Forest Europe 2015 statistics explored the features, inertia and limits of this expansion, and its relationships with countries’ development, forest management and trade, intense in this area of the world. Persisting footprint of past demographic pressure on forests was identified, with opposed traces on their area and growing stock density. Steady growing stock (GS) increases, proportional to GS, not density-limited, and sustained by forest area increases, supported the view of an inflationary forest dynamic. Economic development and liberalism fostered both forest exploitation and production, yielding no significant impact on GS changes. Wood exports exerted a tension on forest exploitation and GS changes, thus lowering GS inflation but providing a resource security margin in the face of expected climate threats. Conflicting a common view, GS inflation and moderate felling-to-increment ratios make increased use of wood resources and C sequestration reconcilable, and GS expansion timely for ongoing EU forest policy processes. Anticipated adverse impacts of ongoing climate change were not clearly identified in these statistics.The current increase in European forest resources forms a singularity across the globe. Whether this trend will persist, and how biological and economic trends feature it form crucial issues to green economy challenges and C sequestration. The present screening of  Forest Principles of the Rio conference, [3 of accumulated wood , and are therefore denser than the world average. In the European agenda, climate change mitigation [Forest Europe since 1990 and increasingly served by national forest inventory programs . Europeference, , 3) withtigation  and bio-tigation . Debatestigation , adoptedms  be identified that would sustain increases in wood availability? Despite debatable methods, recently reported increases in harvested forest areas in Europe [Well-established from global forest reporting, wood consumption is also stimulated by economic development, with major wood consumers being those among most developed countries . Global lopment . Standing living wood volume in forests. Terminology used to qualify the ability of living trees to expand in size.Growing stock density . Spatial density of the growing stock relative to total forest area . “Forest density” as a terminology also used in the Forest Identity metrics . The indicator in 2010 was considered.Annual rate of GS change (%.year-1). Difference in GS at two dates expressed as a fraction of initial GS and annualized. Analysis of rate of GS change 2005-2015 central to this study. “Inflation” is used as a terminology to qualify a strong and continuous increase in GS, mirroring standard uses in economics and cosmology.Felling and net increment rates (%.year-1). Volume of felling and net increment  expressed as a fraction of initial GS and annualized. To comply with GS change definition, indicators in 2010 were considered.Felling-to-net-increment ratio . Conventional forest ratio that measures felling relative to forest production. This indicator should not strongly exceed 100% over the long run. Value of indicator in 2010.Acceleration in GS change (%.year-2). Quantifies the temporal change in the rate of GS change. Computed as a difference of annual rates of GS change between periods 2005-2015 and 1990-2005.Gross domestic product per capita . Standard economic indicator of economic welfare that measures the gross value added (GVA) corrected from subsidies (-) and taxes (+). Value of indicator in 2013.Property Rights Index in Forestry . Novel indicator of freedom of action in the forestry sector based on property rights assessment, aggregating access, withdrawal, management, exclusion and alienation rights. The indicator is exogenous to Forest Europe reporting, and was extracted from Nichiforel et al.  with value in 2015. It is documented for 30 out of 39 countries under study.Contributions of the primary and full forest sector to GVA (%). Economic sectors as defined by UN/ISIC . With respect to the primary forest sector (management and silviculture), the “full” forest sector also encompasses i) the paper and pulp industry, ii) the industrial wood transformation sector. Values of indicators in 2010.Annual import and export rates (%). Annual volumes of wood exports and imports expressed as a fraction of countries’ growing stock. Forest Europe data not distinguishing exports/imports inner/outer Europe. Imports and exports are expressed on a same scale as felling and net increment rates. Values of indicators in 2010.Forest identity. A conceptual quantitative and 4-dimensional approach to forest description developed by Kauppi et al. (2006) and including forest area, forest volume of growing stock, biomass and carbon as descriptors, and translated from one another using forest density (m3/ha), allometric biomass ratio (ton/m3) and C concentration.Malthusian, Boserupian. Qualifies trends in demographic/economic development/growth that rely on resources distributed across space. Malthusian refers to the Thomas Malthus’ view  that agricultural resource availability constrains human development whose growth was viewed as exponential. Boserupian refers to Esther Boserup’ opposed view  that human development will on the contrary foster agricultural resource securing through intensification and technological progress. Concepts have been translated into forest resource analysis by Mather et al. (1999).Green economy strategies advocating resource use intensification are hence recurrently pointed out as conflicting the in situ forest C sequestration option [The emergency of climate change last emphasizes the effective role of forests in sequestrating carbon . Green en option . With EuForest Europe’s regular reporting on forests has been providing an invaluable source of information, with issue 2015 of this report [NFI) programs  increasing the reliability of statistics dedicated to forest status and dynamic. These statistics however remain less accurate in former socialist countries of Eastern Europe, where NFI programs are recent or inexistant, and reporting data largely stem from forest management plans ([S1 Table). In this investigation of the report, the aims were i) to highlight the salient features of current forest dynamic in Europe and track biological limitations of relevance to the inference of future trends (section 2), ii) to explore the role of economic development and trade on growing stock dynamic, and options for the balance of substitution and C sequestration functions of forests (section 3).Since 1990, t plans , attesting a persistent footprint of demographic pressure in the present extension of forests, after decades, sometimes centuries, of reforestation [−4, [GSD, m3.ha-1, Box 1,  stronger with rural population density, +0.64, p < 10−4) suggesting that the amount of wood resources per unit area was related to population concentration. While a Malthusian, or demography-driven forest depletion logic (Box 1) has conditioned early trajectories of the European forest area [Boserupian logic instead [identity of European forests [Box 1) here unmasks an original broad apparent trade-off between the spatial density of forests and their density in wood resources . These effects did not offset each other in countries’ GS, as evidenced by the positive correlation also found between forest area and GS across European countries . Yet, this relationship was found saturating (quadratic) rather than linear .A negative relationship was found between population density and the forest cover fraction across countries . By conest area , 28, a B instead  would th forests , Box 1) -1 . Though much less popularized . This ongoing increase in forest area, through the process of tree development, already confirmed a positive component to GS changes in European forests for the future.Rates of changes in the forest area of European countries over 2005–2015 were found predominantly positive and ranged between –0.3 to +3.2%.yeard (e. g. ), rates Fig 2B) and showed a comparable magnitude (+1.54%.year-1 on average over 1990–2005). GS changes therefore demonstrated a substantial inertia, together with a stationary pace.Rates of GS change compared across the two reporting periods were found positively correlated  with initial GS , without any clear deviation from a proportional assumption. This suggested that large-scale GS changes remain positively conditioned by GS capital.Last, absolute GS changes over 2005–2015 were found strongly correlated , portraFig 2), causal limitations, either endogenous or exogenous to forests, were also investigated.While GS accumulation in European forests was evidenced  effect on biomass accumulation was first scrutinized. This relationship also termed capital-production or density-biomass relationship [3.ha-1, Fig 3A) with a modest average 148 m3.ha-1 across Europe. Only Germany, Switzerland and Austria exceeded 300 m3.ha-1. Relationships between GSD (2005) and rates of GS change (2005–2015), either expressed per unit of volume  or area  showed either null (NS) or even weakly positive  relationships, respectively. Though lower GS changes in countries of greater GSD were detected, this absence of a density limitation on GS changes was consistent with moderate GSD encountered across most countries, thus not prone to stop GS accumulation in a near future.As a biological limitation, growing stock density . A large FIR gradient was encountered  and demonstrated a negative association with GS changes , approaching null GS changes with a FIR close to 100%. Of significance, current levels of FIR thus indicated that European forests are able to support increased fellings while maintaining a positive though reduced in situ forest GS and therefore C sink. This reconciles overly opposed C substitution/sequestration objectives [Management-driven limitations were inquired using the felling-to-net increment rates ratio , we third tracked a possible deceleration in GS changes across periods, using data from period 1990–2005 compared to 2005–2015. Deceleration was measured by the between-period difference of rates of GS change across countries. The mean of this difference was negative (–0.3%.year-1) and insignificant (p = 0.8), yet masking a large continental gradient . Also, the greater the GS changes over 1990–2005 and the greater the deceleration between periods , whereby acceleration decreased by –0.77% per % annual rate of GS change , and was found systematically negative for GS changes beyond +2%.year-1. Previous GS change and FIR in 2010 together formed significant predictors of GS acceleration , the former being of much greater significance . Possible causes of this apparent deceleration include: i) a negative bias in GS change when switching to statistical inventory methods , ii) a pattern of regression to the mean, after extreme changes in GS over the 1990–2005 period, as often found in observational studies  were not contradictory to both the method- and CC-based hypotheses. Alltogether, these findings supported the conclusion that GS dynamic in European forests over 2005–2015 has not been statistically preempted by any density, management, or substantial climate limitation.In view of the strong and locally impressive pace of GS changes over 2005–2015 , iii) c, iv) or was first considered in view of its general significance [Box 1) on GDPc was found positive and very significant . GDPc did not show any correlation with the extent of forest resources, area or GS, ruling out size effects in this relationship. An order of magnitude in the range GDPc across Europe (95% confidence range between 5.3 k€ and 50.5 k€.GDPc-1) hence yielded an increase from +1.3% to +3.1% in the annual felling rate . With GDPc beyond 30 k€.year-1, countries including e.g. Sweden, Finland, Germany, or Ireland both showed felling rates above 2.5%.year-1, while countries with a GDPc around 15 k€.year-1 such as e.g. Turkey, Poland, Ukraine and Romania (>7 Mha forests) showed felling rates below 0.9%.year-1. While illegal logging in former Eastern socialist countries may bias felling statistics downward [Fig 4A) suggested greater pressure on forest resources with increasing countries’ richness, even among more advanced economies.Gross domestic product per capita . The same GDPc gradient yielded an increase from +0.25 to +2.2% in the annual net increment rate , and contrasted between Ireland and Denmark (>5%), Finland, Germany (>3%) and Sweden (>2.5%) on the one hand, and Turkey, Romania, Poland or Ukraine (<2.5%) on the other. This again suggested a Boserupian trend (Box 1) in increasing forest productivity to secure wood resources and meet the economic demand. Both trends however did not counterbalance, and resulted in a weak positive trend in FIR against GDPc , with countries >30 k€ GDPc such as Sweden, Austria and Germany showing FIR >90%, and countries below 25 k€ in GDPc with large forest areas showing FIR <60% . Setting Albania aside , ruling out any broad threat of economic development on wood resources.Mirroring this trend, a much less expected positive dependence of the annual net volume increment rate (2010) on GDPc was also identified, though less intense , FIR raBox 1 for definitions of GVA and forest sectors) were logically found associated with greater forest exploitation , these two indicators did not correlate with GDP , discarding the hypothesis that value added in the forest sector may explain previous relationships of forest intensification with GDPc.Whereas greater contributions to the gross value added–or GVA–of the primary forestry sector and of total sector , to an extent where substituting private ownership fraction to GDPc in previous analyses  yielded comparable outcomes . Relationships with FIR/GS rate of change 2005–2015 were again found in limit of significance /not significant , respectively.In addition, the areal fraction of private forest ownership was found strongly correlated with GDPc  as an integrated measure of freedom in decision making across five right categories ([−7).We therefore hypothesised this association between GDPc and private forest ownership fraction to footprint the existing positive association between economic growth and property rights , 41. Rely  egories  and to a lower extent with GDPc . Again, both the felling and net increment rates were positively associated to this index . Also, GS rate of change 2005–2015 was more negatively associated to PRIF index . The degree of freedom of action–so called liberalism–in forestry was thus found associated to lower GS accumulation in European countries.Remarkably, PRIF index strongly correlated with the fraction of private forest ownership , these were shown to be positively associated with GSD in 2015 . FIR/rate of GS change revealed weak yet confirmatory correlations with the areal fraction of production management plans  both strongly correlated with each other and produced identical patterns of associations with forest dynamic indicators, making them indistinguishable. FIR further remained in wise limits (< 1) across European countries. Adverse effects of development on GS dynamics were therefore not evidenced. Deciphering the apparent virtuous role of forest management plans on GSD and GS dynamic will require more comprehensive delivery of this indicator across countries.Forest Europe reporting arise from the Joint Forest Sector Questionnaire of the UNECE/FAO protocol [Trade data of protocol  and haveprotocol . These sprotocol .While wood imports primarily intend to meet inner demands not satisfied by countries’ forests, and in this case without any prior influence on the felling rate or GS dynamic (debatable approach of ), they mBox 1) were correlated to forest dynamic attributes. Relationships between import and felling rates or GS changes 2005–2015 were found inexistent, ruling out strong protective options among countries. By contrast, a large gradient of export rates  generated a remarkable set of correlations  confirming that part of GS inflation in European countries is readily exported. Of note, net increment rates again increased with greater export rates , stressing the responsiveness of the forest sector among countries, in a context of absence of erosion in GS . Export rates were found greatest in traditional forest countries  and whereby intensive forestry is implemented . In this set of countries, GS changes 2005–2015 were significantly lower than on average (+0.28%/+1.57%). That the total annual volume of exports (475 million m3) roughly matched the annual increase in GS 2005–15 (422 million m3) supports the existence of an ample security margin on wood resource availability in Europe.Volumes of imports/exports of wood and derived products expressed as roundwood equivalent fractions of countries’ GS . That a clear negative relationship between forest cover fraction and population density remains apparent in today’s statistics  attests of the irreversible impact of human expansion on forests. In this context, the pressure towards land sparing for nature recovery [The long-lasting pressure of human demography on forests, and its release following industrial/urban development and rural land abandonment, have been early emphasized in studies of forest transitions , 27, vierecovery  and the recovery , 56, nowrecovery  are not recovery , 59 formFig 1B) and the apparent area-GSD trade-off  remain far less expected, and recall the need for more inclusive descriptions of forests (the forest identity of [Box 1). While one cannot exclude that greater densities in forests of the transitional minimum were favoured by historical protection measures, the 200 m3.ha-1 gradient in GSD observed across countries of the continent suggests that active planning efforts were also implemented to restore timber resources, and within restricted time periods after forest transitions. This is in accordance with Grainger’s [S4 Fig). The predominantly even-aged structure of European forests (70% of area) and its fair distribution across developmental stages  lasts suggest a constant effort at renewing forests after final harvest [In view of this demographic footprint, the positive relationship evidenced between population densities and forest GSD .ainger’s  distinctainger’s . Associa harvest .Stock increase causally requests either the addition of new forests with ongoing tree recruitment, or a positive balance between felling and increment of pre-existing forests, resulting from either restricted wood utilization or increase in forest productivity, with possibly endogenic dynamic or environmental causes. While a full quantitative appraisal of each component’s contribution to GS changes remains a scientific challenge , the preFig 2A), and was more than double in magnitude. GSD has also greatly increased in Europe over recent decades . Such patterns would not be observed early after forest transition, where the contribution of areal extension of forests to GS is minor if any , and shFigs 3 and 4), the contribution of restricted current wood felling levels to GS increases makes no doubt, and conflicts recent views [S4 Fig). Average statistics however hide strong within-country heterogeneities [With an average FIR of 0.65 in 2010 and below unity in almost all countries  in line with an increasing growing stock, the difference in the net increment rate (Box 1) was also found reversed and significant , suggesting a decreasing return of the growing stock. More in-depth scrutiny of this decrease and of whether it will last is thus requested.Environmental enhancement of the growing stock  and more forests , atmosph alleged , therefo alleged . Convers alleged , and may alleged . ConsistFig 2C) and the lack of evidence for density-dependent limitations . The recent analysis of forest dynamic in tall natural forests of New-Zealand [3/ha, and in a range of 300–500 m3/ha at the extremes (S3 Table). These values remain far above the current average GSD in Europe (192 m3.ha-1 in 2015) where only German, Swiss and Austrian forests exceed 300 m3.ha-1. GS change inertia relative to the 1990–2005 time period , where no substantial climate change damage onto forests has been reported, also supports a long-term self-sustained dynamic. While not significant, GS deceleration was negatively associated with GS changes over 1990–2005  and formed a discrete sign, yet not fully elucidated (see 1.3), of a future deflation. Budget-oriented analyses of GS form an appropriate way to quantify endogenous and environmental causes of growth increases. In the single published attempt in this respect to date, Henttonen et al. [Forest dynamics on its own is also able to cause such substantial GS increases, including the increase of forest increment with increasing GS in the absence of density-dependent limitations. This obviously occurs in European forests, as supported by the proportional relationship between GS changes and GS  amount to 310 million m3, i.e. 0.88% of the total GS. A comparable order of magnitude may be reached regarding bark beetle damages in Europe . With undeniable but not yet substantial climatic impact, and a strong inner dynamic, GS increases may therefore slow down but surely persist in the near decades. The tension exerted by wood exports on countries’ forests  attests of a resource security margin for currently exporter countries in the face of future climate threat, albeit export reduction would be prejudicial to international exchanges.With increased confidence in the attribution of severe weather events–droughts, heatwaves and storms–to climatic change , climati, a global-scale association [S2 and ), but non-significantly with decreased GS changes (p < 0.04 with PRIF index). The more causal and direct significance of private forest ownership fraction and PRIF index than GDPc to forest activities however leads us to favour the hypothesis of liberalism influence in the highlighted patterns of forest dynamic. Export rates, not imports, were found associated with attributes of forest dynamic, and positively associated with increased felling rate and decreased growing stock , in a very similar way as with previous indicators. While these fluxes were not distinguished between inner/outer Europe in the European reporting, they indicated that part of GS inflation in exporter countries is not capitalized in the forest. The accuracy of trade data has been inquired and has improved over years [Boserupian perspective on securing resource availability through innovation and technology [S2 and ), not highlighted in Kauppi et al. [Fig 4). The European agenda for more intensive use of renewable resources [in situ forest C sequestration persistence strategies , suggesting that the issue has