Micro-X-ray fluorescence image analysis of otoliths to distinguish between wild-born and stocked river-spawning whitefish captured in the Baltic Sea

Strontium concentrations are low in fresh waters compared to seawaters. Therefore, wild-born river-spawning and stocked freshwater-reared whitefish Coregonus lavaretus L. display regions with low concentrations of strontium in the centre of their otoliths. Strontium in otoliths from wild-born river-spawning whitefish ascending the River Tornionjoki, river-spawning whitefish stocked as one-summer-old fingerlings caught ascending the River Kemijoki, and sea-spawning whitefish caught near the angstrom land Islands was mapped using mu-XRF. The strontium-depleted regions in the centre of the whitefish otoliths, measured using ImageJ, had mean sizes of 0.18 +/- 0.2 mm(2) (River Tornionjoki) and 2.3 +/- 0.3 mm(2) (River Kemijoki), whereas the otoliths from whitefish caught at sea lacked a strontium-depleted region altogether. Measurement of the area of the strontium-depleted region in whitefish otoliths provides a convenient method with which to differentiate between whitefish of different provenance and to determine the origins of whitefish in mixed catches

Micro-X-ray fluorescence image analysis of otoliths to distinguish between wild-born and stocked river-spawning whitefish captured in the Baltic Sea

Strontium concentrations are low in fresh waters compared to seawaters. Therefore, wild-born river-spawning and stocked freshwater-reared whitefish Coregonus lavaretus L. display regions with low concentrations of strontium in the centre of their otoliths. Strontium in otoliths from wild-born river-spawning whitefish ascending the River Tornionjoki, river-spawning whitefish stocked as one-summer-old fingerlings caught ascending the River Kemijoki, and sea-spawning whitefish caught near the Åland Islands was mapped using µ-XRF. The strontium-depleted regions in the centre of the whitefish otoliths, measured using ImageJ, had mean sizes of 0.18 ± 0.2 mm2 (River Tornionjoki) and 2.3 ± 0.3 mm2 (River Kemijoki), whereas the otoliths from whitefish caught at sea lacked a strontium-depleted region altogether. Measurement of the area of the strontium-depleted region in whitefish otoliths provides a convenient method with which to differentiate between whitefish of different provenance and to determine the origins of whitefish in mixed catches.</p

Prevalence of stocked whitefish in River Kemijoki, Finland, inferred by micro X-ray fluorescence analysis of otoliths

Micro X-ray fluorescence (mu-XRF) analysis of otoliths was evaluated as a method to estimate the proportion of stocked one-summer-old whitefish Coregonus lavaretus L. in catches of adult fish (n = 20) ascending the River Kemijoki to spawn. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis was applied as control. Polished otoliths were scanned with mu-XRF to obtain strontium maps that were used to infer visually the provenance of the whitefish. Thirteen of the fish showed signs of being stocked as one-summer-old fingerlings. LA-ICP-MS was applied to determine the elemental composition in a spot outside the core of the otolith. The results were largely consistent with the visual inspection of the mu-XRF mapped otoliths. In conclusion, mu-XRF mapping successfully identified whitefish stocked as one-summer-old fingerlings. The vast majority of whitefish returning to the River Kemijoki to spawn were stocked fish

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced

&lt;p&gt;Background: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers.&lt;/p&gt; &lt;p&gt;Methods: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining.&lt;/p&gt; &lt;p&gt;Results: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (&gt; 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.&lt;/p&gt; &lt;p&gt;Conclusions: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.&lt;/p&gt

Estimating the proportion of river and sea spawning whitefish in catches from the brackish Gulf of Bothnia (Baltic Sea) by gill raker counting, genotyping and otolith chemistry analysis

