore, we used elongatus (326 bp and 304 bp), and the original PCR product remains visible as a single 630-bp band in P. minutus (Fig. 2). Amplifications were conducted in 10 µl reactions, using 1.0 µl of genomic DNA as template. PCR conditions began with 30 sec at 95 °C for denaturation, followed by 40 cycles of 30 sec denaturing at 95 °C, 30 sec annealing at 50 °C, and 1 min extension at 72 °C, with a final extension step of 4 min at 72 °C. Invitrogen Taq polymerase was used at manufacturerrecommended concentrations.

A restriction digest using 3 µl of PCR product was completed using the HhaI restriction enzyme at recommended concentrations (with NEBuffer 4; New England Biolabs). The digestion reaction ran for 3 hours at 37 °C, with a final 20 min denaturation step at 65 °C. Results of the restriction digest were visualized on a 1.5% agarose gel, with a 100-bp ladder used to size the RFLP fragments (Invitrogen). Individuals with ambiguous results, including smeared bands or failed digestion reactions, were amplified and digested a second time to confirm initial results. It should be noted that, given the design of our RFLP protocol, failed digestion reactions could be interpreted as P. minutus. Future studies that intend to use this protocol to examine broader areas of the North Atlantic should include a positive digestion control, or digestion of P. minutus PCR products by a second restriction enzyme to confirm identification of this species. Since only P. acuspes was found in our Baltic material this does not affect the results of the present work.

DNA Sequencing -In order to verify the success of our RFLP protocol and check the haplotype diversity of Pseudocalanus sp. in the Baltic Sea, we obtained DNA sequence data from 83 individuals collected at 7 locations in the southern, central and northern Baltic proper and the Gulf of Finland (Table 1). PCR conditions were as described above, but amplifications were conducted in 25 µl reactions. PCR products were cleaned using an ExoSap protocol prior to sequencing. Shrimp alkaline phosphatase (USB, cat no. 70092Z) and exonuclease I (USB, cat no. 70073Z) were used at manufacturer-recommended concentrations, with PCR products incubated at 37 °C for 30 min followed by a 15 min denaturation step at 95 °C. PCR products were sequenced directly using BigDye Terminator cycle sequencing chemistry, with PCR primers used in sequencing reactions.

Both sequence strands were aligned and checked for accuracy in GeneIous (v4.7, Biomatters), and a consensus sequence was generated for each individual. Multiple sequence alignments, including all individuals, were generated in ClustalX [START_REF] Thompson | The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF].

Results

New sequences obtained from P. minutus, P. elongatus and P. acuspes for primer design were very similar to the existing GenBank sequences, differing by only up to 0.7%. The GenBank P. acuspes sequence differed by only 1 base substitution from our data, with slightly more variation observed in P. elongatus (4 substitutions, 658-bp fragment) and P. minutus (3 substitutions) between our sequences and the haplotypes submitted in prior studies. Having confirmed the accuracy of this prior sequence data, we used our data for primer design of the internal primers PSEUDR and PSEUDF for routine amplification in the restriction fragment length polymorphism (RFLP) protocol.

Using our RFLP approach, we scored 888 Pseudocalanus specimens to species, from material collected throughout the southern, central, and northern Baltic proper regions and the Gulf of Finland (Table 1). All individuals assayed were unambiguously identified to be P. acuspes. Our overall success rate for the method was 96%, with the small number of failed PCRs likely due to poor preservation of specimens or loss of tissue during DNA extraction (922 extracted specimens, 888 successfully scored). In order to confirm the accuracy of our method and the overall result of only a single Pseudocalanus species in the Baltic Sea, we obtained DNA sequence data from 83 specimens collected at 7 sites throughout the Baltic proper and Gulf of Finland. These specimens were chosen to cover all areas of the geographic distribution of our samples, and were collected on a range of dates between 2006 and 2008 (Table 1). Of the 83 individuals sequenced, 82 were of a single haplotype, with the final specimen a singleton that differed by 1 base substitution from the common haplotype (singleton collected in sample G -Gulf of Finland, Table 1). Haplotype diversity (h) for the Baltic Sea was 0.024 [START_REF] Nei | Molecular evolutionary genetics[END_REF][START_REF] Excoffier | Arlequin ver. 3.0: An integrated software package for population genetics data analysis[END_REF], and nucleotide diversity was below 0.00005. The Pseudocalanus acuspes mtCOI nucleotide sequence was translated into protein sequence and no stop codons were observed within the open reading frame.

Similarities to other GenBank Pseudocalanus protein sequences were 99-100%. The amino acid sequence was also highly similar to other calanoid copepods in the database (e.g., >95%; Neocalanus, Ctenocalanus). Finally, given the unexpectedly low level of genetic diversity observed at mtCOI throughout the Baltic Sea, we also obtained DNA sequence from the mt gene 16S rRNA for 16 individuals from 3 samples to verify that low diversity would be observed at other linked mitochondrial loci. Two 16S rRNA haplotypes that differed by a single base were observed in these specimens.

Discussion

Our RFLP results confirm that P. acuspes is the only Pseudocalanus species with a resident population in the Baltic Sea. [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] found only this species in the southern regions of the Baltic proper in his revision of the genus. This study confirms his initial observations with higher spatial and temporal resolution sampling within the Baltic Sea, including material from the northern Baltic proper and Gulf of Finland. Given seasonal sampling within the Gulf of Finland and the north-western Baltic proper, we consider it very unlikely that other Pseudocalanus species (e.g., P. elongatus) ever occur in this region. However, it is still possible that P. elongatus may be occasionally present in the Southern Baltic Sea following major advective inflow events from the Kattegat into the Baltic Sea. These events, although typical, are fairly infrequent (e.g., [START_REF] Matthäus | Natural variability and human impacts reflected in long-term changes in the Baltic deep water conditions -a brief review[END_REF], but may bring ephemeral P. elongatus expatriates into this temporarily higher salinity region.

Our sampling was not designed to capture these inflow events. [START_REF] Grabbert | Species-specific PCR discrimination of species of the calanoid copepod Pseudocalanus, P. acuspes and P. elongatus, in the Baltic and North Seas[END_REF] recently confirmed that P. elongatus (confirmed genetically) can indeed be found in low numbers in the Southern Baltic (Bornholm basin) after major inflows of cold saline water into the Baltic Sea.

Nevertheless, given our results for the broader Baltic region, we conclude that P. acuspes is the only Pseudocalanus species of ecological significance in the Baltic Sea.

