As highly educated individuals tend to be more optimistic than those in less advantaged situations , this may have caused some ceiling effects in our ability to induce optimism. There are also reasons to suspect that the intervention effects may vary depending on socioeconomic status or other sociodemographic factors and that our findings may not be generalizable to a broader population. Most previous research on optimism or related factors like purpose in life and health has found little evidence of effect modification by socioeconomic status or other sociodemographic factors . However, we suspect that limited resources might increase the likelihood of facing barriers that interfere with individuals’ ability to translate their optimism or other facets of positive psychological well-being into action. Limited work has examined these potential sources of effect modification and it will be important for future studies to assess the possibility. Because writing prompts were different in the intervention versus control groups, we cannot be sure that the intervention perse led to higher coder-rated optimism. We did not assess trait characteristics that are likely to attenuate/ exacerbate optimism effects such as levels of learned helplessness or perceived control.
Future study should evaluate how trait characteristics may modify optimism effects. Lastly, our experiment was relatively short,bucket flower and therefore we were unable to determine whether the effects would increase over time or to evaluate how long people would stay with the intervention. Additionally, we did not follow up with participants after they completed the lab visits. As a result, we could not assess if effects of the intervention would persist beyond the study period. One important avenue for future research is to develop a longer optimism experiment with follow up to evaluate the sustainability of the effect. Despite these limitations, our study represents the largest experimental research to date to investigate the effects of induced optimism on physical activity and stress reactivity. Our experimental design allowed us to assess causality from optimism to physical activity and stress reactivity. Additionally, we recruited participants from community-dwelling adults with diverse age, socioeconomic, and racial/ethnic backgrounds. Our research is also among the very few studies that evaluated both participant and coderrated optimism and their effects on physical activity and stress reactivity. The two studies used almost identical writing interventions with different outcomes and were simultaneously conducted at two large research institutions, providing a good example of study replication. In sum, our results suggest that intentional interventions can have beneficial effects on optimism and positive affect, but the size of the effect may not be sufficient to lead to substantial changes in willingness to engage in physical activity and stress reactivity.
Further research should aim to refine such interventions to move beyond creating relatively small changes, investigate what factors make such interventions maximally effective, and assess the level of change required to change downstream behavior and other health-related responses. Furthermore, given findings that coder-rated optimism and affect were more strongly associated with physical activity duration and stress reactivity, behavioral measures of these psychological states may provide novel insight into changes occurring below the threshold of conscious awareness or that may not differ noticeably enough to translate into differences in self-reported levels.Fixed nitrogen is often a limiting nutrient for primary productivity in the surface ocean, and consequently influences the dynamics of oceanic carbon sequestration . Nitrogen fixation by marine cyanobacteria is an important source of oceanic fixed nitrogen, adding an estimated 100–200 Tg-N annually to open ocean ecosystems . This nitrogen fixation is often associated with cyanobacterial trichomes or aggregates colonized by heterotrophic bacteria, picoeukaryotes and metazoans . Respiratory activity within these so-called ‘pseudobenthic’ environments can create ephemeral suboxic to anoxic zones, establishing a niche for facultative anaerobes within otherwise oxygenated surface waters . Emerging evidence suggests that denitrification occurs within these anoxic habitats, coupling processes of nitrogen-fixation and loss at the microscale . While initial studies of marine biological nitrogen fixation focused on colonial filamentous Trichodesmium species and symbiotic, heterocystous Richelia species , more recent work has demonstrated the importance of unicellular diazotrophic cyanobacteria from the order Chroococcales . Diazotrophic UCYN have been studied extensively in the global oceans by surveys of the nitrogenase gene nifH diversity, which revealed three phylogenetically distinct clades .