European whitefish (Coregonus lavaretus) is a commercially and recreationally important species in the Gulf of Bothnia (Figure 1), where the salinity of the surface water increases from ~2 ‰ to ~6 ‰ in the north-south (~725 km) direction. Two sympatric ecotypes with similar outer features occur: stationary sea spawning whitefish and migrating anadromous river spawning whitefish (Lehtonen 1981). The two types mix in sea away from breeding locations and off breeding time. River spawning whitefish can undertake long feeding and spawning migrations, e.g. between the north and the south of the gulf. The river spawning whitefish abundantly occur at feeding grounds in the south where they stay for years, until maturing. Mainly due to anthropogenic destruction of the spawning rivers, the river spawning whitefish has diminished during last decades and is presently listed among endangered species (Helcom 2013). A reliable assessment of the two ecotypes and their subpopulations is a prerequisite for taking effective actions for stock preservation. In order to compare methods for whitefish ecotype identification we undertook genotyping, otolith chemistry analysis and gill raker counting on river and sea spawning whitefish populations. Materials and methods. Whitefish to undergo spawning was sampled from rivers along the Finnish west coast (river spawners) and at sea from known spawning sites close to the southern feeding grounds at the Åland Islands and the Archipelago Sea (sea spawners) (Himberg et al. 2015, Figure 1). Gill rakers were counted on the left outer arch. Data on gill raker number was also collected from literature. Genetic diversity was assessed by nine microsatellite markers (Ozerov et al. 2015). Two methods were used for otolith (sagittae) chemistry analysis. Otoliths were dissolved and analyzed for elemental concentrations with ICP-OES (Hägerstrand et al. 2015), or polished to the core and analyzed for core strontium concentration by PIXE analyses (Lill et al. 2015). Results and discussion. The genetic divergence between river and sea spawning whitefish was low (Ozerov et al. 2015), indicating that genotyping is of little use to differentiate between the two ecotypes on its own. However, genotyping may add to differentiation in combination with other methods such as otolith chemistry analysis and gill raker counting. Otolith chemistry identified naturally reproduced river and sea spawning whitefish based on differences in the strontium concentration in the otolith core region (Lill et al. 2015). Furthermore, bulk otolith elemental concentration analysis (Ba, Sr, Zn, Mn, Mg, Fe) mapped whitefish populations to different regions in the Gulf of Bothnia, and indicated migrating populations (Hägerstrand et al. 2015). Here, barium was shown to be a useful freshwater-seawater (salinity) gradient indicator. Gill raker counting showed that the average gill raker number of river and sea spawning whitefish was significantly different (t=22.50, df=934, p<0.0001), i.e. 29.9±2.14 (n=480) and 26.7±2.21 (n=450), respectively (Himberg et al. 2015). Despite the overlapping distributions of gill rakers between the ecotypes, the average gill raker number can be used to approximate the ratios of the ecotypes and the alterations in them. In conclusion, gill raker counting was verified as an easy, fast and inexpensive method to estimate the spatiotemporal proportion of river and sea spawning whitefish at the southern feeding grounds of the Gulf of Bothnia. The gill raker counting method is well suited to aid in a sustainable management of whitefish stocks. The stability of gill raker numbers in whitefish populations (Himberg et al. 2015) is essential when using this character in approximating the proportion of river and sea spawning whitefish. Interestingly, while the mean number of gill rakers in river spawner populations along the Finnish west coast has stayed stably high (normally ~30) at least during a century (Himberg et al. 2015), the mean gill raker number of sea spawners at the southern Åland Islands (Tengsöda, Geta) has decreased from an average of ~27.5 during 1964-1980 (Himberg 1970, Himberg 1978, Lehtonen 1981, Himberg 1995), to below 26 at today (Table 1, Supplemental Data). This decrease is probably the result of stocking during recent decades of sea spawning whitefish from the local hatchery (Guttorp, Åland Islands), and of the initial selection of brood fish for this activity. The low number of gill rakers of local sea spawning whitefish at the Åland Islands aids in differing it from river spawning migrators. Stocking has substantially increased the abundance of whitefish in the Gulf of Bothnia (Leskelä et al. 2009, Jokikokko and Huhmarniemi 2014). As a result of compensatory stocking to preserve river spawner populations and stocking to increase fisheries catches, a vast amount of the river spawning whitefish “type” fish is released every year. The stocked fish is raised in freshwater ponds. The juvenile fish is released into rivers and river estuaries along the west coast of Gulf of Bothnia, and also into the sea e.g. at the feeding grounds in the southern Archipelago Sea. The fish released into the sea to increase fisheries catches is often of the River Kokemäki whitefish origin since this whitefish is fast growing and gets big (Lehtonen 1981). The proportion in the sea of naturally reproduced and stocked river spawning whitefish is unknown as methods to differentiate between them are not well developed. In the accompanying paper in this series we describe a method that can resolve this issue (Hägerstrand et al. 2015, Front. Mar. Sci. doi: 10.3389/conf.FMARS.2015.03.00113). River spawning whitefish raised in fresh-water ponds and stocked in northern rivers or river estuaries maintain their feeding migration instinct and also return to their home river following maturation (Leskelä et al. 2009, Jokikokko et al. 2014). When stocked in the sea they ascend into rivers close to the site of release (Leskelä et al. 2009). Stocked river spawners can also re-establish populations in rivers from where the river spawning whitefish type has disappeared (Chris Karppinen, personal communication). The homing behavior and spawning characteristics of river spawners stocked in the southern Archipelago Sea far from large rivers are not known. Thus, it remains unclear if these fishes interfere with the spawning of local whitefish and alter their genetic properties