Our DNA sequence results demonstrate extraordinarily low mitochondrial diversity in the Baltic Pseudocalanus acuspes population. Studies on other planktonic copepods, including Pseudocalanus congeners in other ocean regions, typically report the proportion of unique mtCOI haplotypes to be in the range of 0.15-0.78, with haplotype diversities between 0.6-0.98 (with the exception of a few outliers, Table 2A). Nucleotide diversities also often greatly exceed the value reported here for Baltic P. acuspes (<0.00005), up to a maximum of 0.1467 for the circumglobal species Eucalanus hyalinus (Table 2A). This near absence of mtCOI gene diversity in the Baltic P. acuspes population is highly unusual for planktonic copepods, especially given the large population sizes maintained by this species within the Baltic Sea. However, if we compare P. acuspes to other non-planktonic invertebrates, we find that low mitochondrial diversity is a common feature of other species that inhabit the Baltic Sea region (Table 2B). Studies of benthic mysids [START_REF] Remerie | Phylogeography of an estuarine mysid, Neomysis integer (Crustacea, Mysida), along the north-east Atlantic coasts[END_REF][START_REF] Audzijonyte | Phylogeographic analyses of a circumarctic coastal and boreal lacustrine mysid crustacean, and evidence of fast postglacial mtDNA rates[END_REF], molluscs [START_REF] Nikula | Phylogeography of Cerastoderma glaucum (Bivalvia: Cardiidae) across Europe: a major break in the Eastern Mediterranean[END_REF][START_REF] Luttikhuizen | Disjunct distribution of highly diverged mitochondrial lineage clade and population subdivision in a marine bivalve with pelagic larval disersal[END_REF] and amphipods [START_REF] Kelly | Contrasting patterns in genetic diversity following multiple invasions of fresh and brackish waters[END_REF] all report low proportions of unique haplotypes (0.05-0.37) and low haplotype diversities, in some cases with the Baltic Sea samples appearing monotypic in mtCOI composition. The low salinity Baltic Sea is a marginal ecosystem for marine species, such as P. acuspes, and Baltic Sea populations often have reduced genetic diversity relative to Atlantic populations of the same species [START_REF] Johannesson | Life on the margin: genetic isolation and diversity loss in a peripheral marine ecosystem, the Baltic Sea[END_REF]. Mitochondrial markers, including mtCOI, are particularly susceptible to the loss of diversity due to the smaller effective population size of the mitochondrial genome. Prior work on the genetics of Baltic marine species has focused on commercially harvested fish, marine mammals, and benthic invertebrates (e.g., [START_REF] Johannesson | Life on the margin: genetic isolation and diversity loss in a peripheral marine ecosystem, the Baltic Sea[END_REF]. Our results for P. acuspes are some of the first reported for planktonic organisms, and they suggest that planktonic species will be similarly characterized by reduced genetic diversity in Baltic Sea populations, despite large population sizes and very high dispersal capacity. Low genetic diversity, as observed in P. acuspes, may limit the capacity of this species to adapt to altered environmental conditions. Baltic P. acuspes also live under suboptimal conditions for growth and reproduction (at least with respect to salinity: [START_REF] Hessle | Undersökningar över plankton och dess växlingar i Östersjön under åren 1925-1927. Svenska Hydrografisk-biologiska kommisionens skrifter[END_REF][START_REF] Ackefors | Zooplankton[END_REF], which may further limit the ability of this species to track changing environmental conditions. If this results in lower population abundance, such a scenario could have detrimental effects on the trophodynamics of the Baltic pelagic food web, including fish production.

Our results also are broadly congruent with the hypothesis that the Baltic Sea population of P. acuspes is a glacial relict. [START_REF] Peters | Trophodynamics and seasonal cycle of the copepod Pseudocalanus acuspes in the Central Baltic Sea (Bornholm Basin): evidence from lipid composition[END_REF] found that the Baltic population of P. acuspes had a similar life cycle to the Arctic population of the same species and hypothesized that the life cycle and lipid storage strategies of the Baltic population of P. acuspes originates from extant adaptations to high latitudinal habitats. The Baltic P. acuspes population is isolated [START_REF] Bucklin | Molecular systematics and phylogenetic assessment of 34 calanoid copepod species of the Calanidae and Clausocalanidae[END_REF] and is likely a member of the glacial relict fauna of the Baltic Sea [START_REF] Peters | Trophodynamics and seasonal cycle of the copepod Pseudocalanus acuspes in the Central Baltic Sea (Bornholm Basin): evidence from lipid composition[END_REF][START_REF] Grabbert | Species-specific PCR discrimination of species of the calanoid copepod Pseudocalanus, P. acuspes and P. elongatus, in the Baltic and North Seas[END_REF]. Low genetic diversity would be expected in isolated, glacial relict populations, as was observed in P. acuspes in this study.

When studying the geographic distribution of all Pseudocalanus species, [START_REF] Frost | A taxonomy of the marine calanoid copepod genus Pseudocalanus[END_REF] found that two or more species of this genus commonly co-occur. Therefore, due to the difficulty of visually discriminating between Pseudocalanus species, the reported biology and ecology of single species is fairly unreliable in the literature. Nevertheless, significant differences in biology and ecology can be found among co-occurring species (e.g., [START_REF] Mclaren | Life cycles of four species of Pseudocalanus in Nova Scotia[END_REF][START_REF] Mcgillicuddy | Intermingling of two Pseudocalanus species on Georges Bank[END_REF]. In ecosystems containing multiple species of Pseudocalanus, use of genetic methods such as the RFLP protocol described here, will greatly improve our understanding of the ecological significance of individual species. Protocols of this kind are easy to develop, and provide an excellent tool for discriminating between morphologically indistinguishable species. Letters indicate 'Sample ID' listed in Table 1.

3

  Pseudocalanus sp. (or spp.

  our new DNA sequence data to design internal primers PSEUDF [5' -CHT TAT ACT TAA TAG CYG GG -3'] and PSEUDR [5' -CYC CAC CTC CAC CTA CRT CA -3'], for amplification of a 630-bp fragment of mtCOI in all three Pseudocalanus species.Routine Polymerase Chain Reaction (PCR) and RFLP -Routine PCR amplification used the newly-designed primers PSEUDF and PSEUDR (630-bp fragment). The final fragment lengths obtained in the restriction digest were verified using NEBcutter (New England Biolabs). The HhaI digestion results in two fragments for P. acuspes (488 bp and 142 bp), two fragments for P.

  , and the numbers (N) of females (adult and copepodite stage five) screened from each sample using the RFLP protocol and DNA sequencing. Table 2. Literature summary of genetic diversity observed at the mitochondrial gene COI in (A) marine planktonic copepods, and (B) Marine invertebrate species in the Baltic Sea. Listed are: the Taxon and Area of study, Number of individuals sampled (N), Fragment length (bp), Number of haplotypes (H), Number of unique haplotyes (H/N), Haplotype diversity (h), Nucleotide diversity (π, per nucleotide site), and the original reference of each study. N.r. = not reported. Note: Most of the studies reported in B surveyed a larger distribution: we only report results for the Baltic Sea region in this Table.

Fig. 1 .

 1 Fig. 1. Geographic distribution of collecting locations in the Baltic proper and Gulf of Finland.

Fig. 2 .