a somewhat ideologic dimension. Continued increasing GS also implies greater wood availability at constant felling rates [GDPc, the share of private forest ownership, and PRIF as an indicator of freedom of action in forestry were found undistinguishable ociation  interpreociation . Recent ociation . Yet, puociation . Both prer years . Kastnerer years  have draer years  and Foreer years , but alser years . This cochnology , 89. Howi et al. . On the i et al. . On a gli et al. , stressii et al. ) in lineFig , a globaesources  and otheesources , and theng rates . Yet, anAlong with increasing climatic pressure on forests, current statistics–while supporting the inflationary perspective–suggest that the history of the forest transition and environmental Kuznet curves, figuring out impacts of human development on the environment , 92 has 3/ha) that does not biologically limit volume expansion. Despite there exist strong evidences for a stimulating influence of past environmental changes on forest growth, the time horizon of forest reporting prevented its direct scrutiny.Increasing forest GS in Europe is multicausal and results from sustained increases in forest area after the forest transitions, from wood felling rates that remain below increment rates (FIR < 1), and from a still moderate capitalization of forests  indicators, whereby increased felling rates also come along with increased increment rates. The degree of richness and liberalism is therefore not identified as a threat on forest resources.Increased exports nevertheless yield significantly lower GS increases. This exported fraction of GS expansion reveals a security margin in the face of the growing climatic threat posed on forests. Over the studied period, a slightly lower net increment rate in 2010 than in 2005, and a GS change deceleration between 1990–2005 and 2005–2015 formed the single and still discrete candidate indices of increasing environmental stress.Forest Europe reporting, in spite of their large assumed heterogeneities across countries and indicators.Remarkably, all relationships identified were found unambiguous and consistent between each other, and stress the overall remarkable quality of data assembled in Forest Europe reporting encompasses the 46 countries of Europe. Forest area is strongly variable across these countries, encompassing a gradient of several orders of magnitude. Forests of the Russian federation that extend up to nearly two orders of magnitude the importance of most significant forest resources in Western Europe were discarded from the analysis, in view of the awkward stationarity of official statistics in both the forest area and growing stock , and the intrinsic difficulties of a federal reporting both homogeneous across space and constant over time [S1 Table.The ver time . Also, t. While NFI programs covered the full reporting period 1990–2015 in only 11 countries under scrutiny, the later period 2005–2015 was covered in 13 additional ones. Consequently, 54%/72% of the European forest area were covered by NFI programs by 1990/2005, respectively . Countries’ forested area  and economic richness  appeared to be fundamental determinants of early implementation of NFI programs . The effects of NFI forest coverage (S1 Table) on areal and GS rates of changes at the two reporting periods were screened systematically and did not reveal any significant bias. Yet, lower annual rates of change in GS 2005–15 were observed with an NFI program , more acute with NFI implemented prior to 1990 . Also areal changes were found smaller in presence of NFI  as a likely consequence of implementing a forest definition [NFI programs are based on fundamental principles of statistical sampling and inference and constitute the most achieved form of forest resource inventory . After efinition . Owing tFig 2B) and GS change deceleration . Some indicators published in Forest Europe 2015 [The reporting period 2005–2015 was therefore privileged in view of the restricted forest cover ensured by NFI programs over 1990–2005  and the biases highlighted. For some countries, reporting data on forest fluxes could not result from NFI programs alone, as either launched late after 2005 (e.g. UK) or attesting of only one running NFI cycle (e.g. Greece). The period 1990–2005 was however included in the investigation of GS change inertia (property rights index in forestry (PRIF) indicator is a measure of freedom of action in forestry, designed by Nichiforel et al. [The country-wise l et al. , and updl et al. . The indhttps://www.R-project.org/). A preliminary and systematic matrix correlation analysis (Pearson’s correlations and associated test) was performed on all quantitative indicators published in Forest Europe 2015 and systematically to fairly delivered by European countries. The correlation matrix (Pearson correlations) is provided in S2 Table. This process was intended to ensure inclusiveness of the analysis and detect unanticipated relationships, meaningful to the issue of current forest inflation. Weighted correlations and weighted regression analyses were systematically computed, with countries’ forest area in 2015 as the weighting vector across countries most able to fulfil the requirements of the international UN/FAO forest definition owing to the increased implementation of NFI programs across Europe [weights package of R. Weighted regressions and associated t-tests and R2 were computed with the standard lm function of R. P-values delivered all along the manuscript refer to these tests. Those associated to regression model comparison (GS change deceleration against FIR 2010 and GS change 1990–2005) result from nested F-tests. Significance was assessed at a 0.01 level. Graphical representations were computed with function symbols of R, allowing dot sizing with forest area . For some relationships , weighting was unnecessary as soon as the x-indicator was a measure of countries’ forest extension.All analyses and figures were performed and drawn with R software version 3.5.1 and packages (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL s Europe . WeighteS1 Fig(a) Fraction of the European forest area covered by a national statistical forest inventory program in 1990 and 2005, (b) influence of countries’ richness and importance of forest area on their existence.(JPEG)Click here for additional data file.S2 Fig−4), (b) +0.55 (p < 10−3), (c) +0.36 (p < 0.05), -0.24 (p = 0.16).Relationships between private ownership rate (% forest area) and felling (a) and net increment (b) rates, felling-to-net-increment  and resulting changes in the growing stock over 2005–2015 (d) across 39 European countries under study. Private ownership rate in 2010. Albania was set aside (see Methods) Weighted correlations (forest area in 2015): (a) +0.60 (p < 10(JPEG)Click here for additional data file.S3 Fig−4), (c) +0.04 (NS), -0.37 (p = 0.04).Relationships between the Property Rights Index for Forests  and felling and net increment rates , felling-to-net-increment  and resulting changes in the growing stock over 2005–2015 (d) across a subset of 30 out of 39 European countries under study. PRIF index in 2015, as defined in Nichiforel et al. 2018, and updated for 33 European countries in Nichiforel et al. 2020. Private ownership rate in 2010. PRIF in countries including Turkey, Ukraine, Belarus, Georgia, Montenegro, Albania, Moldova, Cyprus and Luxembourg was not documented. Weighted correlations (forest area in 2015): (a) +0.53 (p < 0.001), (b) +0.69 (p < 10(JPEG)Click here for additional data file.S4 Fig3/ha), (b) felling-to-net increment ratio (%), and (c) growing stock change over 2005–2015 for 16 countries reporting on the indicator. FIR in 2010. GSD in 2015. Weighted correlations (forest area 2015) are (a) +0.71 (p < 0.01), (b) -0.17 (NS), (c) +0.43 (< 0.1). 12/16 countries with NFI programs covering the reporting period 2005–2015, 10/16 as EU members, 11/16 located in Eastern Europe: Ukraine, Belarus, Turkey, Bulgaria, Poland, Hungary, Croatia, Slovakia, Slovenia, Latvia and Estonia.Relationship between forest area subjected to a production forest management plan (% forest area) and (a) growing stock per hectare Click here for additional data file.S1 Tablea Data from Forest Europe 2015, b 1: 1990–2015, 2: 2005–2015, c C1 = 1st cycle of inventory, C2 = 2nd cycle of inventory. Reporting coverage by NFI statistics assumes that results of a previous NFI cycle were available prior to the starting year of a reporting period. d Information were obtained from Tomppo et al. 2010  and Vidal et al. 2016  syntheses of European COST actions dedicated to national forest inventories. Links to additional resources are indicated when found.See paper for country selection. (DOCX)Click here for additional data file.S2 Tablet-test <0.01) or green (level of significance <0.05). The test statistics of a Pearson correlation is given by t = r2/(1-r2)1/2 (n-2)1/2 where r is the correlation and n the number of sampling units , yielding: r = t / (t2 + n– 2)1/2. Associated correlations thresholds: |r| > 0.32 (p < 0.05) and |r| > 0.41 (p < 0.01). Indicators: A_country = country area (km2), EU28/NFI: binary variables indicating EU members and implementation of an NFI program, PopDens/RurPopDens = population density/rural population density (hab/km2), GDP = annual gross domestic product (€/capita), %Priv, %MgtPlan, %ProdMgtPlan = share of country forest area private/under a management plan/under a production management plan (%), A = forest area (km2), %AFor = country afforestation rate (%), GS = country forest growing stock (m3), GSD = country forest growing stock density (m3/ha), NI = forest net increment (m3), Fell = felling volume (m3), FIR = felling-to-net-increment ratio (%), %NI, %Fell = expressed as a percentage of total previous GS, ChgA/ChgGS = absolute changes in A/GS, %ChgA/%ChgGS = changes in A/GS expressed as a percentage of total previous GS, ChgGSha = change in GS expressed per unit of previous forest area. AccGS %/ha = acceleration in GS changes, computed as the difference between annual changes in GS over the two study periods 1990–2005 and 2005–15. Notations _YY/_YYYY refer to the calendar year (state variable) or calendar period (flux).Significant correlations are highlighted in grey , in red (level of significance of correlation (DOCX)Click here for additional data file.S3 TableForest Ecosystems (2021): 8:34. These forests are broadleaved with a Fagus genus as a dominant tree species, thus a wood specific gravity of 600 kg/m3 was used for biomass to volume conversion, using Fagus sylvatica data from Kerfriden B. et al., 2021, Plant Ecology 222:289–303, DOI: 10.1007/s11258-020-01106-0. The ratio of total aerial tree volume to total stem volume has been estimated at 1.27 in broadleaved semi-natural forests of France including 49 tree species and it was used for stem volume conversion using Saint-André L. et al., 2010, in Loustau D. (ed) Forests, carbon cycle and climate change, Quae ed, Paris, p79, Table 4.1. Last, conversion of total C stock  to aboveground C stock in Paul et al. arise from the constant 0.6 fraction established from this reference, Aboveground forest carbon stocks stem from Paul T., Kimberley M.O., Beets P. N. Natural forests in New Zealand–a large terrestrial carbon pool in a national state of equilibrium. (DOCX)Click here for additional data file.