Short feeding migration associated with a lower mean size of whitefish in the River Tornionjoki, northern Baltic Sea

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Prevalence of stocked whitefish in River Kemijoki, Finland, inferred by micro X-ray fluorescence analysis of otoliths

Micro X-ray fluorescence (mu-XRF) analysis of otoliths was evaluated as a method to estimate the proportion of stocked one-summer-old whitefish Coregonus lavaretus L. in catches of adult fish (n = 20) ascending the River Kemijoki to spawn. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis was applied as control. Polished otoliths were scanned with mu-XRF to obtain strontium maps that were used to infer visually the provenance of the whitefish. Thirteen of the fish showed signs of being stocked as one-summer-old fingerlings. LA-ICP-MS was applied to determine the elemental composition in a spot outside the core of the otolith. The results were largely consistent with the visual inspection of the mu-XRF mapped otoliths. In conclusion, mu-XRF mapping successfully identified whitefish stocked as one-summer-old fingerlings. The vast majority of whitefish returning to the River Kemijoki to spawn were stocked fish

Proportion of river- and sea-spawning whitefish in catches at the Åland Islands (Baltic Sea), estimated from gill raker counts

The Åland Islands are feeding grounds for river- and sea-spawning whitefish (Coregonus lavaretus), including also hatchery-reared released whitefish. Management of fisheries requires knowledge of whitefish origin from their catches, but such information is deficient at the Åland Islands. The numbers of gill rakers had unique (discretized) Gaussian frequency distributions in all three forms studied. This justified the usage of Gaussian finite mixture models on gill raker data for estimating the proportions of the forms in mixed catches during the non-spawning season. From 14 catches at the Åland Islands from 2012–2013, on average 54.8% were river-spawning whitefish, while the presence of hatchery-reared, sea-spawning whitefish was low but uncertain. Using a hierarchical variant of the model, we showed that the proportion of river-spawning whitefish decreased from 69.6% in June to 38.3% in August. Our study provides feasible methods for monitoring spatio-temporal variation in the proportions of river- and sea-spawning whitefish

Potential of Otolith Microchemistry to Distinguish Nursery Areas of Salmon within River Simojoki

Draining into the northern Baltic Sea, River Simojoki is an important spawning river for Atlantic salmon. The present study aimed to preliminary explore the potential of analysing the elemental composition of otoliths to distinguish the within-river nursery area of origin for salmon. Parr were sampled at three nursery areas in the river and smolts of unknown origin were sampled near the river mouth during the migration to the sea. The concentrations of multiple elements and the strontium 87Sr/86Sr isotopic ratio in the otoliths were analysed using single- and multi-collector laser ablation inductively coupled plasma mass spectrometry, respectively. Based on the otolith elemental variables, parr could be reclassified to the sampled nursery areas using discriminant function analysis and random forest with a success rate of 53.3% and 63.3%, respectively. However, for parr sampled at the uppermost nursery area in the river, the success rates were 90% and 100%, respectively. Applying the classification models trained on the parr samples to determine which nursery area sampled smolt originated from was constrained by the limited sampling of parr, both in sample sizes and the coverage of the nursery areas found in the river