 2 Fig.2. Illustration of the Restriction Fragment Length Polymorphism (RFLP) approach to species discrimination of Pseudocalanus acuspes, P. minutus, and P. elongatus. Restriction digest of PCR products using the Hha1 enzyme (see methods), visualized on a 2% agarose gel with a 100-bp ladder (Invitrogen Cat. No. 10488-058). The 630-bp fragment visible for P. minutus is the same length as the original PCR product (lanes 1-4), P. elongatus has two fragments 326 and 304 bp in length, which appear as a single, smeared band slightly above 300-bp in length (lanes 5-8), and P. acuspes has two fragments of 488 and 142 bp (lanes 9-12).

Table and Figure legendsTable 1 .

 1 Collection locations for Pseudocalanus spp. in the Baltic proper and Gulf of Finland

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Acknowledgements

We thank Leif Lundgren, Stefan Svensson, Stella Mudrak, Jörg Dutz, and Jasmin Renz for providing us with samples. We also thank three anonymous reviewers whose comments improved the manuscript. This research was initiated as part of the 2007 Nordic Marine Academy graduate course 'The challenge of pelagic feeding: from prey detection to secondary production in contrasting pelagic food webs', and was partially financed by the Estonian Ministry of Education and Research (grant no SF0180005s10).

Tables


Janet Athene Lane

Liam J Murray

Ian M Harvey

Jenny L Donovan

Consultant Gastroenterologist Prakash Nair

Richard F Harvey

Jenny ; Donovan

Richard ; Harvey

Jenny L Donovan

Consultant Gastroenterologist Richard F Harvey

associated with a significantly increased body mass index in a placebo-controlled trial

Keywords: Functional dyspepsia < Disease-based, Stomach and duodenum < Organ-based, H. pylori < Topics, Screening < Helicobacter pylori, body mass index, weight, randomised controlled trial, obesity, dyspepsia

Aim

To determine the impact of H. pylori eradication on BMI in a European population. Methods 10,537 unselected people aged 20-59 years were screened for H. pylori. 1558 of the 1634 infected participants were randomised to intervention (eradication therapy: ranitidine bismuth citrate 400 mg and clarithromycin 500 mg twice daily) or placebo for two weeks with follow-up at six months (92%) for weight and dyspepsia symptoms (epigastric pain).

Results

The mean weight of participants in the intervention group increased from 77.7 kg at baseline to 78.4 kg at six months (unadjusted increase 0.7 kg) and [REMOVE: the participants mean weight changed] from 76.8 kg to 77.2 kg (0.5 kg) in the placebo group. The adjusted difference between [REMOVE: the] randomised groups [REMOVE: at six months] was statistically significant at 0.6 kg (95% confidence interval (CI) 0.31, 0.88)). Significantly more participants gained [REMOVE at least] ≥3 kg in the intervention group (138/720, 19.2%) compared to the placebo group (92/706, 13.0%) (odds ratio (OR) 1.57 (95% CI 1.17, 2.12)). The mean BMI increased from 27.5 kg/m 2 to 27.8 kg/m 2 at six months in the intervention group compared with 27.0 kg/m 2 to 27.2 kg/m 2 in the placebo group (adjusted difference between groups was statistically significant at 0.2 kg/m 2 (95% CI 0.11, 0.31)). Dyspepsia was less frequently reported by intervention group participants (168/736, 22.8%, placebo group 209/711, 29.4%), OR 0.71 (95% CI 0.55, 0.93).

Conclusions

BMI increased significantly following randomisation to H. pylori eradication therapy, possibly due to dyspepsia resolution.

INTRODUCTION

A meta-analysis of observational studies investigating the role of H. pylori infection and cardiovascular risk factors concluded that infected individuals had a slightly but significantly higher body mass index (BMI) [START_REF] Danesh | Risk factors for coronary heart disease and infection with Helicobacter pylori: meta-analysis of 18 studies[END_REF]. Some recent studies with representative populations have also observed an association of H. pylori infection with BMI [START_REF] Rosenstock | Association of Helicobacter pylori infection with lifestyle, chronic disease, body-indices, and age at menarche in Danish adults[END_REF][START_REF] Russo | Determinants of Helicobacter pylori seroprevalence among Italian blood donors[END_REF][START_REF] Kopacova | Body indices and basic vital signs in Helicobacter pylori positive and negative persons[END_REF], whereas others, such as the NHANES III study, have not [START_REF] Cho | Helicobacter pylori and overweight status in the United States: data from the Third National Health and Nutrition Examination Survey[END_REF]. These conflicting results highlight the difficulties in identifying an association with crosssectional study designs where individuals differ by factors other than H. pylori infection status, for example age and gender, so potentially confounding the association.

The impact of H. pylori eradication on BMI and weight has been investigated previously in Japan in four cohort studies and one randomised controlled trial. BMI increased by 0.8 kg/m 2 over three years in a trial with 90 non-ulcer dyspepsia patients [START_REF] Kamada | The longterm effect of Helicobacter pylori eradication therapy on symptoms in dyspeptic patients with fundic atrophic gastritis[END_REF]. However, the analysis only included those participants in whom H. pylori eradication had been successful. The four observational studies comprised one cohort of industrial workers and three of patients with peptic ulcer disease or gastritis. BMI and weight were increased after H. pylori eradication in these cohort studies when compared to either individuals where eradication had failed, [START_REF] Azuma | Eradication of Helicobacter pylori infection induces an increase in body mass index[END_REF][START_REF] Furuta | Effect of Helicobacter pylori infection and its eradication on nutrition[END_REF][START_REF] Fujiwara | Long-term effect of Helicobacter pylori eradication on quality of life, body mass index, and newly developed diseases in Japanese patients with peptic ulcer disease[END_REF] or to controls matched for age and sex [START_REF] Kamada | Eradication of Heliocobacter pylori increases the incidence of hyperlipidaemia and obesity in peptic ulcr patients[END_REF].

We have investigated the impact of H. pylori eradication on BMI, weight and dyspepsia in a large population-based randomised controlled trial. This trial utilises a robust placebo-controlled design and also explores the role of a potential causal mechanism, namely dyspepsia.

MATERIALS AND METHODS

Study population

This study is nested in a large randomised controlled trial of the effects of H. pylori eradication on the symptoms, treatment and costs of dyspepsia (described fully elsewhere) (11;12). In brief, 26,203 individuals unselected for dyspepsia symptoms, aged 20-59 years and registered at seven general practices in South West England were invited by letter to participate between 1996 and 1999. 10,537 attenders were screened for H. pylori infection using the 13 C-labeled urea breath test (UBT) and infected participants were randomised to an intervention of H. pylori eradication therapy or placebo. The local research ethics committee approved the study and written informed consent was obtained from participants (trial registration ISCTRN44816925).

Study design

This is a randomised placebo controlled trial (Figure 1). Participants who tested positive for H. pylori with the UBT (using a standard test meal of citric acid and orange juice with a cutoff of Cδ 13 3.5 per cm 3 ) (13) were randomised either to H. pylori eradication therapy (ranitidine bismuth citrate 400 mg and clarithromycin 500 mg twice daily for two weeks) or to placebo. Randomisation was stratified by gender and age (20-29, 30-39 40-49 and 50-59 years). Staff independent of the study prepared the randomisation sequence, which was generated by computer with a block size of 10. Medication packs were prepared by pharmacists who had no contact with participants and were dispensed by research nurses who were blind to the allocation.