Grapsus albolineatus. It is a typical metazoan mitochondrial genome. The total size is 15,583 bp, contains the entire set of 37 genes, and has an AT-rich region. Then, 23 of the 37 genes were encoded by the heavy (+) strand while 14 are encoded by the light (−) strand. Compared with the pan-crustacean ground pattern, two tRNA genes (tRNA-His and tRNA-Gln) were rearranged and the tandem duplication/random loss model was used to explain the observed gene rearrangements. The phylogenetic results showed that all Grapsidae crabs clustered together as a group. Furthermore, the monophyly of each family was well supported, with the exception of Menippidae. In general, the results obtained in this study will contribute to the better understanding of gene rearrangements in Grapsidae crab mitogenomes and provide new insights into the phylogeny of Brachyura.Complete mitochondrial genomes (mitogenomes) can provide useful information for phylogenetic relationships, gene rearrangement, and evolutionary traits. In this study, we determined the complete mitochondrial DNA sequence of the herbivorous crab  However, the phylogenetic relationships among members of Brachyura and their evolutionary origin continue to be controversial due to the high morphological similarity and ecological diversity4. Initially, Brachyura was divided into Podotremata, Heterotremata, and Thoracotremata5. Subsequently, it was segmented into Dromiacea and Eubrachyura 6. However, the latest classification scheme divides Brachyura into Cyclodorippoida, Eubrachyura, Dromicea, and Raninoida8. Although the phylogenetic relationship within Brachyura is still uncertain, the current classification system has been recognized by most scholars.Brachyura crab is the largest clade in the Decapod crustacean group, with more than 7250 known species, including 98 families of marine, freshwater, and terrestrial habitats, most of which are economically importanthttp://www.marinespecies.org/), the family Grapsidae has 8 genera and 49 species in total. However, only five species sequences of Grapsidae have been published12. The herbivorous crab  is one of the marine crustaceans that live on rocky shores which belongs to the phylum Arthropod, subphylum Crustacea, order Decapoda, infraorder Brachyura, clade Thoracotremata, family Grapsidae, genus Grapsus. They are mainly distributed in Japan, Hawaii, Australia and China’s Guangdong, Hainan Island, Xisha Islands, Taiwan. So far, most studies of this species have focused on the morphology and growth14. Although there are few studies on the molecular level, most of them were based on partial mitochondrial and nuclear ribosomal RNA gene sequences15.According to WoRMS , 2 ribosomal RNA genes (rrnl and rrns), 22 transport RNA genes (tRNAs), and an AT-rich region  which contains some initiation sites for transcription and replication of the genome16. Mitochondrial DNA forms a separate unit of genetic information that evolved independently from the nuclear genome. Due to its haploid properties, matrilineal inheritance, limited recombination, and rapid rate of evolution17, the mitogenome is increasingly being used in evolutionary and phylogenetic studies. With the rapid development of sequencing technology, next-generation sequencing has become a fast and low-cost method to provide complete mitotic genomes18.The mitochondrial genome (mitogenome) of metazoans is usually 14–20 kb in size and encoded with a set of 37 genes, including 13 protein coding genes  model are more commonly accepted. Recombination models are involved in the breaking and reconnecting of DNA strands21. The TDRL model assumes that the rearranged gene order occurs via tandem duplications followed by random deletion of certain duplications22. This model has been widely used to explain the translocation of genes encoded on the same strand23. Model tRNA mis-priming model and the tandem duplication/non-random loss model (TDNL) are less commonly used.Gene rearrangements in the mitogenomes of crabs are relatively commonG. albolineatus and used existing complete mitogenomes to compare it with other Brachyura species. In addition, a phylogenetic analysis of 70 brachyuran species was conducted based on the nucleotide sequences of 13 PCGs (Protein-coding gene). These results will help us to understand features of the G. albolineatus mitogenome and the evolutionary relationships within Brachyura.In this study, we successfully sequenced the complete mitogenome of G. albolineatus is a typical closed-circular molecule of 15,583 bp in size (GenBank accession number MZ262276), which is similar in length to the published Grapsidae mitogenomes12, a size range from 15,406 to 15,920 bp , as well as a brief non-coding region, all the genes were identified , 8 tRNAs , and 2 rRNA which are located on the light (L-) strand , trnL1 (25 bp), and cox2/trnS2 (20 bp)) and the other 10 shorter than 10 bp (nad4 (7 bp), atp8 (4 bp), cox3/atp6/rnK/nad6/trnW (1 bp), trnG (3 bp), and nad3/nad2 (2 bp)) , T (34.04%), G (12.02%), and C (20.54%), with a high A–T bias. The A + T (%) content of the mitogenomes was 66.74%. The AT-skew and GC-skew value are calculated for the chosen complete mitogenomes , 3 cytochrome c-oxidases (cox1–cox3), two ATPases , and cytochrome b (cob). The total length of the 13 PCGs is 11,323 bp. The length of the 13 PCGs range from 303 to 1371 bp  use TAG as the stop condon , UCU (Ser2), and GUA , whereas GCU (Ala) is the least common codon. Based on CDspT and RSCU, comparative analyses showed that the codon usage pattern of G. albolineatus is conserved. The codon usage patterns of 13 PCGs are similar to those of other Grapsidae species.The average A + T content is 65.26%, ranging from 39.63% (ND5) to 74.21% (ATP8)  and the rest are encoded by the light strain (L-). In the whole mitogenome, the size of tRNAs range from 50 to 73 bp and have a total length of 1402 bp, with an obvious AT bias (71.54%) . The AT-skew and GC-skew are 0.173 and −0.203, respectively  than the critical values . The genes are not saturated, so the reconstructed phylogeny was reliable.The 12S and 16S rRNA genes are 1331 and 827 bp, respectively, which are typically separated by 26. A large number of studies and results have shown that gene rearrangements in metazoan mitochondrial genomes are conserved20 and the occurrence of gene rearrangements is relatively random and rare27. However, it can be used as direct evidence of evolutionary relationships between species28. Mapping the gene layout based on the complete mitochondrial sequences of 70 species. Through comparison and analysis with the ancestor of Decapoda  had the same gene rearrangement, which are consistent with the ancestral of Brachyura  and Macrophthalmidae (Ocypodoidea) have the same high level rearrangementa  tree and Bayesian Inference (BI) tree) resulted in identical topological structuring with different supporting value. Then, only one topology (ML) with both support values was presented displayed  + Metapograpsus). It is obvious that G. albolineatus had the closest relationship with G. tenuicrustatus, and that these two species form a sister clade with high support values , constituting a Grapsus group. However, recent molecular studies, including our dataset, have not reached an agreement about closest relatives in Grapsidae. Our phylogenetic tree showed that Grapsidae and Dotillidae form a sister clade, which was in concordance with Wang et al.10. While Wang et al. and Ng, N. K. et al. found that Grapsidae do not have any close relatives35, Li et al.36 found that Grapsidae and Ocypodidae form a sister clade.In the present study, the phylogenetic relationships were analyzed based on the sequences of the 13 PCGs to clarify the relationships in Brachyura. yed Fig. . Both tr37. It showed that Grapsidea was divided into three clades (((Seasamidae + Gecarcinidae + Xengrapsidae) + Grapsidae) + Varunidae), Ocypodoidea was divided in three clades ((Ocypodidae + Dotillidae) + Macrophthalmidae + Mictyrisae) and Eriphioidea was divided into two clades (Oziidae + Menippidae). Within Thoracotremata, the superfamilies Ocypodoidea and Grapsoidea supported paraphletic and 9 families showed the following relayionship: ((((Seasamidae + Gecarcinidae) + Xengrapsidae) + Ocypodidae) + (Grapsidae + Dotillidae) +  + Mictyrisae)  Fig. .33. However, previous researchers revealed that Macrophthalmidae and Varunidae were grouped into one clade, then into another clade with Varunidae  + Mictyridae)39, which was conflict with our results. The classification of Grapsoidea and Ocypodoidea has long been controversial. Previous studies based on morphological characteristics considered them to be monophyletic branches. However, an increasing number of molecular studies, including ours, challenge the inconsistent views on the traditional classification system that are put forward. Although the polyphyly of Grapsoidea, Ocypodoidea, and Eriphioidea is well supported, the phylogenetic relationships of these superfamilies need to be further analyzed by integrating additional molecular data36. Previous studies on mitochondrial phylogeny have confirmed the importance of mitochondrial genomic data in elucidating the Grapsidae phylogeny19. On the contrary, many families contained only one representative, which may produce unstable phylogenetic relationships. Therefore, it is necessary to perform further mitogenome sequence studies to obtain a more comprehensive taxon sampling and understand the phylogeny and evolution of Grapsidae.The main phylogenetic structure of our tree is consistent with previous results, but some controversial findings were observed. Here, the families Macrophthalmidae and Varunidae were grouped into one clade, and Mictyridae as basal group which supports the previous findings revealed in Wang et al. and Zhang et al.G. albolineatus was collected from Yangjiang, Guangdong Province, China . The specimen was immediately preserved in absolute ethanol after collection and then stored at −20 °C. This specimen was identified by morphology and fresh tissues were dissected from the operculum and preserved in absolute ethanol before DNA extraction. The total genomic DNA was extracted using the salt-extraction procedure with a slight modification40 and stored at −20 °C.A specimen of G. albolineatus was sequenced by Origin gene Co. Ltd., Shanghai, China and was sequenced on the Illumina HiSeq X Ten platform. HiSeq X Ten libraries with an insert size of 300–500 bp were generated from the genomic DNA. About 10 Gb of raw data was generated for each library. Low-quality reads, adapters, and sequences with high “N” ratios and length less than 25 bp were removed. The clean reads were assembled using the software NOVOPlasty (https://github.com/ndierckx/NOVOPlasty)42, annotated, and manually corrected on the basis of the complete mitogenome sets assembled de novo by using MITOS tools (http://mitos2.bioinf.uni-leipzig.de/index.py)43. To confirm the correct sequences, we compared the assembled mitochondrial genes with those of other Grapsus species and identified the mitogenomic sequences by checking the cox1 barcode sequence with NCBI BLAST43. The abnormal start and stop codons were determined by comparing them with the start and stop codons of other marine gastropods. Then, the reads were reconstructed using the de novo assembly program. The complete mtDNA was annotated using the software Sequin version 16.0 (https://trace.ncbi.nlm.nih.gov/Traces/sra). The mitogenome map of the G. albolineatus was drawn using the online tool CGView Server (http://cgview.ca/)45. The secondary structures predicted of the tRNA genes were plotted by using MITOS Web Server. The relative synonymous codon usage (RSCU) values and substitution saturation for the 13 PCGs, calculated by DAMBE 545, were analyzed with MEGA 746. The GC-skews and AT-skews were used to determine the base compositional difference and strand asymmetry among the samples. According to the following formulas46, composition skew values were calculated as AT-skew = A − T/A + T and GC skew = G − C/G + C. Substitution saturation for the 13 PCGs was calculated by DAMBE 545.The mitogenomes of https://www.ncbi.nlm.nih.gov/genbank/) and adding two species of Paguridae to serve as the outgroup . BI was performed in MrBayes 3.2, and the best-fit evolutionary models were determined using MrMTgui50. MrMTgui was used to associate PAUP, ModelTest, and MrModelTest across platforms. MrBayes settings for the best-fit model (GTR + I + G) were selected by Akaike Information Criterion (AIC) in MrModelTest 2.352. The Bayesian phylogenetic analyses were performed using the parameter values estimated with the commands in MrModelTest or ModelTest 53. With three hot chains and one cold chain, they were run simultaneously twice by Markov Chain Monte Carlo (MCMC) sampling, and the posterior distribution was estimated. The MCMC chains were set for 2,000,000 generations and sampled every 1000 steps, with a relative burn-in of 25%. The convergence of the independent runs was evaluated by mean standard deviation of the split frequencies (< 0.01). The phylogenetic trees were visualized and edited using Figure Tree v1.4.3 software54.The phylogenetic relationships within Brachyura were reconstructed using the sequences of the 13 PCGs of a total of 57 complete mitogenome sequences downloaded from the GenBank database (G. albolineatus was sequenced by next-generation sequencing, thereby generating new mitochondrial data for Grapsidae and confirming its ancestral gene order. The G. albolineatus mitogenome is a typical closed-circular molecule including 13 PCGs, 22 tRNA genes, two rRNA genes, and a CR. The AT-skew and GC-skew are both negative in the mitogenome of G. albolineatus, showing an obvious bias towards the use of T’s and C’s, consistent with published findings in most Brachyura crabs. G. albolineatus exhibits a novel gene rearrangement, which is similar to G. tenuicrustatus, P. crassipes, P. marmoratu, M. frontalis, and M. quadridentatus. Compared with the pan-crustacean ground pattern, the trnH of G. albolineatus shifted into trnE and trnF instead of the usual location between nad5 and nad4. By adding 62 Brachyura mitochondrial genomes, rearrangement and the phylogeny of Brachyura was reanalyzed. The phylogenetic analyses indicated that G. albolineatus has close relationships with G. tenuicrustatus, P. crassipesand, P. marmoratu, M. frontalis, and M. quadridentatus, belonging to Grapsoidea, part of the Grapsidae family.In this study, the mitogenome of


