Dinoflagellates within the genus Symbiodinium can form unique symbioses with various marine invertebrate species, including jellyfish, sea anemones, giant clams, soft corals, and hard corals or scleractinians. These symbioses are pivotal for the ecological success of many of these organisms as they provide a major carbon source to their hosts via translocation of energy rich photosynthate (Muscatine et al., 1984; Muscatine, 1990). This genus contains a high degree of genetic diversity which is currently organized into nine broad clades (designated by the letters A–I), some of which contain hundreds of distinct species (LaJeunesse, 2001). Importantly, different Symbiodiniums can have significant influence on the host's response to changes in the environment (Baker, 2003). For example, in corals, thermal tolerance is often associated with the specific symbiont type (Kemp et al., 2006; LaJeunesse et al., 2009; Silverstein et al., 2011; Ortiz et al., 2012). However, matching physiological characteristics with specific symbiont types can be difficult and time consuming. There is a real need within the coral reef community to match physiological variability with genetic variance across symbiont types to better understand what/how these symbioses will respond under future climate conditions.

Such phenotypic assessment (Phenomics) is gaining momentum within other photobiological fields where comprehension of genetic variability has far outpaced phenotypic descriptions (Furbank and Tester, 2011). Phenotypic structure across different symbiont types grown in culture has been documented using photophysiological metrics as measured using active chlorophyll fluorometric measurements (Suggett et al., 2015). However, environmental variables such as light intensity and the spectral characteristics inherent to the coral host in which the symbiont resides can also influence photobiological behavior (Iglesias-Prieto and Trench, 1997b; Iglesias-Prieto et al., 2004; Wangpraseurt et al., 2012, 2014). The potential for phenotypic variance within a single species also increases the difficulty of matching physiology with genotype in symbiodinium (Iglesias-Prieto and Trench, 1997b; Iglesias-Prieto et al., 2004).

The genus Symbiodinium contains a number of different photopigments, some involved with light harvesting, while others act as accessory pigments where they can either channel additional energy toward the photosynthetic reaction centers, or dissipate excess light energy (Iglesias-Prieto and Trench, 1994, 1997b). The light harvesting compounds; peridinin-chlorophyll-a-protein (PCP) and chl a-chl c(2)-peridinin-protein (acpPC) have been particularly well-studied and comprise the primary light harvesting units within Symbiodinium (Iglesias-Prieto et al., 1991, 1993; Niedzwiedzki et al., 2013, 2014). The relative abundance of these different photopigments can differ across symbiont types (Hennige et al., 2009) and potentially offer another biological parameter that can be utilized to study species specific phenotypes within the genus Symbiodinium. As was demonstrated with symbionts housed within Porites and Montipora, host coral specimens found in Kaneohe Bay, absorbance based measurements can under certain circumstances provide some information on the abundance of photopigment concentrations (Hochberg et al., 2006). However, additional instrumentation is needed to understand the active role of these pigments within the photosynthetic apparatus.

Single turnover (ST) active chlorophyll-a fluorometers utilize a short (<300 μs) saturation light pulse to induce charge separation and reduction of the primary electron acceptor Q_a within the PSII reaction center (Kolber and Falkowski, 1998). A major advantage of ST fluorometery is the additional measurement of the functional absorption cross section of PSII (σ_PSII) (Suggett et al., 2003), which measures the proportion of light captured by light harvesting proteins attached to a reaction center and utilized by PSII. In these instruments, saturation/excitation lamps are typically centered at a 450–500 nm wavelength to target peak absorbance of chlorophyll-a. However, by adjusting the excitation wavelength, it is possible to preferentially target other light harvesting photopigments. For example, by utilizing LEDs with peak emission centered at 447, 505 or 530 nm, we are able to preferentially excite at the peak absorption “soret” bands for the pigments Chl a/c₂, diadinoxanthin, and peridinin, respectively (Niedzwiedzki et al., 2013, 2014). Longer wavelengths such as 625 nm may transfer energy directly to the Chl a molecule (Niedzwiedzki et al., 2014). Preferential excitation may provide further insight into how pigment orientation and connectivity to the PSII reaction center may differ across environmental conditions or across symbiont types. However, such systems come at a high cost and alternative systems could be highly beneficial toward improving our understanding of Symbiodinium photobiology.