H pylori status was assessed in both groups by a UBT six months after randomisation.

All participants and research staff were blinded to the allocation and the UBT results until trial completion at two years. Two year follow-up in randomised participants was by postal questionnaire to collect symptoms and medical note review for the primary outcome (without no repeat of anthropometric measures). The primary outcome of the overall trial was consultations in primary care at two years. There was

Data collection and outcome measures

Height and weight (without coats or shoes) were measured at enrolment by research nurses using a stadiometer and a beam balance. Weight was measured at six months only in randomised participants. Participant socio-demographics and lifestyle characteristics (e.g. smoking status) were collected at baseline. Dyspepsia frequency (defined as epigastric pain) over the previous three month period was measured with five-point Likert scales rated from 1 (none) to 5 (daily) in validated participantcompleted questionnaires (14;15). Regular or frequent symptoms were defined by the questionnaire developers as occurring on two or more occasions over each of the previous three months. Dyspepsia symptoms were measured at enrolment and six months in all randomised participants. Participants who did not attend follow-up appointments or had moved from the area were posted questionnaires.

Statistical analyses

Baseline data

Analyses included all H. pylori positive participants (1634) and a computer-generated random sample of the uninfected participants (3268/8901, 36.7%). The sampling strategy was devised to generate a 2:1 ratio of H. pylori negative to positive participants as a higher ratio may not have increased the precision of estimates and would have substantially increased study costs. BMI was calculated as the weight (kg) divided by the square of the height (metres) whilst obesity was defined as a minimum BMI of 30 kg/m 2 . Mean weight and BMI were compared in turn between infected and uninfected individuals using regression models, the crude differences in means being presented with 95% confidence intervals (95% CI). Adjusted differences of means were obtained by controlling for confounders of BMI and weight by adding the following measures as covariates in the regression models, namely age (bands 20-39, 40-49, 50-59 years with the lowest two groups combined as few participants were in and II = professional, III = skilled, IV and V = partly or unskilled), childhood social class (owner-occupied accommodation or rented/other), smoking status (never, past and current) and alcohol consumption (low = less than 14 units/week for women or 21 units for men and high = greater than 13 units for women or 20 units for men).

Ethnicity was excluded from the analyses as the study population was predominantly white (97%). Similar logistic regression models estimated the associations of dyspepsia and obesity with H. pylori infection as odds ratios (OR). Adjustment was also made for potential clustering by residential location by including general practice in the models (Stata version 10; College Station, Texas, USA).

Randomised trial data

The primary outcome of the nested study was the impact of randomisation to H. pylori eradication on BMI and weight at six months. The overall trial sample size calculation of 1550 participants was based on rates of consultation for dyspepsia in primary care at two years [START_REF] Lane | A placebocontrolled randomized trial of eradication of Helicobacter pylori in the general population: study design and response rates of the Bristol Helicobacter Project[END_REF]. There were five planned secondary analyses in this trial [START_REF] Lane | Impact of Helicobacter pylori eradication on dyspepsia, health resource use, and quality of life in the Bristol helicobacter project: randomised controlled trial[END_REF]. No power calculation or analysis plan existed for this nested study due to the exploratory nature of the hypothesis and the absence of prior high quality evidence. Differences in means were the effect measure estimates calculated by the ANCOVA [START_REF] Vickers | Analysing controlled trials with baseline and follow up measurements[END_REF] technique that incorporates baseline covariates in the analytic model to account for chance baseline differences between randomised groups. Analyses were also adjusted for the randomisation strata of age and gender. We also explored whether a paired data method comparing the mean individual weight differences between the two randomised groups altered the effect estimates. Data are presented with 95% CI.

Analyses (unless specified otherwise) were conducted on an intention-to-treat basis (ITT) with all randomised individuals included in the analysis regardless of subsequent H pylori status. Analyses utilised an approach based on levels of evidence rather than p value thresholds [START_REF] Sterne | Sifting the evidence-what's wrong with significance tests[END_REF]. Dyspepsia symptoms and the numbers of obese participants at six months were each compared on an ITT basis between the two groups as randomised with the intervention effect estimate presented as ORs. An exploratory sub-group analyses in the intervention group investigated the weight of individuals with dyspepsia at six months compared with those without symptoms (including participants whose symptoms had resolved from baseline measurement). The lowest two age strata (20-29 and 30-39 years) were combined for all the analyses as there were fewer participants due to the inverse relationship of H. pylori prevalence and age [START_REF] Lane | A placebocontrolled randomized trial of eradication of Helicobacter pylori in the general population: study design and response rates of the Bristol Helicobacter Project[END_REF].

RESULTS

Study population

The 10,537 participants had a mean age of 45.8 years (SD 8.95) and 46 % were male.

Participants infected with H. pylori were heavier than uninfected individuals although this was no longer apparent with confounding controlled (0.4 kg, 95% CI -0.47, 1.22)(Table 1). BMI also did not differ by H. pylori status (0.3 kg/m 2 following adjustment, 95% CI 0.0, 0.57). Obesity was more prevalent in H. pylori infected participants compared with uninfected participants with an adjusted odds ratio of 1.19 (95% CI 1.02, 1.38) (Table 1). Dyspepsia symptoms did not differ by H. pylori status (805/3156, 25.5% negative: 402/1564, 25.7% positive, OR 0.988, 95% CI 0.86, 1.83).

H. pylori prevalence and eradication

1636 participants were positive for H. pylori (15.5%). 1558 participants agreed to be randomised (95%) with 787 randomised to the intervention and 771 to placebo (Figure 1). Random allocation resulted in two groups that were comparable for the measured baseline variables (Table 2). Follow-up of randomised participants at six months was 92% (1427). The rate of H. pylori eradication in the intervention group was 90.7% (659/727). At two years participants reported what sort of tablets they thought they had received or whether they were unsure (266 participants guessed that they had received active treatment at two years in the eradication group compared with 241 expected by chance (χ 2 = 1.90, p = 0.1683), indicating that blinding had been successful.

BMI, weight and dyspepsia following H. pylori eradication therapy

The differences in the unadjusted weight and BMI over six months in both randomised groups is shown in Figure 2. The overall difference in mean weight between the randomised groups was 0.6 kg (95% CI 0.31, 0.88) at six months (Table 3). The paired data analysis also gave an effect estimate of 0.6 kg, 95CI 0.30, 0.87).

The overall difference in mean BMI was 0.2 kg/m 2 (95% CI 0.11, 0.31). In the intervention group more participants gained at least 3 kg over six months following randomisation (138/720, 19.2%) compared to the placebo group (92/706, 13.0%), There was also a greater proportion of obese participants in the eradication group (26.2%) compared to the placebo group (22.1%) but this was not statistically significant (Table 3).