The risks associated with achieving a high peak systolic blood pressure (SBP) during clinical exercise testing remain controversial, although this issue has not been evaluated in relation to predicted SBP standards. This cohort study aimed to evaluate the long-term risk of all-cause mortality in males in relation to reference values of peak SBP and the increase in SBP during exercise from the Fitness Registry and the Importance of Exercise: A National Database (FRIEND).We followed 7164 males (mean age: 58.2±10.6 years) over 95 998 person-years of follow-up (mean 13.4±5.4 years), who performed a maximal treadmill exercise test at baseline. SBP was measured at rest and at peak exercise. Risk of all-cause mortality over 20 years (Cox regression) was determined in relation to reference percentiles of peak SBP and increase in SBP with exercise: <10th (low), 10th–90th, >90th (high) percentiles.A high peak or a large increase in SBP with exercise was not associated with all-cause mortality. Subjects with a low peak SBP had a 20% higher unadjusted risk for all-cause death compared with those with a normal value (1.20 (1.11–1.31)), and a statistically non-significant 7% higher risk after adjustment for all baseline risk factors (1.07 (0.97–1.18)). The corresponding unadjusted and adjusted risks associated with a low increase in SBP were 1.24 (1.15–1.35) and 1.11 (1.02–1.21), respectively.A low—but not high—peak SBP is associated with increased unadjusted risk of all-cause mortality. The FRIEND percentiles of exercise SBP can aid clinicians in individualising risk assessment. Systolic blood pressure rises during progressive ramp exercise to maximal values affected by age, sex and exercise workload.Previous studies using arbitrary thresholds for exercise blood pressure have found both increased and decreased risk of mortality with an exaggerated systolic blood pressure with exercise.This study evaluated the association between exercise systolic blood pressure and mortality, using published normative values for exercise systolic blood pressure, accounting for age and sex.We found that an exercise systolic blood pressure in the lower 10th percentile—but not in the upper 90th percentile—was associated with increased mortality.It is thus important to identify individuals with an inability to adequately increase systolic blood pressure during exercise, which could be facilitated by using reference standards.A failure to significantly increase systolic blood pressure (SBP) above resting levels during an incremental exercise test, or a subsequent drop in SBP are both acknowledged markers of underlying cardiovascular disease and are associated with future mortality.14 15In the most recent scientific statement on exercise standards for testing and training from the American Heart Association (AHA) published in 2013,This study sought to examine the long-term risk of all-cause mortality in US male Veterans referred to clinical exercise testing, using recent reference percentiles for SBPpeak and the SBP increase from rest to peak exercise (ΔSBP), derived from the Fitness Registry and the Importance of Exercise National Database (FRIEND).18From 9831 exercise tests performed at the Veterans Affairs (VA) Health Care System in Palo Alto, California between 1987 and 2007, we considered the 9079 (92.3%) tests performed by males on a treadmill. We excluded 758 subjects with missing data in terms of SBP at rest and/or peak exercise, exercise capacity ), birth date, test date or date of death. We also excluded 422 subjects with any of the following pre-existing comorbidities: (1) heart failure; (2) congenital heart disease; (3) hypertrophic cardiomyopathy; (4) implanted pacemaker or cardiac defibrillator; (5) heart transplant or cardiac support device and (6) severe valvular heart disease. We also excluded the following subjects: (1) those with a follow-up time <26 weeks (n=102); (2) subjects with an SBP standing at rest <80 mm Hg or >250 mm Hg (n=19); (3) subjects reaching a peak MET value <3 (n=157) or a peak Borg rating of perceived exertion <15 in combination with <70% of age-predicted maximal heart rate (n=79); (4) those with a drop or no increase (0 mm Hg) in SBP from rest to peak exercise (n=244) and (5) subjects≥80 years, as the age span for the reference standards used in the current study was 20–79 years (n=134).18From each subject’s computerised medical record, demographic, clinical and medication information was obtained prior to the exercise test. We ascertained vital status of the participants as of 20 July 2018. Date of death was verified using the VA Beneficiary Identification and Record Locator System File.This study was originally initiated in 1987, and patients or the public were not involved in planning of this study.Each subject underwent a standardised treadmill exercise test using an individualised ramp protocol with standard criteria for termination, including signs of inducible cardiac ischaemia or a sustained drop in SBP.10.1136/bmjsem-2021-001106.supp1Supplementary dataBlood pressure was measured by auscultation before and during the treadmill test in accordance to current AHA guidelines for exercise testing laboratories.Each subject’s SBPpeak and ΔSBP value were categorised using age-specific reference percentiles recently published from the FRIEND registry.22The SBPpeak and ΔSBP values for each subject were categorised as: (1) ‘Low’ when falling below the 10th percentile in FRIEND; (2) ‘Normal’ when within the 10th–90th percentile and (3) ‘High’ when above the 90th percentile. As an 2 test to determine statistically significant differences between groups. Two-sided statistical significance was set at <0.05.We used SPSS software V.26.0 (IBM) for database management and statistical analysis. Survival analysis was performed using R Studio V.1.1.456  with the survival (V.2.38) and survminer (V.0.4.3) packages. Continuous variables were presented as mean (SD), using one-way ANOVA or the χOutcome analyses were performed with 20-year all-cause mortality as the endpoint. The Kaplan-Meier method with the log-rank (Mantel-Cox) method was used to compare survival per FRIEND reference percentile of the variables of interest. Unadjusted and adjusted Cox proportional HRs were calculated. Natural cubic spline modelling was used to characterise the risk associated with SBPpeak and ΔSBP as continuous variables, using three knots placed at 25th, 50th and 75th percentiles. The adjusted models included (a) age, (b) plus SBPrest and exercise capacity (in METs), (c) plus baseline cardiovascular risk factors  coronary artery disease). Use of antihypertensive medication and statin use were included in the definition of hypertension and hyperlipidaemia, respectively. Details on how each covariate was defined is provided in In total, 7164 males between 21 and 79 years of age (58.2±10.6 years) were included. Of these, 23% had a low and 6% had a high SBPpeak, according to the reference percentiles, while 25% had a low and 3% had a large ΔSBP from rest to peak exercise .There was a difference in both SBP and DBP at rest (standing) between SBPpeak categories (both p<0.001), with successively greater values from subjects with low to normal and high SBPpeak groups . One-thiDuring a mean follow-up of 13.4±5.4 years (truncated at 20 years), 3034 (42%) of subjects died . The relative risk of dying increased continuously with lower absolute values of peakSBP and ΔSBP, in both unadjusted and adjusted analyses . There wAs seen in Being in the lower 10th percentile reference category of SBPpeak was associated with a 14% higher risk of all-cause mortality (1.14 (1.04–1.25)) after adjusting for age, exercise capacity and SBPrest. When also adjusted for baseline risk factors and medication, the risk was no longer statistically significant (1.07 (0.97–1.18)). For ΔSBP, a similar association of higher risk with lower ΔSBP from rest to peak exercise was seen, although low ΔSBP was still significantly associated with higher mortality after full adjustment: 1.11 (1.02–1.21). Of note, high peakSBP or high ΔSBP were not associated with risk of all-cause mortality in unadjusted or adjusted analyses. Similar results (but with wider CIs) were seen when applying the 5th and 95th reference percentiles from FRIEND .In total, 1139 males had an exercise SBPpeak ≥210 mm Hg . Of these, 714 subjects (63%) were categorised as within the 10th–90th percentile according to FRIEND, while 425 (37%) exceeded the upper 90th percentile. In contrast to peakSBP , there wTo our knowledge, the current study is the first to evaluate the prognostic value of exercise SBP in relation to reference standards. We found that both a low SBPpeak and a small ΔSBP from baseline to peak exercise (in the lower 10th reference percentile) were associated with increased risk of long-term all-cause mortality. In contrast, a comparatively high SBPpeak or a large ΔSBP did not infer a greater risk of death during the follow-up.Repeated measurements of SBP before, during and following exercise are an integral part of clinical exercise testing.The negative prognostic implications of a drop in SBP or a failure to increase SBP above resting levels during exercise are well established.In this context, at a minimum, age and sex appear to be important considerations when analysing future risk of events in relation to SBP. As exercise SBP is related to exercise intensity through cardiac output, incorporating exercise intensity or exercise capacity in either reference equations for SBPpeak or in the outcome analysis is important. Recent European reference equations for bicycle ergometry suggest including sex, age, SBP at rest and exercise workload (in Watts) for predicting SBPpeak.et alet alThe combined effect of poor exercise capacity and low SBPpeak has previously been shown to be particularly negative for prognosis.First, this study only included male subjects, due to a very small proportion of females in the overall cohort, and further studies including females are required. Second, we lacked data on incident hypertension, cardiovascular disease and cardiovascular specific death which could add further insight in the prognostic implications of the SBP response to exercise. Third, given that the cohort represents clinical referrals, the results may not necessarily apply to a general population. Finally, the study included subjects over a span of 20 years, and medications as well as subject characteristics may have changed during the study period.Published reference percentiles for SBPpeak and ΔSBP predict long-term all-cause mortality in males, independent of age, exercise capacity, SBP at rest and age. Lower than predicted SBPpeak or a limited increase in SBP with exercise should be considered negative prognostic markers and could reflect underlying cardiovascular disease requiring additional follow-up.


