Mortality processes of Trichodesmium – incubation
results
Grazer and virus influence
Our microcosm incubations allowed us to specifically focus on the loss
factors and show the involvement of biotic and abiotic stressors in inducing
PCD and mechanistically impacting the demise and fate of a natural
Trichodesmium bloom. We recognize that the enclosure and incubation of collected biomass
in bottles and carboys may accelerate cellular processes compared to the natural
lagoon setting. However, the published rates of Trichodesmium mortality
from field studies (Rodier and Le Borgne, 2010) indicate that these can
parallel our loss rates with natural bloom demise occurring 24–48 h after
peak of biomass.
We focused initially on biotic factors that could impact the incubated
Trichodesmium biomass. The low number of harpacticoid zooplankton
specific to Trichodesmium (O'Neil and Roman, 1994; O'Neil, 1998) in
the lagoon (Hunt et al., 2016) and especially those in the bottles (personal
observation) refutes the possibility that grazing caused the massive mortality
of Trichodesmium biomass in our experimental incubations.
Viruses have been increasingly invoked as key agents terminating
phytoplankton blooms (Brussaard et al., 2005; Jacquet et al., 2002; Lehahn et
al., 2014; Tarutani et al., 2000; Vardi et al., 2012). Infection by phages
has been invoked as the mechanism of Trichodesmium bloom crashes,
but it has yet to be unequivocally demonstrated (Hewson et al., 2004; Ohki,
1999); indeed, no specific Trichodesmium phage has been isolated or
characterized to date (Brown et al., 2013). Here, total VLP abundance was
highest at the time of sampling from the surface Trichodesmium bloom
and at the start of the incubation (∼ 8 × 106
VLP mL-1). It actually declined 2-fold in the first 8 h of incubation
before increasing over the next 32 h (Fig. 4a). While our method of analysis
cannot distinguish between phages infecting Trichodesmium from those
infecting other marine bacteria, it argues against a massive, phage-induced
lytic event of Trichodesmium. Such an event would have yielded a
notable burst of VLP upon bloom crash, especially considering the high
Trichodesmium biomass observed. The coincidence between the maximal
abundance of VLP and highest Trichodesmium biomass is counter to
viruses serving as the mechanism of mortality in our incubation experiments.
Nonetheless, virus infection itself may be a stimulant for community N2
fixation perhaps by releasing key nutrients (i.e., P or Fe) upon lysis of
surrounding microbes (Weitz and Wilhelm, 2012). Although we did not
characterize them here, it is indeed possible that
Trichodesmium-specific phages were present in our incubation
experiments and they may have exerted additional physiological stress on
resident populations, facilitating PCD induction. Virus infection has been
shown to increase the cellular production of reactive oxygen species (Evans
et al., 2006; Vardi et al., 2012), which in turn can stimulate PCD in algal
cells (Berman-Frank et al., 2004; Bidle, 2015; Thamatrakoln et al., 2012).
Viral attack can also directly trigger PCD as part of an antiviral defense
system activated to limit virus production and prevent massive viral
infection (Bidle and Falkowski, 2004; Bidle, 2015; Georgiou et al., 1998).
Stressors impacting mortality
Nutrient stress can be acute or chronic to which organisms may acclimate on
different timescales. Thus, for example, the consistently low DIP
concentrations measured in the lagoon during the 22 days preceding the
Trichodesmium surface bloom probably enabled acclimation responses
such as induction of APA and other P acquisition systems.