UCYN-A are small , metabolically streamlined, uncultured cyanobacteria that lack the oxygen-producing photosystem II and live as endosymbionts within haptophytes, a lineage of eukaryotic algae . UCYN clades B and C are larger , free-living cyanobacteria and include cultured representatives, such as Crocosphaera watsonii and Cyanothece sp. ATCC51142. Studies of aggregate-associated nitrogen fixation have focused predominantly on Trichodesmium sp. colonies and rafts , or filamentous heterocystous cyanobacterial colonies . However, some Crocosphaera watsonii strains have been observed to produce copious quantities of exopolysaccharides and have been linked to the formation of transparent exopolymer particles . These gel-like particles provide microhabitats for other microorganisms, and thus have the potential to play an important role in marine biogeochemical cycling . Here, we report a new species of uncultured, unicellular cyanobacteria from the order Chroococcales which forms millimeter-sized aggregates together with diatoms and other putatively heterotrophic bacteria. These macroscopic aggregates, which we call “green berries,” are found in the muddy, intertidal pools of Little and Great Sippewissett salt marshes . They are found interspersed with previously described, sulfur-cycling “pink berry” consortia . Using a combination of metagenomic sequencing and ecophysiological measurements, we demonstrate that the green berries are characterized by diazotrophy and rapid rates of photosynthesis and respiration that produce steep oxygen gradients. Heterotrophic bacteria within the green berries are closely related to other marine epiphytic marine strains and encode key genes in the denitrification Pathway.The green berries are found in the same organic-rich, intertidal pools of Little Sippewissett salt marsh on Cape Cod where both multicellular magnetotactic bacteria and pink berries have been previously studied . Though less abundant than the pink berries found in these pools , the green berries form similar irregular ellipsoid aggregates measuring 1–8 mm in diameter, with an average equivalent spherical diameter of 1.7 mm ± 0.1 mm . Green berries were dense and compact aggregates that were typically observed at the sediment-water interface, but were occasionally found to float at the water surface when suspended by bubbles. Microscopic observation of the green berries revealed abundant coccoid unicellular cyanobacteria 5–7 µm in diameter , interspersed with pennate diatoms . Filamentous cyanobacteria were observed occasionally, but were rare compared to the unicellular GB-CYN1 morphotype. A clear, extracellular matrix coated these aggregates of phototrophic cells, and was colonized by a variety of smaller bacteria .
GB-CYN1 exhibited absorption maxima at 620, 660, and 680 nm corresponding to thepresence of phycocyanin,cut flower bucket allophycocyanin and chlorophyll a, respectively. Sequencing of 18S rRNA genes from the green berries indicated that the eukaryotic community was predominantly made up of two different pennate diatom species related to Navicula cari strain AT-82.04c and Amphora pediculus strain AT-117.11 . These same diatom species were also the dominant eukaryotic 18S rRNA gene sequences recovered from pink berry aggregates, though diatoms were more abundant in green berries than in pink berries, as observed by microscopy and the relative abundance of 16S rRNA chloroplast sequences . Bacterial 16S rRNA gene sequences amplified from the green berries were dominated by sequences related to either diatom chloroplasts or Chroococcales unicellular cyanobacteria . Unassembled metagenomic sequence reads assigned to rRNA sequences and protein-coding regions support the observed abundance of Chroococcales , but did not recover comparable proportions of diatom chloroplasts . FIGURE 2 | Comparison of green berry bacterial diversity estimates from16S rDNA PCR amplified clones library with unassembled Roche 454 metagenomic sequence reads. Taxonomic assignment of metagenomic reads matching ribosomal RNA reads was conducted using the M5RNA database in MG-RAST . A similar taxonomic assignment was conducted with metagenomic reads matching protein coding sequences in the M5NR database . Note that the 16S rRNA clone library abundance data for the Bacillariophytawas obtained from diatom chloroplasts sequences, which are likely present in multiple copies in the cell and thus not directly comparable to metagenomic 18S rRNA sequences for this group . The overall bacterial community structure of the green berries was significantly different from coexisting pink berry consortia . Some abundant taxa from the pink berries co-occurred in the green berries as rare OTUs, such as the purple sulfur bacterial species Thiohalocapsa sp. PB-PSB1 , and a Winogradskyella species . The persistence of these distinct, co-occurring pink and green berry consortia suggests that the process of macroscopic aggregation enables niche partitioning between oxygenic and anoxygenic phototrophs in these marsh pools. Most of the non-cyanobacterial sequences in the green berry consortia are related to aerobic and facultatively anaerobic marine heterotrophs from the Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria . Many of these sequences were most closely related to environmental 16S rRNA sequences associated with aggregates of oxygenic phototrophs. Examples of such habitats included phytodetrital aggregates collected from euphotic and hadal environments , and epiphytes of marine macroalgae . The occurrence of related phylotypes in such environments suggests that taxa may be well adapted to an attached lifestyle, degradation of photosynthate, and the fluctuating oxygen conditions in an aggregate environment. Metagenomic data indicate that the orders Rhizobiales and Rhodobacterales of the Alphaproteobacteria are abundant in the green berry consortia. While these groups were rarely detected in the PCR-based 16S rRNA survey, we have previously observed this same PCR bias from the 8F primer during studies of the pink berry consortia . We find the abundance of these clades in the green berries particularly interesting as they include lineages of marine denitrifying bacteria.