The aim of this study was to observe changes in different components  in lung cancer associated with cystic airspace following treatment with neoadjuvant chemotherapy (NC), using computerized tomography (CT). We analyzed serial  clinical data and CT imaging in six patients treated with NC. The diameters, areas, and volumes of different tumor components  were measured. Delta (Δ) was used to represent changes in these parameters between two examinations: Δ1(%) represents the change from baseline to first follow-up after NC, and Δ2(%) represents the change from baseline to last follow-up after NC. We used the intra-group correlation coefficient (ICC) to test for consistency between parameters as measured by two radiologists. The diameter of solid components in all lesions showed a trend of continuous reduction compared with baseline . For cystic airspace and entire tumors, different lesions showed different trends over the course of treatment. For diameter, area, and volume, Δ1 of changes in the solid component ranged from − 8.3 to − 46.9%, − 19.4 to − 70.8%, and − 19.1 to − 94.7%, respectively; Δ2 ranged from − 30.8 to − 69.2%, − 50.8 to − 92.1%, and − 32.7 to − 99.8% in diameter, area, and volume, respectively. Results were inconsistent between different components of lung cancer associated with cystic airspace that was treated with NC, but the diameter, area, and volume of solid components were continuously reduced during treatment. Furthermore, area and volume measurements showed more-significant variation than diameter measurements. Tumors with mixed cystic-airspace and solid components account for about 3.7% of all lung cancers3. Mascalchi et al.4 classified these tumors by their different imaging manifestations into four types . The most common pathological categories include adenocarcinoma (AC) and squamous-cell carcinoma (SCC)6.Lung cancer associated with cystic airspace is a unique type of lung cancer10. Some studies have reported on treatment modalities and survival outcomes12. However, few discuss the criteria by which to evaluate treatment for cystic-airspace lung cancer. Oncologists do not know whether the Response Evaluation Criteria in Solid Tumors (RECIST) apply equally to cystic-airspace lesions, or which component of the tumor  is most suitable for evaluation. Accordingly, detailed serial changes in the solid component, cystic airspace, and total tumor might suitably indicate treatment response in this subtype of lung cancer. Furthermore, serial results of imaging parameters other than tumor diameter  at different time points  must also be evaluated. This information will aid clinicians in evaluating treatment response and help them optimize follow-up protocol.At present, most research on cystic-airspace lung cancer focuses on morphological features as shown on computed tomography (CT), pathological characteristics, and mechanism of tumor formationThe aim of this study was to observe serial parameter changes in different components of lung cancer associated with cystic airspace after treatment with neoadjuvant chemotherapy (NC).This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Affiliated Hospital of Xuzhou Medical University . As it was a retrospective study, the Ethics Committee exempted it from informed-consent requirements.Patient inclusion criteria included (1) tumor confirmed as primary lung cancer by pathological examination, (2) NC treatment, (3) no previous history of other malignant tumors or related anti-tumor treatment, and (4) consent to undergo CT imaging. Patient exclusion criteria included (1) incomplete NC treatment and (2) lack of serial CT imaging.13.Forty-seven lung cancer patients with cystic airspace were treated at our institution between November 2017 and June 2020. Only 6 (12.8%) out of 47 patients who were treated with NC were analyzed. We retrieved their clinical and CT imaging data from the institution’s electronic medical record system (EMRS) and picture archiving and communication system (PACS). Their cancers were staged using the tumor–node–metastasis (TNM) system in accordance with the guidelines of the Union International Cancer Control (UICC), 8th editionAll patients were included for follow-up, which was conducted over the telephone or from the EMRS and PACS by a radiologist (P.P.D.) with 2 years’ experience in chest CT. The last follow-up date in this study was September 1, 2020. The study endpoint was changed in different components of lung cancer  after chemotherapy.We used different CT scanners in this retrospective study; detailed scanning parameters are listed in Supplementary Table We analyzed CT images from three examinations during NC treatment: baseline, first follow-up examination, and last follow-up examination. Maximum diameters, maximum cross-sectional areas, and volumes of different components  of the lesion were measured. The cystic airspace was defined as the gaseous-density shadow, the solid components as soft-density or ground-glass opacity (GGO), including mural nodules, cyst wall, and GGO lesions.https://www.fsf.org/) in DICOM format for image segmentation. Two radiologists (Y.K.M. and K.X.) with more than 20 years’ combined lung cancer research experience selected the region of interest (ROI) and sketched it layer by layer along the edges of the lesions. The radiologists did not know the details of clinical treatment and survival data. The average measurements they recorded were used in this study’s analysis.We imported the CT imaging data into ITK-SNAP software 14  represents changes in different parameters from examination to examination:16. An ICC value of 0–0.2 indicated poor consistency, 0.21–0.40 indicated average consistency, 0.41–0.60 indicated moderate consistency, 0.61–0.80 indicated relatively good consistency, and 0.81–1.00 indicated good consistency.Descriptive results were expressed as percentages (%). We used the intra-group correlation coefficient (ICC) to test the consistency of the quantitative parameters measured by the two radiologistsAll six patients  had cough, and four (66.7%) had asthma. Four (66.7%) had a history of smoking. Two (33.3%) had hypertension and diabetes. Three (50%) showed elevated serum carcinoembryonic antigen (CEA) levels.4. We detected a total of six lesions, five (83.3%) of which were in the right lung. Lesion size ranged from 17.9 to 49.5 mm. Three (50%) patients had ACs, and three (50%) had SSCs. One (16.7%) patient had brain metastasis (M1 stage). Only one (16.7%) patient died during the study. Range of follow-up was 7.0–33.0 months (Table Three (50%) patients were classified as type I, one 16.7%) as type II, and two (33.3%) as type III6.7% as tSupplementary Table The diameter of solid components in all lesions showed a trend of continuous reduction compared with baseline parameters. Δ1 ranged from − 8.3 to − 46.0% and Δ2 from − 30.8 to − 69.2% , whereas that of the other three lesions had increased (Δ1 ranged from 0.5 to 81.9%). At the last CT examination after treatment, the cystic airspaces of only two lesions were reduced .The maximum whole-lesion diameters of four lesions were reduced at the first CT examination during treatment and at the last one afterward  is recommended for such evaluation15. However, the relevant evaluation criteria for cystic lung cancer are rarely discussed, which can confuse oncologists’ attempts to assess the criteria for tumor treatment response and therefore influence treatment strategies for individual patients in clinical practice. Our results demonstrated that different measurements of tumor components showed different results and that only the solid component was reduced by NC treatment. We speculate that the change in the solid component might be more correlated with clinical treatment response. In cystic-airspace lung cancer patients treated with NC, this component was more suitable for evaluation than were the cystic-airspace component and total tumor. However, we observed only six patients, and our study did not discuss the correlations of different component results with tumor long-term outcomes. Therefore, these correlations must be further evaluated by survival outcomes in a larger cohort over a longer follow-up time. The results of such studies could help guide the treatment for lung cancer associated with cystic airspace.Lung cancer associated with cystic airspace is a rare manifestation of the disease. At present, CT is one of the main modalities for cancer staging and for evaluating treatment response and outcomes23. This could be because small changes in linear size are amplified by corresponding changes in area and volume. Therefore, area and volume might more comprehensively reflect tumor burden by objective three-dimensional measurement, and so measuring these two parameters in lung cancer associated with cystic airspace could be a useful supplement to RECIST criteria. However, the measurement error of area and volume is large, and the working procedure is complicated, which might limit its clinical application.Furthermore, when comparing the degrees of variation in the three measurement parameters , we found that area and volume changes in treatment were more significant than diameter changes. Some studies show that analysis of the largest cross-sectional area and of volume might more accurately depict the burden of complex objects compared with traditional linear size measurement, which has great clinical implications25. The small bronchial stenosis prevents partial air exchange, resulting in increased residual-air volume in alveolar space. In our study, some solid components decreased, while some cystic lesions increased, over the course of NC treatment. Solid and cystic-airspace component parameters had contrary tendencies in some lesions. We thought that perhaps the reduction in the solid components resulted in enlargement of the “unidirectional valves.” The parameters of the cystic-airspace component including the total tumor in the lesion were increased. Therefore, we speculate that changes in the cystic-airspace component did not accurately and objectively reflect treatment response.Some studies that have investigated the formation of cystic airspace have shown that tumor cell proliferation can lead to the formation of valves in the bronchial lumenThis study had some limitations. First, the number of cases was small; we analyzed the serial CT images of only six patients. Second, patients’ NC regimens were inconsistent, which might have affected treatment efficacy. Furthermore, two patients were T1N1M0, and one was T2N1M0. We speculate that perhaps the oncologist used an aggressive treatment strategy in clinical practice. Third, such patients’ actual conditions and attitudes toward treatment might influence the oncologist’s treatment decisions. Finally, the pathological lung cancer types of the enrolled patients were not consistent, so we did not perform different subgroup analyses for different pathological types.In conclusion, after neoadjuvant chemotherapy in lung cancer with cystic airspace, measurements of different components showed inconsistent results, with only the solid component reduced during treatment. Area and volume measurements changed significantly compared with diameter measurements over the course of treatment.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Table 6.