Trichodesmium has the ability to obtain P via inorganic and organic
sources, including methylphosphonate, ethylphosphonate, and
2-aminoethylphosphonate (Beversdorf et al., 2010; Dyhrman et al., 2006), and via a phosphite uptake system (PtxABC) that accesses P via the reduced
inorganic compound phosphite (Martínez et al., 2012; Polyviou et al.,
2015). Our metatranscriptomic data demonstrated upregulated expression of
genes related to all three of these uptake systems (DIP, phosphonates,
phosphites) 8 and 22 h after incubation began, accompanying biomass demise
(Fig. 5a). This included one gene for phosphite uptake (ptxA) and several
genes from the phosphonate uptake operon (phnDCEEGHIJKLM) (Hove-Jensen et
al., 2014). Upregulated expression of phnD, phnC, phnE, phnH, phnI, phnJ,
phnK, phnL, and phnM occurred as the Trichodesmium biomass
crashed (Fig. 5a, Table S1), consistent with previous results demonstrating
that phnD and phnJ expression levels increased during DIP depletion
(Hove-Jensen et al., 2014). It is likely that, during bloom demise, the C-P
lyase pathway of remaining living cells was induced when DIP sources were
extremely low, while POP and DOP increased along with the decaying organic
matter. The ability to use phosphonates or phosphites as a P source can
provide a competitive advantage for phytoplankton and bacteria in P-depleted
waters (Coleman and Chisholm, 2010; Martinez et al., 2010). Thus, it is
puzzling why dying cells would upregulate phn genes or phoA transcripts
after 22 h incubation (Fig. 5a). A more detailed temporal resolution of the
metatranscriptomic analyses may elucidate the expression dynamics of these
genes and their regulating factors. Alternatively, in PCD-induced
populations, a small percentage of cells remain viable and resistant as either cysts
(Vardi et al., 1999) or hormogonia (Berman-Frank et al., 2004) that can serve
as the inoculum for future blooms. It is plausible that the observed
upregulation signal was attributable to these subpopulations.
The concentrations of dissolved and bioavailable Fe were not measured in the
lagoon water during the experimental period as Fe is typically replete in the
lagoon (Jacquet et al., 2006). However, even in Fe-replete environments such
as the New Caledonian lagoon, dense patches of cyanobacterial or algal
biomass can deplete available resources and cause limited microenvironments
(Shaked, 2002). We obtained evidence for Fe stress using several proxy genes
demonstrating that enhanced cellular Fe demand occurred during the bloom
crash (Table S1). Trichodesmium's strategies of obtaining and
maintaining sufficient Fe involves genes such as isiB. isiB
was highly expressed when biomass accumulated on the surface waters,
indicative for higher Fe demand at this biomass load (Bar-Zeev et al., 2013;
Chappell and Webb, 2010), yet expression declined significantly with the
dying biomass. Transcripts for chlorophyll-binding, Fe-stress-induced protein
A (IsiA) increased (albeit not significantly) 3-fold over 22 h of bloom
demise (Fig. 5b, Table S1). In many cyanobacteria, isiA expression
is stimulated under Fe stress (Laudenbach and Straus, 1988) and oxidative
stress (Jeanjean et al., 2003) and functions to prevent high light-induced
oxidative damage by increasing cyclic electron flow around the photosynthetic
reaction center photosystem I (Havaux et al., 2005; Latifi et al., 2005;
Michel and Pistorius, 2004). Dense surface blooms of Trichodesmium
are exposed to high irradiance (on day 23 average photosynthetically active
radiation was 3000 µmol photons m-2 s-1). It is
possible that high Fe demand combined with the oxidative stress of the high
irradiance induced the higher expression of isiA (Fig. 5b). As cell
density and associated self-shading of Trichodesmium filaments
decreased during bloom crash, light-induced oxidative stress is likely the
principal driver for elevated isiA expression.
The gene idiA is another environmental Fe stress biomarker that allows
acquisition and transfer of Fe through the periplasm into the cytoplasm
(Chappell and Webb, 2010). In our incubation, upregulated expression of
idiA (an ABC Fe+3 transporter) was evident after 8 h. This is
consistent with increasing Fe limitation, as Trichodesmium abundance
(measured via 16S rRNA gene sequencing) was still high at T6 (after 6 h
of incubation) (replicate 1). These findings are consistent with proteomics
analyses from depleted iron (0 µM Fe) Trichodesmium
cultures which revealed an increase in IdiA protein expression (Snow et al.,
2015). Lastly, our metatranscriptomic data highlighted a reduction in Fe
storage and utilization, as the expression of Fe-rich ferritin-like DPS
proteins (Castruita et al., 2006), encoded by dpsA, decreased ∼ 5-fold by the time that most of the biomass had crashed (T22) (Fig. 5b,
Table S1). dpsA was also downregulated under Fe-replete conditions in
Synechococcus (Mackey et al., 2015), but the downregulation observed
here is more likely related to Trichodesmium cells dying and
downregulating Fe-demanding processes such as photosynthesis and N2
fixation.