Dyspepsia symptoms were less frequently reported by the intervention group participants (168/736, 22.8%) compared with those in the placebo group (209/711, 29.4%). The OR for dyspepsia reduction in the intervention group was 0.71 (95% CI 0.55, 0.93). In a sub-group analysis of participants in the intervention group with and without dyspepsia at six months the adjusted effect estimate for weight between the two groups was 0.6 kg (95% CI 0.23, 0.90). There are several strengths to this study, notably the randomised study design which enrolled a large community-based population of both sexes over a wide age range.

The UBT detection method minimised H. pylori misclassification by identifying only those individuals with current infections (most of the previous cross-sectional studies were based on ELISA tests which also detect past infections). An efficacious H. pylori eradication regime reduced contamination of the intervention group through individuals with failed eradication whilst the successful concealment of treatment allocation and blinding of participants and study staff ensured unbiased assessment of outcomes [START_REF] Lane | Impact of Helicobacter pylori eradication on dyspepsia, health resource use, and quality of life in the Bristol helicobacter project: randomised controlled trial[END_REF]. Nevertheless, limitations exist to this study because it was nested within a longer term trial so these results warrant confirmation in other populations and countries. The principal limitation is the duration of follow-up as weight and BMI gains have been observed previously for up to three years after H. pylori eradication (6) but the overall trial follow-up at two years was by postal questionnaire and medical note review with no anthropometry. Lifestyle changes which could have influenced BMI e.g. smoking cessation, increased alcohol consumption and levels of exercise were not measured again at six month follow-up, but successful blinding of participants and trial staff with a placebo comparator should have ensured that any such effects were distributed by chance across the two groups. Blood samples were not taken to maximise trial participation but they could have been used to investigate biological intermediates more recently identified on the putative causal pathway.

There was also no opportunity to measure the weight and BMI of the H. pylori negative participants again as trial follow-up was restricted to randomised individuals due to logistics and funding. pylori concluded that BMI was slightly greater in infected individuals(1). This study found no difference in BMI by H. pylori status which concurs with some recent population-based studies (5;8;18;19) although several other studies showed an association,(2-4) which may reflect the various geographical origins of the populations or the degree of adjustment for confounding.

All previous studies on the impact of H. pylori eradication on BMI were conducted in Japan where the mean population BMI is markedly lower than in western countries and obesity is classified at 25 kg/m 2 rather than 30 kg/m 2 as in the rest of the world [START_REF] Kagawa | Differences in the relationship between BMI and percentage body fat between Japanese and Australian-Caucasian young men[END_REF]. There are also other differences between Asian and non-Asian countries in the patterns of H. pylori and gastritis, the relative frequency of different H. pylori sub-types and the incidence of gastric cancer [START_REF] Mccoll | Helicobacter pylori and oesophageal cancer--not always protective[END_REF]. The analysis of a randomised trial of H. pylori eradication in non-ulcer dyspeptic patients was not conducted on an ITT basis so the gain in BMI was only measured in those individuals with successful eradication so is difficult to interpret more generally [START_REF] Kamada | The longterm effect of Helicobacter pylori eradication therapy on symptoms in dyspeptic patients with fundic atrophic gastritis[END_REF]. In four cohort studies with industrial workers or patients with peptic ulcer disease, non-ulcer dyspepsia or gastritis, BMI increased following H. pylori eradication compared to individuals where eradication therapy failed [START_REF] Kamada | The longterm effect of Helicobacter pylori eradication therapy on symptoms in dyspeptic patients with fundic atrophic gastritis[END_REF][START_REF] Azuma | Eradication of Helicobacter pylori infection induces an increase in body mass index[END_REF][START_REF] Furuta | Effect of Helicobacter pylori infection and its eradication on nutrition[END_REF][START_REF] Fujiwara | Long-term effect of Helicobacter pylori eradication on quality of life, body mass index, and newly developed diseases in Japanese patients with peptic ulcer disease[END_REF] or to age and sex matched controls [START_REF] Kamada | Eradication of Heliocobacter pylori increases the incidence of hyperlipidaemia and obesity in peptic ulcr patients[END_REF]. However, no adjustment was made for confounding in any cohort study.

The improvement in dyspepsia symptoms observed in this study at six months in the intervention group and previously reported at two years [START_REF] Lane | Impact of Helicobacter pylori eradication on dyspepsia, health resource use, and quality of life in the Bristol helicobacter project: randomised controlled trial[END_REF] may have stimulated the appetite and subsequently caused the gain in weight and BMI through increased food intake. An exploratory sub-group analyses of intervention group individuals who had resolution of dyspepsia symptoms did not show a greater weight gain than the overall comparison but the lack of statistical power for this analysis does not make this a robust finding. Hyperlipidaemia was also shown to increase following H. pylori eradication in two Japanese studies (8;10). Some of these effects may be mediated through hormonal systems by ghrelin and leptin, two peptides located in the stomach wall which regulate food intake and appetite (ghrelin negatively and leptin positively) (22;23). Studies in healthy volunteers, (5;19;24) however, found no association of serum leptin with H. pylori status nor changes following H. pylori eradication in patients with chronic gastritis or in healthy volunteers (7;25). However, [START_REF] Azuma | Gastric leptin and Helicobacter pylori infection[END_REF]. Plasma ghrelin levels were lower in H. pylori positive individuals in some studies, (19;27;28) although two small studies differed as to whether levels altered following eradication. (25;27) Recently, H. pylori eradication increased weight and decreased plasma ghrelin levels (but increased gastric levels) in men participating in a Japanese gastric cancer surveillance programme [START_REF] Osawa | Changes in plasma ghrelin levels, gastric ghrelin production, and body weight after Helicobacter pylori cure[END_REF]. Large studies now are required to clarify the role of these peptides to mediate the influence of H. pylori eradication on BMI.

Randomisation to H. pylori eradication therapy increased BMI in a large populationbased randomised trial after six months. These changes could become clinically significant if the weight gain continued as indicated by previous cohort studies. H. pylori eradication may possibly be contributing in part to the recent dramatic rise in obesity in Western populations given the concurrent decline in prevalence but this hypothesis relies on the far less robust ecological evidence and requires further investigation (30;31). Currently, patients being treated with H. pylori eradication therapy should be advised of the possibility of weight gain following eradication given that obesity is a risk factor for many other diseases, including GORD(32) and cardiovascular disease [START_REF] Danesh | Risk factors for coronary heart disease and infection with Helicobacter pylori: meta-analysis of 18 studies[END_REF].

Financial support

This study was funded by the UK National Health Service Research and Development programme (South and West) and GlaxoSmithKline (GSK).

Competing interests statement

The randomisation sequence was generated by one of the funders (GSK) but the sponsors had no further role in the study design, data collection, analyses or writing up of reports and publications. JAL and RH received financial support from GSK to attend the American Gastroenterology Association meeting in May 2000. * Social class; I and II = professional, III = skilled occupation, IV and V = partly or unskilled occupations + Non-white participants grouped due to low numbers enrolled ** Greater than 20 units per week for men and 13 units for women $$ Epigastric pain symptoms on two or more occasions in each of the previous three months Specific objectives and hypotheses.