Suicide ideation has increased since the outbreak of Covid-19 in many countries. The present longitudinal study investigated potential predictors of suicide ideation.M = 27.69, SD = 6.88) were assessed via online surveys in spring 2020  and in spring 2021 .Data of 406 participants from Germany , sense of control, and physical health significantly decreased. Depression symptoms (BL), PMH (BL), and consciously enhanced physical activity since the pandemic outbreak (FU) significantly predicted 12-month suicide ideation (FU). In a moderated mediation analysis, the positive relationship between depression and suicide ideation was significantly mediated by PMH. Consciously enhanced physical activity significantly moderated the negative association between PMH and suicide ideation.The context of Covid-19 could negatively impact mental health and physical health. This might increase the risk for suicide ideation. However, PMH and physical activity might serve as protective factors. The protective effect of physical activity could be especially important in people with high depression symptoms and low PMH, such as clinical patients. Potential ways of how PMH and physical activity could be enhanced in the Covid-19 context to prevent suicide ideation are discussed. Suicide is a leading cause of death among young people . Available literature mainly focused on risk factors that could increase suicide ideation in the context of Covid-19, such as depression, anxiety, and stress . At BL, 58.9% of the participants were students, 39.4% were employed, 1.5% were unemployed, and one person was retired; at FU, 51% were students, 47.3% were employed, 1.5% were unemployed, and one person was retired. Participants were instructed and gave informed consent to participate via an online form. The study’s implementation was approved by the responsible Ethics Committee. Power analyses using the G*Power program, version 3.1, indicated that of all analyses in the present study the moderated mediation analysis required the largest sample size of at least N = 311 for valid results  was sent to 450 current or former students at a large university in the Ruhr region of Germany who had agreed to be contacted for research investigations. In March 2021, an e-mail invitation for the second survey  was sent to the 423 individuals who completed the first survey. Participation was voluntary and compensated by course credits for students. There were no specific requirements for participation. The final sample included 406 people who participated in both surveys . Higher sum scores indicate higher negative symptoms. Current scale reliability: depression: Cronbach’s αBL = .905, αFU = .926; anxiety: αBL = .846, αFU = .920; stress: αBL = .881, αFU = .906.The Depression Anxiety Stress Scales 21 . The higher the sum score, the higher the level of PMH. Current scale reliability: αBL = .927, αFU = .935.The unidimensional Positive Mental Health Scale . The higher the sum scores, the lower the sense of control. Current scale reliability: αBL = .827, αFU = .878.Following Niemeyer et al. , the twoworst imaginable health state) to 100 . Higher scores reveal higher levels of physical health. Previous research demonstrated the validity of this short instrument  at FU. This statement was formulated for the present study by the authors.To measure consciously enhanced physical activity since the Covid-19 outbreak, participants rated their agreement with the statement “Compared to the time before the Covid-19 outbreak, I consciously do more physical activity ” on a 7-point Likert-type scale . Twelve-month suicide ideation is revealed for a SBQ-R score > 1. The use of this instrument for screening purpose in clinical and nonclinical samples is well-established  of the Suicide Behaviors Questionnaire – Revised  was used as the effect-size measure. All post-hoc comparisons were Bonferroni-corrected .Statistical analyses were conducted using SPSS 26  and age (BL) as control variables; symptoms of depression, anxiety, and stress , PMH (BL), sense of control (BL), physical health (BL), and consciously enhanced physical activity (FU) were added in Step 2. There was no violation of the multi-collinearity assumption  as the outcome. Step 1 included gender  assessed the moderated mediation effect. Depression symptoms (BL) served as the predictor, PMH (BL) as the mediator, consciously enhanced physical activity (FU) as the moderator, and 12-month suicide ideation (FU) as the outcome; controlling for the covariates age (BL) and gender (BL). The relationship between depression symptoms (BL) and PMH (BL) was denoted by path a; path b denoted the link between PMH (BL) and suicide ideation (FU); the relationship between depression symptoms (BL) and suicide ideation (FU) after the inclusion of PMH (BL) and consciously enhanced physical activity (FU) in the model was denoted by path c’ (the direct effect).Then, a moderated mediation analysis was run (Process: model 14). The model included a conditional indirect effect see Fig. . The boon = 122) of the sample: rarely (one time): 13.3% (n = 54), sometimes (two times): 9.1% (n = 37), often (three to four times): 3.4% (n = 14), and very often (five or more times): 4.2% (n = 17). Table p < .001 and small effect). The level of PMH (large effect), sense of control , and physical health (medium effect) significantly decreased between both measurement time points .Twelve-month suicide ideation (SBQ-R suicide ideation >1) was found in 30% (p < .001), PMH , and consciously enhanced physical activity  served as significant predictors of the 12-month suicide ideation (FU)  = 12.129, p < .001. The explained variance of the overall model was R2 = .225. The direct effect (path c’) of depression symptoms (BL) on suicide ideation (FU) was significant (p = .002) after controlling for PMH (BL), consciously enhanced physical activity (FU), and their interaction. The conditional indirect effect of depression symptoms (BL) on suicide ideation (FU) through PMH (BL) was significant in people with low  and medium  levels of consciously enhanced physical activity (FU) . Notably, this change revealed the highest statistical effect in comparison to the other investigated variables. Positive emotions experienced in social interactions, a regular sleep rhythm, and a high sense of control belong to the positive predictors of PMH . Considering available literature, this might be directly due to the infection by the coronavirus but also to reduced sleep hygiene, increased tendency to less regular and less healthy eating behavior, and enhanced sedentary behavior since the pandemic outbreak  and physical health at BL (contradiction of Hypothesis 2d) did not predict suicide ideation over the following year. One might speculate that the insignificance is due to the specificity of the Covid-19 situation. The introduced governmental measures limit the sense of control in general for many people . Physical activity is an important protective factor of mental health and physical health . Furthermore, consciously enhanced physical activity assessed at FU moderated the association between PMH at BL and the 12-month suicide ideation assessed at FU (partly confirmation of Hypothesis 3b). However, the direction of the moderation effect was unexpected. The enhancement of physical activity did not directly increase the protective effect of PMH on suicide ideation (contradiction of Hypothesis 3b). Rather the closeness of the association between PMH and suicide ideation decreased with higher levels of consciously enhanced physical activity. These findings can be interpreted as follows: PMH is an important protective factor of suicide ideation during the Covid-19 pandemic. Nevertheless, a high level of depression at the beginning of the pandemic could contribute to a low PMH level and therefore increase the risk for suicide ideation during the pandemic. However, conscious enhancement of physical activity could reduce this negative effect and specifically protect individuals with high levels of depression. Engagement in physical activity fosters positive emotions that people with high levels of depression often lack . Second, the 12-month suicide ideation was measured only at FU. Thus, no conclusions about the change in suicide ideation during the Covid-19 outbreak can be drawn. Third, we assessed the consciously enhanced engagement in physical activity compared to the time before the Covid-19 outbreak at FU with only one self-constructed item. Thus, no direct comparisons of the level of physical activity are possible. Also, no information about the type of physical activity was assessed. Therefore, it remains unclear whether the found effects of physical activity are common for physical activity in general or for specific activities . In addition, the item used to assess consciously enhanced engagement in physical activity was constructed for the present study. Thus, its validation is an important issue for future research. Furthermore, it is important to note that both the consciously enhanced engagement in physical activity and the suicide ideation were retrospectively assessed at FU for a 12-month period. Against this background, conclusions on the direction of the relationship between both variables that was investigated by Hypothesis 2e and Hypothesis 3b in the present study should be considered with caution. Fourth, the data were assessed by self-report measurements that are prone to social desirability and same-source bias (Musch et al. In conclusion, the present study confirms an increase in depression, anxiety, and stress symptoms since the Covid-19 outbreak. In contrast, the level of positive mental health, sense of control, and physical health decreased. Furthermore, individuals with high levels of depression symptoms at the onset of the pandemic seem to be at enhanced risk for suicide ideation over one year. Positive mental health and consciously enhanced engagement in physical activity could reduce this risk. Public governmental communication should present ways to foster both protective factors while maintaining social distancing.