Programmed cell death (PCD) and markers for increased export
flux
The physiological and morphological evidence of PCD in Trichodesmium
has been previously documented in both laboratory (Bar-Zeev et al., 2013;
Berman-Frank et al., 2004) and environmental cultures collected from surface
waters around New Caledonia (Berman-Frank et al., 2004). Here, we confirmed
characteristic features of autocatalytic PCD in Trichodesmium such
as increased caspase-specific activity (Fig. 6a), globally enhanced
metacaspase expression (Fig. 6b), and decreased expression of gas vesicle
maintenance (Fig. 7). Metatranscriptomic snapshots interrogating expression
changes in all of the annotated Trichodesmium metacaspases (Fig. 6b)
generally portrayed upregulated expression concomitant with biomass decline.
Our results are consistent with previous observations that Fe-depleted
PCD-induced laboratory cultures of Trichodesmium IMS101 had higher
expression levels of TeMC1 and TeMC9 compared to healthy Fe-replete
cultures (Bar-Zeev et al., 2013; Berman-Frank et al., 2004). To our
knowledge, this is the first study examining expression levels of
metacaspases in environmental Trichodesmium samples during a natural
bloom. Eleven of the 12 metacaspases in Trichodesmium were expressed
in all three metatranscriptomes from the surface bloom. To date, no specific
function has been determined for these metacaspases in Trichodesmium
other than their association with cellular stress and death. Efforts are
underway to elucidate the specific cellular functions, regulation, and
protein interactions of these Trichodesmium metacaspases (Bar-Zeev
et al., 2013; Pfreundt et al., 2014; D. Spungin, personal communication,
2016).
In cultures and isolated natural populations of Trichodesmium, high
caspase-like specific activity is correlated with the initial induction
stages of PCD with activity declining as the biomass crashes (Bar-Zeev et
al., 2013; Berman-Frank et al., 2004, 2007). Here, caspase-like activity
increased with the crashing populations of Trichodesmium (Fig. 6a).
Notably, maximal caspase activities were recorded at T22, after which
most Trichodesmium biomass had collapsed. The high
protein-normalized caspase-specific activity may be a result of a very
stressed and dying subpopulation of Trichodesmium that had not yet
succumbed to PCD (Berman-Frank et al., 2004). Alternatively, the high
caspase-like activity may be attributed to the large population of
Alteromonas bacteria that were associated with the remaining
detrital Trichodesmium biomass. However, currently, we are unaware
of any publications demonstrating high cellular caspase-specific activity in
clades of γ-Proteobacteria.
Gas vesicles are internal structures essential for maintaining buoyancy of
Trichodesmium populations in the upper surface waters enabling them
to vertically migrate and respond to light and nutrient requirements (Capone
et al., 1997; Walsby, 1978). Mortality via PCD causes a decline in the number
and size of cellular gas vesicles in Trichodesmium (Berman-Frank et
al., 2004) and results in an enhanced vertical flux of trichomes and colonies
to depth (Bar-Zeev et al., 2013). Our metatranscriptomic data supported the
subcellular divestment from gas vesicle production during bloom decline, as
the expression of vesicle-related genes was downregulated (Fig. 7). In
parallel, TEP production and concentration increased to
> 800 µg GX L-1, a 2-fold increase from pre-bloom
periods (Figs. 1d and 4c). When nutrient uptake is limited, but CO2 and
light are sufficient, uncoupling occurs between photosynthesis and growth
(Berman-Frank and Dubinsky, 1999), leading to increased production of excess
polysaccharides (such as TEP) and corresponding with high TEP found in bloom
decline phases rather than during the increase in population density (Engel,
2000; Smetacek, 1985). In earlier studies we demonstrated that PCD-induced
demise in Trichodesmium is characterized by an increase in excreted
TEP (Berman-Frank et al., 2007) and enhanced sinking of particulate organic
matter (Bar-Zeev et al., 2013). TEP may be positively buoyant
(Azetsu-Scott and Passow, 2004), yet their stickiness causes aggregation and
clumping of cells and detritus, ultimately enhancing sinking rates of large
aggregates, including dying Trichodesmium (Bar-Zeev et al., 2013).
Changes in microbial community with Trichodesmium
decline
In the incubations, other diazotrophic populations succeeded the declining
Trichodesmium biomass as indicated by increasing N2-fixation
rates, POC, and PON (Fig. 4b). In experiment 2, based on qPCR of targeted
diazotrophic phylotypes, the diazotroph community composition shifted from
being dominated by Trichodesmium spp. and unicellular groups
UCYN-A1, UCYN-A2, and UCYN-B (T0) to one dominated by diatom–diazotroph
associations Het-1 and Het-2 (T72) (Bonnet et al., 2016b; K. Turk-Kubo,
personal communication, 2016). In experiment 1 heterotrophic bacteria thrived
and increased in abundance as the Trichodesmium biomass crashed
(Fig. 3).