4-5 Outcomes 6

Clearly defined primary and secondary outcome measures and, when applicable, any methods used to enhance the quality of measurements (eg, multiple observations, training of assessors, &c).

4-5

Sample size 7

How sample size was determined and, when applicable, explanation of any interim analyses and stopping rules. Statistical methods used to compare groups for primary outcome(s); methods for additional analyses, such as subgroup analyses and adjusted analyses.

5

Results

Participant flow 13

Flow of participants through each stage (a diagram is strongly recommended). Specifically, for each group, report the numbers of participants randomly assigned, receiving intended treatment, completing the study protocol, and analysed for the primary outcome. Describe protocol deviations from study as planned, together with reasons.

2006 Fig 1

Recruitment 14

Dates defining the periods of recruitment and follow-up.

Baseline data 15

Baseline demographic and clinical characteristics of each group.

Numbers analysed 16

Number of participants (denominator) in each group included in each analysis and whether the analysis was by "intention to treat". State the results in absolute numbers when feasible (e.g., 10/20, not 50%).

table 2

Outcomes and estimation 17

For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precision (e.g. 95% confidence intervals)

  index (BMI) increased following Helicobacter pylori eradication in several Japanese cohorts which requires further investigation.

  follow-up of H. pylori negative participants after the initial screening phase.

  group[START_REF] Lane | A placebocontrolled randomized trial of eradication of Helicobacter pylori in the general population: study design and response rates of the Bristol Helicobacter Project[END_REF]), gender, adult social class (categorised as social class I

  of 1.58 (1.19, 2.10).

11 DISCUSSION

 11 Randomisation to H. pylori eradication therapy resulted in a mean weight gain of 0.6 kg and an increase in BMI of 0.2 kg/m 2 within six months compared with participants who had received placebo. A higher proportion of individuals gained at least 3kg over the six months in the intervention group. Dyspepsia symptoms were less frequent in the intervention group at six months and may provide a potential casual pathway for the recorded increases in BMI and weight.

12 A

 12 meta-analysis of 18 observational studies of cardiovascular risk factors and H.

  were elevated in H. pylori infected individuals and decreased following eradication

  presented at the American Gastroenterology Association meeting in May 2003 and was subsequently published in abstract form (Gastroenterology 2003; 124 S4:A622). Contributors JAL ran the project on a daily basis, analysed the results and wrote the paper. RH conceived and designed the study and helped write the final version of the paper. PN helped initiate the project. LM, IH and JD helped plan the project and produce the final version of the paper. JAL wrote the initial draft of the paper and all authors contributed to the final version. JAL is the guarantor.

Figure 2

 2 Figure 1 Trial profile

2 Scientific background and explanation of rationale. 3 Participants 3 Eligibility criteria for participants and the settings and locations where the data were collected 4 Interventions 4

 23344 full details reported in the 2006 BMJ paper (332:199-204) Randomised controlled trial of Helicobacter pylori eradication and dyspepsia, health resource utilisation and quality of life: Bristol Helicobacter project J Athene Lane, LJ Murray, S Noble, M Egger, IM Harvey, JL Donovan, P Nair, RF Harvey Introduction Background Methods Precise details of the interventions intended for each group and how and when they were actually administered 4 Objectives 5

  generate the random allocation sequence, including details of any restriction (eg, blocking, stratification). implement the random allocation sequence (e.g., numbered containers or central telephone), clarifying whether the sequence was concealed until interventions were assigned.2006 Implementation 10Who generated the allocation sequence, who enrolled participants, and who assigned participants to their groups. participants, those administering the interventions, and those assessing the outcomes were aware of group assignment. If not, how the success of masking was assessed.

  by reporting any other analyses performed, including subgroup analyses and adjusted analyses, indicating those prespecified and those exploratory. naAdverse events 19All important adverse events or side-effects in each intervention group 2006 results, taking into account study hypotheses, sources of potential bias or imprecision and the dangers associated with multiplicity of analyses and outcomes. the results in the context of current evidence.[START_REF] Azuma | Eradication of Helicobacter pylori infection induces an increase in body mass index[END_REF][START_REF] Furuta | Effect of Helicobacter pylori infection and its eradication on nutrition[END_REF][START_REF] Fujiwara | Long-term effect of Helicobacter pylori eradication on quality of life, body mass index, and newly developed diseases in Japanese patients with peptic ulcer disease[END_REF]

Table 1

 1 BMI, weight and frequency of obesity in H. pylori positive and negative individuals prior to randomisation of infected individuals

		H. pylori	H. pylori	Effect	Unadjusted	Adjusted effect
		positive	negative	measure	effect	(95% CI) a
		(n = 1632)	(n = 3260)	estimate	(95% CI)
	Mean BMI	27.2 (4.6)	26.7 (4.5)	Difference	0.6 (0.28, 0.82)	0.3 (0.00, 0.57)
	kg/m 2 (SD)			in means
	Mean weight	77.2 (15.4)	75.9 (14.9) Difference	1.2 (0.34, 2.13)	0.4 (-0.44, 1.28)
	(kg)(SD)			in means
	Number of	382 (23.4)	638 (19.6) Odds ratio 1.26 (1.09, 1.45)	1.19 (1.02, 1.39)
	obese (%)
	a Adjusted for age, gender, adult and childhood social class, general practice, smoking
	status and alcohol consumption.

Table 2

 2 Characteristics of H. pylori infected participants at randomisation

		Intervention group (%)	Placebo group (%)
		(n = 787)	(n = 771)
	Sex
	Men	385 (49.0)	378 (49.0)
	Women	402 (51.1)	393 (51.0)
	Mean age in years (SD)	48.4 (8.0)	48.6 (7.9)
	Socioeconomic status*
	I and II	233 (30.0)	233 (30.7)
	III	387 (50.0)	363 (47.8)
	IV and V	155 (20.0)	164 (21.5)
	Ethnic origin
	White +	758 (97.1)	748 (97.9)
	Non-white	23 (2.9)	16 (2.1)
	Smoking status
	Never	405 (52.8)	389 (50.9)
	Past smoker	179 (23.3)	190 (24.9)
	Current smoker	183 (23.9)	185 (24.2)
	Alcohol consumption
	None	145 (19.5)	123 (17.0)
	Low alcohol intake	541 (72.8)	534 (74.0)
	High alcohol intake **	57 (7.7)	65 (9.0)
	NSAID usage
	None	558 (76.5)	526 (72.7)
	Less than daily	132 (18.1)	154 (21.3)
	Daily	39 (6.0)	43 (6.0)
	Dyspepsia symptoms $$	185 (24.3)	194 (26.6)
	Anthropometry
	Height (cm) (SD)	167.7 (9.2)	168.3 (8.9)
	Weight (kg) (SD)	77.7 (15.9)	76.8 (15.1)

Table 3

 3 BMI, weight and frequency of obesity six months after randomisation to H. Adjusted for age, gender and baseline values of the outcome.