Ircinia oros were tested through a multi-bioassay integrated approach to assess their antifouling potential. Tests were performed using three common species, associated with three different endpoints: the marine bacterium Aliivibrio fischeri (inhibition of bioluminescence), the marine diatom Phaeodactylum tricornutum (inhibition of growth), and different development stages of the brackish water serpulid Ficopomatus enigmaticus -inhibitory activity). The effects of extracts were species specific and did not vary among different extraction methodologies. In particular, no significant reduction of bioluminescence of A. fischeri was observed for all tested samples. By contrast, extracts inhibited P. tricornutum growth and had toxic effects on different F. enigmaticus’ developmental stages. Our results suggest that the proposed test battery can be considered a suitable tool as bioactivity screening of marine natural products.The phylum Porifera and their symbionts produce a wide variety of bioactive compounds, playing a central role in their ecology and evolution. In this study, four different extracts (obtained by non-polar and semi-polar extraction methodologies) of the Mediterranean sponge The online version contains supplementary material available at 10.1007/s11356-021-15683-8. Marine sponges (Porifera) are an ancient metazoan group and a major component of benthic fauna throughout temperate, tropical, and polar environments Bell . SpongesIn addition, sponges and their symbionts can exhibit chemical defence mechanisms against predation or epibiont overgrowth, often referred to as epibiosis or biofouling  and inhibition of growth of Phaeodactylum tricornutum (diatom), were used to assess the toxicity of Ircinia oros extracts. Both organisms, considered as target model species, were used as surrogates to evaluate extracts potential to reduce the colonization of benthic biofilms, dominated by bacteria and diatoms  and non-target developmental stages (adult). Specifically, sperm motion and vitality, inhibition and cellular damage, larval development, and neuro-enzyme inhibitory activity at the adult stage were investigated. In addition, we assessed differences in the effectiveness of non-polar and semi-polar extraction.In this study, two bioassays canonically used in ecotoxicological testing, namely inhibition of bioluminescence in Ircinia oros specimens were collected at the end of September 2020 by SCUBA diving at 10-–12.5-m depth along the north-east coast of Elba Island . Two fragments (15×10 cm2 each) were inserted into a sterile plastic bag filled in with seawater and immediately transported to the laboratory. Samples were photographed in situ for better species characterization and a small piece was used for taxonomy confirmation.Ircinia spp. Briefly, a first batch of dried sponges (20–40 g), obtained after 2-day oven-drying at 40°C, was macerated with MeOH/CH2Cl2 1:1 for 24–48 h. After maceration, the solution was filtered and evaporated to dryness on a rotatory vacuum evaporator set at a temperature of 40 °C for few minutes. Distilled H2O was then added to the organic residue. The aqueous phases were collected, redissolved subsequently in hexane (non-polar extract) and then in ethylacetate (AcOEt) (semi-polar extract), and concentrated under vacuum at low temperature of 40 °C. A second batch of wet sponges (102–120 g) was macerated and partitioned in the same way as described for the first batch. The remaining organic residue containing non-polar and semi-polar extracts obtained from the same sample was resuspended in DMSO (10 mg/mL) and stored at −20 °C until the use. Stock solutions were then prepared in artificial seawater (ASW), according to ISO 10253 (2016). For each of the four extracts (dry sponges portioned with hexane (DH) and AcOEt (DA); wet sponges portioned with hexane (WH) and AcOEt (WA)), nominal concentrations were as follows: 100–50–25–10–5–1–0.5–0.25–0.1–0.05 μg/mL. For each extract, the range of concentrations was based on the following: (I) on a previous study by Hellio et al. , the marine diatom Phaeodactylum tricornutum (endpoint: inhibition of growth), and the brackish water serpulid Ficopomatus enigmaticus at different development stages -inhibitory activity).The antifouling screening assays were performed through ecotoxicological evaluations using three common species associated with three different endpoints: the marine bacterium A. fischeri’ suspensions to highest concentrations of all extracts (100 μg/mL) measuring differences in bioluminescence emission (percentage of bioluminescence inhibition – I %) with control (ASW) after 30 min of exposure. Ultraviolet filters showing an I % > 20% were investigated with a full test in order to calculate EC10/20/50. Then, A. fischeri bacteria were exposed to dilution series (see the “Preparation of extracts” section)  and bioluminescence was again determined after 30 min of exposure. Tests were carried out in triplicate, at 15 °C, with sample pH within the operative range (6–8). All measurements were performed with a M500 luminometer, acquisition, and data processing  were performed with the Microtox® Omni 1.16 software. The reference toxicant was zinc sulfate eptahydrate  and results obtained for this assay fell into the laboratory control chart (6.88–12.45 mg/L Zn2+).The luminescent bacteria test was performed in accordance with the ISO 11348, 2007 methodology. The bacteria  was purchased as freeze-dried bacterial cells. Dried bacteria were resuspended in 1 mL of reconstitution solution, acquired together with bacteria vials, in order to reactivate them. At first, a screening test was performed exposing reconstituted P. tricornutum was performed following ISO procedures, with slight changes to the base protocol . Briefly, P. tricornutum Bholin (CCAP 1052/1A) was the test strain and purchased from the reference center CCAP (Culture Collection of Alga and Protozoa Scottish Association for Marine Science/SAMS Research Services Ltd). Enriched saltwater medium  was used for culturing P. tricornutum algae. The algal batch was prepared 72 h before the test, obtaining a logarithmic-phase algal culture. After 72 h, the batch was diluted to obtain a concentration of 106 cell/mL. A sample of each sponge’ extract was diluted in ASTM-ESM to obtain all working concentrations, as reported in the “Preparation of extracts” section. Using 24-well plates , 20 μL of diluted algal batch were inoculated in each of three 2 mL replicates for both samples and controls (ASTM-ESM). Plates were left at 20 ± 2 °C, under continuous illumination (6000-8000 lx) and slow shaking (80 rpm) for 72 h. Growth after 72 h of exposure was the evaluated endpoint. Absorbance was measured spectrophotometrically at 670 nm (Abs670). Algal concentration (Cells×mL−1) was calculated from absorbance using the following equation:The assessment of growth inhibition in 10/20/50 for all extracts were calculated with the Linear Interpolation Method for Sublethal Toxicity software . The reference toxicant was potassium dichromate , and results obtained for this assay fell into the laboratory control chart (1.94–4.36 mg/L Cr2+).Values of ECF. enigmaticus was collected in S. Rossore-Migliarino Regional Park – Fiume Morto  at the end of October 2020. The reef was transferred to the laboratory covered in a wet towel. Water from the sampling site were used to set up the aquaria. In laboratory, the salinity was increased up to a maximum of 5 points/day, until reaching 30‰ .The brackish water serpulid. F. enigmaticus was induced mechanically by breaking the calcareous tube. According to Oliva et al. . Briefly, the incubated sperm suspensions were added with bovine serum albumin (BSA) 1% (v/v 1:1), placed on microscope glass slides and then examined under a Leica DMi1 inverted microscope, equipped with a MC120-HD camera, using a 40× magnification objective. The video camera recorded for 5 s (100 frame/s) the moving images (three videos per condition) of sperm cells and the computer digitized them. Digitized images consisted of pixels whose changing locations were recorded frame by frame after the exposure. The images were then processed using the CASA system. The values for input parameters in the CASA dialogue box were set according to a protocol developed by evo-devo-eco network , with modifications of the sperm size and FPS (number of frames/s) in order to accurately detect motile spermatozoa. CASA system performs its motion analysis through four main steps: (1) capturing image sequences of sperm, (2) object detection, (3) object tracking, and (4) calculation of motion characteristics such as curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), and linearity (LIN), which is the result of the rate between VSL and VAP; wobble (WOB), which is a measure of sperm head side to side movement and it is the result of the rate between VAP and VCL; progression (PROG) which represents the distance sperm travelled on VAP path; and beat cross frequency (BCF). The total percentage of sperms that were actively swimming (percentage of motility) were also calculated with CASA systems and the differences between control and exposed spermatozoids at each of the extraction method was performed.After gamete emission , lipid peroxidation (LPO), intracellular reactive oxygen species (ROS), and DNA damage) was adopted to assess the spermiotoxic effect of all tested extracts at all concentrations in o et al. . Ten-folo et al. .10/50 values and their 95% confidence intervals were calculated via PROBIT analysis , weighted, and pooled in three replicates . Each pooled sample was pulverized and then homogenized in a potter Elvejem in 100 mM phosphate buffer . Homogenates were centrifuged for 20 min at 9000 × g (S9) at 4°C and stored at −80 °C until used. AChE (acetylcholinesterase) activity was measured according to Ellman et al. (50 plus 95% confidence limits) was estimated for each extract.Individuals of n et al.  in 100 mn et al.  using BSwww.graphpad.com).Results of ecotoxicological assays were reported as mean ± standard deviation (SD). Data were tested for normal distribution and homogeneity of variance by using the Kolmogorov-Smirnov and Bartlett’s tests, respectively. Since the two assumptions were met, the effects of sponge extract methodology and concentration were tested by means of a two-way ANOVA , mean EC10/20/50 with respective 95% confidence limits are not reported.Since there was no inhibition of 10/20/50 values compared those portioned with AcOEt (Table 50 (9.70 μg/mL) followed by WH (12.17 μg/mL). WA and DA extracts (EC50 of 38.26 and 42.99 μg/mL respectively) had weaker effects. Calculated values revealed the following increasing order of inhibition effects: DA > WA > WH > DH.Sponges portioned with hexane (both dry and wet) induced lower ECAs a consequence of high abnormal sperm morphology and mortality at 100, 50, and 25 μg/mL induced by all tested extracts, the estimation of sperm motion characteristics was performed from a range of concentrations of 10 to 0.5 μg/mL  as reported in Online Resource Table Although there was an interaction between concentrations and extracts, sperm quality significantly varied only according to concentrations  was not affected by any of tested extracts Fig. . Finally10/50 values compared the ones portioned with AcOEt (50 (0.89 μg/mL) followed by DH (1.69 μg/mL). WA and DA at EC50 of 1.21 and 5.12 μg/mL induced weaker effects on larval development, respectively. Calculated EC50 values revealed the following decreasing order of effects: DA > WA > DH > WH.Sponges portioned with hexane (both dry and wet) generally showed lower ECth AcOEt . In detaThe ANOVA showed significant effects of extract concentrations on the proportion of larva successfully developed, consistently among extracts. Data are reported in F. enigmaticus fractions incubated at concentrations ranging between 0.05 and 1.0 μg/mL of all extracts, the estimation of the inhibitory activity on the enzyme was examined from 5 to 100 μg/mL. Inhibition of the AChE activity varied significantly with extract concentration .Four different extracts obtained from the sponge e et al. , who obsHaliclona spp. inhibited the growth of several bacteria, including Bacillus cereus, B. pumilus, B. megaterium, Pseudoalteromonas haloplanktis, Pseudomonas chlororaphis, P. putida, and P. aeruginosa. In this study, none of tested extracts reduced the bioluminescence in the marine bacteria A. fischeri in comparison with controls. Although this bioassay has been widely applied for evaluating compound toxicity since it is cost-effective and easy to use  than semi-polar extracts (AcOEt). Significant bioactivity on microalgae inhibition has also been reported for other screening strategies, such as extracting I. oros metabolites with a polar solvent (ethanol)  and non-target developmental stages (adult). Although marine invertebrate gametes and larvae are several orders of magnitude more sensitive than adults, assessing the sensitivity at the adult stage is necessary for determining the effects of a compound throughout a species life cycle , even if not concentration dependent. Many substances extracted from marine organisms have been shown to have a profound influence on the reproductive behaviour and strategy’s function (Silvestre and Tosti 2−), hydrogen peroxide (H2O2), and nitric oxide (NO−), are chemically reactive molecules resulting from oxygen consumption. At certain concentrations, ROS are of extreme importance to sperm function . As a matter of fact, the imbalance in the rate of ROS generation leads to oxidative stress. The products of lipid peroxidation chain reactions display high biological activity destroying DNA, proteins, and enzymes as well as activating signaling pathways which cause cell death  was greater  than that to AcOEt extracts. The toxicity assessments of natural mixtures and emerging contaminants were recently applied to F. enigmaticus , found significant effects on the development of larvae of the nematode, H. contortus; in, particular, a dose-dependent inhibition of the motility of third-stage larvae as well as of the development of the fourth-stage larvae.In this work, the larval development assay as a target developmental stage in ecotoxicological studies was also used as endpoint of extract toxicity. The selected assay was focused on the first developmental stage, which brings zygotes to a planktonic and planctotrophic trocophore stage  inhibition in vitro. The substance was first tested for its effect on larval development of A. Amphitrite confirming the ability of Poly-APs to prevent the development of sessile organism by impinging on AChE activity. Our results are in agreement with previous evidence of sponge extract-induced neurotransmitter inhibition, confirming that AChE is an enzyme associated to the cholinergic signal system, and is also involved in cell-to-cell communication, driving embryonic development