Trichodesmium colonies host a wide diversity of microorganisms,
including specific epibionts, viruses, bacteria, eukaryotic microorganisms,
and metazoans (Hewson et al., 2009; Hmelo et al., 2012; Ohki, 1999; Paerl et
al., 1989; Sheridan et al., 2002; Siddiqui et al., 1992; Zehr, 1995).
Associated epibiont bacterial abundance in dilute and exponentially growing
laboratory cultures of Trichodesmium is relatively limited (Spungin
et al., 2014) compared to bloom conditions (Hewson et al., 2009; Hmelo et
al., 2012). Proliferation of Alteromonas and other γ-Proteobacteria during biomass collapse (Fig. 3) confirms their reputation
as opportunistic microorganisms (Allers et al., 2008; Hewson et al., 2009;
Frydenborg et al., 2014; Pichon et al., 2013). Such organisms can thrive on
the influx of organic nutrient sources from the decaying
Trichodesmium as we observed (Fig. 3). Furthermore, the increase in
organic matter including TEP produced by the stressed Trichodesmium
(Figs. 1d and 4c) probably stimulated growth of these copiotrophs. Moreover,
as the Trichodesmium biomass declined in the carboys, the high
concentrations of NH4+ (> 5000 nmol L-1) (Fig. 4b)
sustained both autotrophic and heterotrophic organisms (Berthelot et al.,
2015; Bonnet et al., 2016b, c). Thus, the increase in volumetric N2
fixation and PON that was measured in the incubation bottles right after the
Trichodesmium crash in experiment 2 (Fig. 4b) probably reflects both
the enhanced activity of other diazotrophs (see above and Bonnet et al.,
2016b) and the resistant residual Trichodesmium trichomes (Berman-Frank
et al., 2004) with increased cell-specific N2 fixation. This scenario is
consistent with the hypothesis that PCD induction and death of a fraction of
the population confers favorable conditions for survival and growth of
individual cells (Bidle and Falkowski, 2004; Bidle, 2015).
Implications for the lagoon system and export flux
Phytoplankton blooms and their dense surface accumulations occur under
favorable physical properties of the upper ocean (e.g., temperature,
mixed-layer depth, stratification) and specifically when division rates
exceed loss rates derived from grazing, viral attack, and sinking or export
from the mixed layer to depth (Behrenfeld, 2014). Although physical drivers
such as turbulence and mixing may scatter and dilute these dense
accumulations, the rapid disappearance of biomass in large sea-surface
Trichodesmium blooms (within 1–2 days in the lagoon waters) (Rodier
and Le Bourne, 2010) suggests loss of biomass by other mechanisms. The lack
of Trichodesmium developing within the VAHINE mesocosms and the
spatial–temporal variability in the surface bloom in the lagoon prohibited
in situ sampling of the same biomass for several days and prevented
conclusions regarding in situ mortality rates and export flux. Furthermore,
within these dense surface populations, as well as in the microcosm and
carboy experiments, Fe availability was probably extremely limited due to
high cellular demand and competition (Shaked, 2002). PCD induced by
Fe-depletion experiments with laboratory cultures and natural populations
results in rapid biomass demise, typically beginning after 24 h, with
> 90 % of the biomass crashing 3 to 5 days after induction
(Bar-Zeev et al., 2013; Berman-Frank et al., 2004; Berman-Frank et al.,
2007). In similar experiments with P depletion, Trichodesmium
biomass did not crash rapidly. Rather, limitation induced colony formation
and elongation of trichomes (Spungin et al., 2014) and the cultures could be
sustained for another couple of weeks before biomass declined significantly
(unpublished data). The responses we quantified from the dying
Trichodesmium in the carboys and bottles (Figs. 3–7) were similar
to those obtained from controlled laboratory experiments where P and Fe
stress was validated individually. However, the rapid response here probably
reflects an exacerbated reaction due to the simultaneous combination of
different stressors and the presence of biotic components that can compete
for and utilize the organic resources (carbon, nitrogen, phosphorus)
generated by the dying Trichodesmium. In the lagoon, production of
TEP by stressed biomass combined with the degradation of gas vesicles and
enhanced aggregation will cause such surface accumulations or blooms to
collapse, leading to rapid vertical export of newly fixed nitrogen and carbon
in the ocean.