	pylori eradication therapy or placebo
		Placebo	Intervention	Effect	Unadjusted	Adjusted
		group	group	measure	effect	effect
		(n = 706)	(n = 721)	estimates	(95% CI)	(95% CI) a
	Mean BMI in	27.2 (4.5)	27.8 (4.8)	Difference	0.6	0.2
	kg/m 2 (SD)			in means	(0.15, 1.11)	(0.11, 0.31)
	Mean weight	77.2 (15.2)	78.4 (15.8) Difference	1.2	0.6
	in kg (SD)			in means	(-2.8, 0.41)	(0.31, 0.88)
	Number of	156 (22.1)	189 (26.2)	Odds ratio	1.25	1.30
	obese (%)				(0.98, 1.60)	(0.81, 2.10)

a

Alimentary Pharmacology & Therapeutic

Acknowledgements

We thank all participants, the general practice staff, the nursing team of Lynne Bradshaw, Julie Watson, Tina Critchley, Jo Lee, Carol Everson-Coombe, Penny Nettlefield, and Joanne Smith; Judy Millward, Helen Davies, Amy Hawkins and Sarah Pike for secretarial support; Erwin Brown, Phil Hedges and Nick Pope of the microbiology department and Pete Spurr, Martin Bullock and Fiona Greenwood of the pharmacy department, Frenchay Hospital for help with the breath tests and the study medication respectively and Dr Chris Metcalfe of the School of Social and Community Medicine for statistical advice.


L Denis

E Thiébaut

F Soulez

FAST MODEL OF SPACE-VARIANT BLURRING AND ITS APPLICATION TO DECONVOLUTION IN ASTRONOMY

Keywords:

Image deblurring is essential to high resolution imaging and is therefore widely used in astronomy, microscopy or computational photography. While shift-invariant blur is modeled by convolution and leads to fast FFT-based algorithms, shiftvariant blurring requires models both accurate and fast. When the point spread function (PSF) varies smoothly across the field, these two opposite objectives can be reached by interpolating from a grid of PSF samples.

Several models for smoothly varying PSF co-exist in the literature. We advocate that one of them is both physicallygrounded and fast. Moreover, we show that the approximation can be largely improved by tuning the PSF samples and interpolation weights with respect to a given continuous model. This improvement comes without increasing the computational cost of the blurring operator.

We illustrate the developed blurring model on a deconvolution application in astronomy. Regularized reconstruction with our model leads to large improvements over existing results.

INTRODUCTION

Image deconvolution is widely used to enhance the resolution, signal-to-noise ratio and contrast of blurred images. In many cases, blur is space-variant and thus can no longer be modeled by a convolution. Accurate modeling of the point spread function (PSF) is essential for restoration. Depending on whether PSF variations across the field are smooth or discontinuous, PSF are either interpolated, or the field is segmented into regions inside which PSF are invariant. In the former case, effort is put in finding a good tradeoff between approximation quality and speed. The most challenging aspect of the latter case is the segmentation step.

In astronomy, blurring due to atmospheric turbulence varies in the field of view. Even after adaptive optics correction, the PSF is shift-variant since the correction quality decreases away from the guiding star [START_REF] Cresci | Accounting for the anisoplanatic point spread function in deep wide-field adaptive optics images[END_REF]. Optical aberrations and obstructions also lead to space-variant degradations [START_REF] Anderson | Toward High-Precision Astrometry with WFPC2. I. Deriving an Accurate Point-Spread Function[END_REF]. In 3D microscopy, the 3-D PSF varies with the depth from the coverslip [START_REF] Preza | Depth-variant maximumlikelihood restoration for three-dimensional fluorescence mi-croscopy[END_REF]. In all these applications, PSF vary smoothly.

Except for very compact PSF, the storage and application of a different PSF for each pixel [START_REF] Ozkan | POCS-based restoration of space-varying blurred images[END_REF] is not computationally tractable for iterative deblurring methods. It has then been proposed [START_REF] Trussell | Image restoration of space-variant blurs by sectioned methods[END_REF] to decompose the image into patches where blur can be considered approximatively invariant (isoplanatic regions). After deconvolution of each patch, the reconstructed regions must be tied together which leads to border artifacts [START_REF] Aubailly | Approach for reconstructing anisoplanatic adaptive optics images[END_REF][START_REF] Preza | Depth-variant maximumlikelihood restoration for three-dimensional fluorescence mi-croscopy[END_REF][START_REF] Boden | Massively parallel spatially variant maximum-likelihood restoration of Hubble Space Telescope imagery[END_REF]. It is therefore preferable to model smooth blur variations and then, to deblur the whole image.

Smooth PSF variations can be decomposed on a subspace of PSF [START_REF] Flicker | Anisoplanatic deconvolution of adaptive optics images[END_REF][START_REF] Arigovindan | A parallel productconvolution approach for representing the depth varying point spread functions in 3D widefield microscopy based on principal component analysis[END_REF]. The cost of this modeling increases linearly with the number of basis PSF used. It has been noticed independently by several authors that PSF variations could be modeled by interpolation and yet lead to a fast blurring model [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF][START_REF] Gilad | A fast algorithm for convolution integrals with space and time variant kernels[END_REF][START_REF] Hirsch | Efficient filter flow for space-variant multiframe blind deconvolution[END_REF]. Two formulations have been proposed, equivalent in terms of computational complexity but leading to different PSF models. We discuss in section 2.1 these two approximations and their implications in terms of PSF modeling. We show that one of them is superior both in approximation quality and modeling properties. In section 2.2, we further improve PSF approximation error by tuning the PSF samples and interpolation weights for a target PSF model. The space-variant blurring model is then applied to deconvolution of Hubble Space Telescope (HST) simulated data. We show improved reconstructions compared to the results reported on the same dataset in [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF].

APPROXIMATION OF SMOOTHLY VARYING PSF

During the formation of an image g, the original (crisp) distribution f undergoes distortions due to atmosphere turbulence, object/camera relative motion, the instrument (limited aperture, optical aberrations). These degradations are typically modeled by a linear transform: where h denotes the point spread function (PSF). When the PSF does only depend on the difference rs, equation ( 1) is a convolution and the system is said isoplanatic.

g(r) = h(r, s) f (s) ds, (1)

PSF interpolation

Smoothly varying PSF can be approximated by interpolating PSF at locations s i with interpolation kernel ϕ int :

h(r, s) ≈ i h i (r -s) ϕ int (s -s i ), (2)
with h i (u) ≡ h(s i + u, s i ) the PSF for a source located at s i . The image formation model is then approximated by:

g(r) ≈ i h i (r -s) ϕ i (s) f (s) ds ≡ [H•f ](r), (3)
where ϕ i (s) = ϕ int (ss i ) and H denotes the linear operator of our approximate model. This operator and its adjoint (which is needed for image reconstruction) expand as:

H = i H i •W i and H ♯ = i W i •H ♯ i , (4)
with W i a scaling operator corresponding to the pointwise multiplication by ϕ i , H i and H ♯ i a convolution and a correlation by the ith sampled PSF. In words, Eq. ( 3) expresses the degraded image as the sum of convolutions between PSF samples and weighted versions of the crisp image.