For example, pelagic Rhizobialeshave been linked to denitrification when found in association with macroscopic Trichodesmium sp. aggregates in oxic waters bordering oxygen minimum zones .The cyanobacterial 16S rRNA gene sequences from the green berries grouped into two closely related OTUs , GB-CYN1a and GB-CYN1b, that can be confidently placed in the order Chroococcales . The GB-CYN1 monophyletic cluster formed a clade basal to the UCYNA clade . Using 29 concatenated single-copy phylogenetic marker genes assembled from the metagenome , we reconstructed a phylogenetic tree that placed the GB-CYN1 within a clade including Crocosphaera watsonii and Cyanothece sp. ATCC 51142 as a sister taxa to “Candidatus Atelocyanobacterium thalassa” isolate ALOHA . A phylogenetic tree inferred from nifH gene sequences reveals that the near full-length nifH gene recovered from the GB-CYN1 metagenomic data affiliated with the UCYN-B clade, and was most closely related to Cyanothece sp. 8801/8802 and Crocosphaera watsonii . We conclude that the observed discordance between 16S rRNA, concatenated, and nifH gene phylogenies involving species such as Cyanothece sp. 8801, Gloeothece sp. KO68DGA, and the cyanobacterial endosymbiont of Rhopalodia gibba is most likely due to lateral gene transfer of the nifH gene. Lateral transfer of nifH has been observed in many other species, including mat-forming filamentous cyanobacteria . A full suite of nitrogenase genes were found in the green berry metagenome and were consistently assigned to GB-CYN1, indicating the metabolic potential for nitrogen fixation characteristic of other members of the UCYN A-C clades . Diazotrophy in the green berries was detected in whole aggregates by acetylene reduction. In two separate experiments , we measured rates of 11 and 20 nanomoles acetylene reduced per hour per milligram of aggregate dry weight . These rates are comparable, though faster than the rates of 3–6 nmol mg−1 hr−1 reported in macroscopic aggregates of filamentous cyanobacteria from Bogue Sound, North Carolina . Rates measured from actively growing Cyanothece cultures were two orders of magnitude larger than the rates in the green berries. The total aggregate elemental composition was analyzed using elemental analyzer isotope ratio mass spectrometry . The mean observed C:N ratio in the green berries, 7.1 ± 0.6 , falls within range of the Redfield molar ratio . This observed C:N ratio is higher than that the ratio of 5.4 ± 0.4 observed in similarly large, anoxic, diazotrophic Nodularia spumigena aggregates from the Baltic Sea . Cultures of Crocosphaera watsonii exhibit wide diel fluctuations in C:N ratios as a result of temporal partitioning of carbon and nitrogen fixation activities . Our samples, collected in the late afternoon on a 14 h light/10 h dark photoperiod, are comparable to reports of C:N = 7 from C. wastsonii at similar late afternoon times in a 16 h light/8 h dark photoperiod . Future studies investigating the temporal partitioning for such activities in the green berry aggregate would be informative to elucidate the dynamics of carbon and nitrogen flow in the consortia.