An alternative model has been proposed by [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF] who interpolate the result of convolving the original image by the PSF samples. The corresponding operator writes:

H † = i W i •H i .
(5) [START_REF] Hirsch | Efficient filter flow for space-variant multiframe blind deconvolution[END_REF] uses formulation H and [START_REF] Gilad | A fast algorithm for convolution integrals with space and time variant kernels[END_REF] give both. Note that H † is similar to the adjoint H ♯ of our operator (using convolutions instead of correlations); hence the computational burden are the same for both. Though closely related, the operators H and H † are however different in their ability to correctly approximate the shift-variant PSF. Figure 1 gives a monodimensional illustration of the difference between the two. We consider a system with Gaussian PSF whose standard deviation increases linearly with the position from left to right. Several of these Gaussian PSF are drawn in light gray on last row of the figure. The target PSF at the center of the field is drawn in thick blue stroke. This PSF is approximated using PSF samples at locations depicted by red dots: coarse sampling at the top row, and refined sampling at the next rows until continuous sampling for the last but one. A linear interpolation kernel is used. The formulation of H is used for sub-figure 1(a), while H † is used for sub-figure 1(b). Approximation errors are drawn in green, and the relative norm of the error is given at the right of each curve. Several properties of the approximations are noticeable: (i) PSF interpolation (H) preserves PSF symmetry, while interpolation of convolution results (H † ) does not; (ii) when the PSF supports are small compared to the distance between two PSF sample locations, the two approximations are comparable; (iii) H achieves exact interpolation (i.e., PSF approximation error tends to zero when the PSF grid is ever more refined) while H † does not;

(iv) PSF positivity and normalization are preserved by linear interpolation (which is not the case with H † , even when a linear interpolation kernel is used). Based on the difference between the two models, we recommend the use of formulation H rather than H † for smoothly varying blurs. As noted in [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF][START_REF] Gilad | A fast algorithm for convolution integrals with space and time variant kernels[END_REF][START_REF] Hirsch | Efficient filter flow for space-variant multiframe blind deconvolution[END_REF], operators H and H † can be efficiently computed using fast Fourier transforms (FFT). Each term of the sum in Eq. ( 4) is a convolution of a PSF (size H 2 ) with a patch whose size B 2 corresponds to the support of the interpolation kernel (for a grid of K 2 PSF samples, an N 2 image, and linear interpolation, B = 2N/K). Each discrete convolution requires O[(B + H) 2 log(B + H)] operations, thus the whole operator requires O[K 2 (B +H) 2 log(B +H)] operations. If the PSF support H is much smaller than the support of the interpolation kernel, the computational cost is O[4N 2 log N ] operations, i.e., 4 times the cost of a single convolution of the whole image. Table 1 reports the computation time of our parallel implementation of operator H (based on FFTW and Linux pthread libraries, running on an Intel Xeon 3.3GHz with 6 cores). When the PSF support is large compared to the support of the interpolation kernel, most of the computational effort is spent computing the boundaries of each patch and the complexity raises as illustrated by the last rows of table 1.

Improvement of the approximation

There are several options to improve the approximation error: (i) refine the interpolation grid (costly when the PSF support is large); (ii) increase the interpolation order (huge cost since the support of the interpolation kernel is proportional to the interpolation order); (iii) use generalized interpolation, i.e., refine interpolation weights ϕ i (with constant interpolation support) and PSF samples h i to fit a target PSF. Option (iii) is the most interesting since it improves the approximation without increasing the computational cost of H. We achieve this by minimizing the quadratic difference:

nb
ǫ 2 = 1 N 2 h(r, s) - i h i (r -s) ϕ i (s) 2
dr ds [START_REF] Aubailly | Approach for reconstructing anisoplanatic adaptive optics images[END_REF] with respect to the PSF {h i } K 2 i=1 and the weights {ϕ i } K 2 i=1 . Starting with the PSF samples, we alternately solve for the best weights, and then for the best PSF samples. Using this algorithm, the approximation can be largely improved (the rms error ǫ is divided by 4, which is better than when using a 6 × 6 grid with linear interpolation), as illustrated by figure 2.

Application to deconvolution in astronomy

We used the same dataset as in [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF] to illustrate the application of our model on a realistic astronomical case. Contrary to [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF] who used truncated preconditioned conjugate gradients, we performed regularized inversion with an ℓ 1 sparsityinducing prior and a positivity constraint (minimization done with FISTA algorithm [START_REF] Beck | A fast iterative shrinkagethresholding algorithm for linear inverse problems[END_REF]). Figure 3 presents the obtained results: rms error is 3.6% when using 5 × 5 PSF grid, 6.2% when using the mean PSF, to be compared with the 16% error reported in [START_REF] Nagy | Restoring Images Degraded by Spatially Variant Blur[END_REF]. The use of a regularization largely improves the reconstruction. The spatially-variant PSF model further improves the results.

CONCLUSION

We have shown that PSF interpolation is physically more justified and leads to a better approximation of space-variant PSF. The computational cost remains modest (4× that of a single convolution). We proposed to further improve the model accuracy while keeping the same computational cost. We applied our model to a realistic astronomical case. Our next step will be to extend our approach to blind or myopic deconvolution, in wide-field adaptive optics and microscopic imaging.

Fig. 1 .

 1 Fig. 1. Comparison of (a) H and (b) H † for PSF approximation (blue: target PSF, red: PSF sample locations, green: approximation error).

Fig. 2 .

 2 Fig. 2. PSF approximation: (a)-(c) using bilinear interpolation; (d)-(f) generalized interpolation with optimal PSF and weights; (a), (d): PSF grid; (b), (e): interpolation weights on each of the 9 patches; (c), (f): rms error with respect to target PSF.

Fig. 3 .

 3 Fig. 3. Deconvolution with shift-variant PSF: (a) simulation of an observation of a star field with Hubble's Wide Field Camera before corrective optics; (b) provided 5 × 5 grid of PSF; (c) recovered stars after deconvolution with sparsity prior, gray: good detection, blue: non-detected star (4.9%), red: false-detection (7.2%)

Table 1 .

 1 Average time to compute operator H: (a) 512 2 pixels image, with 31 × 31 pixels PSF; (b) 1000 2 pixels image, with 101 × 101 pixels PSF (Intel Xeon Processor with 6 cores).

	of threads	1	2	4	6
	PSF grid	time in ms (relative to a convolution)
	5 × 5	71 (3.7)	37 (1.9)	19 (1)	14 (0.7)
	(a) 10 × 10	46 (2.4)	22 (1.2)	11 (0.6)	8 (0.4)
	20 × 20	60 (3.2)	30 (1.6)	15 (0.8)	11 (0.6)
	5 × 5 182 (4.2)	97 (2.3)	57 (1.3)	47 (1.1)
	(b) 10 × 10 636 (15) 325 (7.6) 170 (4) 117 (2.7)
	20 × 20 1680 (39) 850 (20) 435 (10) 293 (6.8)

This work has been supported by project MiTiV funded by the French National Research Agency (ANR DEFI 09-EMER-008-01).