Hardware and software platforms designed to help teach coding and programming languages to the next generation of engineers are becoming increasingly popular within the scientific community. These platforms offer sophisticated microcontrollers as a base with which to design customized instrumentation at a low cost. The high cost of commercially available research instrumentation can be heavily restrictive to many laboratories. Research questions/initiatives often must cater to the available instrumentation, thereby limiting the scope of possible questions or experimental options. Open-sourced hardware and software platforms have the potential to revolutionize how scientists conduct research by allowing for cheap purpose built instrumentation. Sophisticated instrumentation can be cheaply designed and built around a specific research question/topic. For example, the MultispeQ is an instrument developed to compare photophysiological parameters in plants across the world in an effort to tie in physiological differences with known genetic variance (Kuhlgert et al., 2016). The OpenROV project (www.openrov.com) represents another open-source platform which utilizes both the Arduino and Beaglebone hardware to construct a low cost remotely operated underwater vehicle which can be adapted with different instrumentation for both recreational and/or research purposes. These low-cost, yet highly sophisticated instruments represent an emerging trend in research that have great potential moving forward.

In this study, we utilize open source platforms to construct a low-cost multispectral ST fluorometer to characterize potential photo-physiological differences across four symbiont types from two clades (A and B) living within the anemone Exaptasia pallida. We further characterized the light acclimation process within two of these symbionts and describe a spectrally dependent response which differed across the Symbiodinium types. This study serves as an example of the potential for furthering scientific inquiry through customized, purpose built instruments via the open source concept and designed for specific research questions.

Our results demonstrate wavelength dependent responses in both effective quantum yield and the functional absorption cross section of PSII across different symbionts. Symbiodinium have two major light harvesting complexes, the membrane bound Chla a-Chla C₂-peridinin-protein-complex (acpPC) and the soluble Peridinin-Chlorophyll Protein (PCP) complex, both of which absorb light predominantly between 400 and 550 nm (Iglesias-Prieto et al., 1991, 1993; Niedzwiedzki et al., 2013, 2014). Importantly, Iglesias-Prieto and Trench (1997b) demonstrated how the relative abundance of these different light harvesting compounds could vary across symbiont types growing in culture and/or with respect to high vs. low light acclimation. This was further refined by Hennige et al. (2009) whom used high performance liquid chromatography (HPLC) to compare the relative photopigment concentrations across eight different symbiont types. Such differences may give rise to the spectrally dependent differences across algal species observed here (Figure 2). Data from this study largely agree with previous work that used a commercially available multi-spectral pulse amplitude modulation (PAM) fluorometer with a symbiotic coral, that also documented clear wavelength dependencies in Symbiodinium photochemical activity (Szabó et al., 2014). In particular, the A4-b (strain: KLAp2) symbiont displayed a higher absolute PSII functional absorption cross section in the light activated state (σ_PSII′) relative to the other symbiotic algae, and similar to the results of Szabó et al. (2014) (albeit in their dark acclimated samples), higher (σ_PSII′) in lower excitation wavelengths (447–530 nm) which then decreased toward red excitation (Figure 2A). In contrast, the light acclimated PSII absorption cross section of the A4-a symbiont steadily decreased as excitation light moved from the blue to the red spectrum. Because the photopigments diadinoxanthin and peridinin have peak absorbance values near 505 and 530 nm, respectively, such divisions in σ_PSII′ could be driven by different levels of Chl a:diadinoxanthin or Chl a:peridinin ratios as well as light harvesting protein content. The decline in functional absorption cross section toward the red excitation spectrum is also naturally due to lower light harvesting complex absorption in the higher wavelength range. In corals, this also correlates to a higher scalar irradiance at higher wavelengths (Wangpraseurt et al., 2012), however, it is not known if a similar irradiance pattern exists in a symbiotic sea anemone that lacks a calcium carbonate skeleton.

The significant drop in chlorophyll a concentration in the high light acclimated A4-a and B1-a symbionts is a common photoacclimation response which significantly decrease the amount of light captured by the cell, thereby reducing the potential for excess excitation energy under high irradiance levels. Photoacclimation in Symbiodinium has been documented among a number of different species and the overall response is well-understood (Iglesias-Prieto and Trench, 1994, 1997b; Hennige et al., 2009; Finney et al., 2010). Similar to that observed during stress conditions, total organism absorbance may also decline, as symbionts reduce the amount of light that is absorbed and channeled toward photosynthesis (Rodriguez-Román et al., 2006; Hoadley et al., 2016). Although absorbance decreased in the high light acclimated anemones harboring the A4-a alga, there was no significant change in absorbance between the two acclimation light levels for the anemones harboring the B1-a symbiont. Despite the decline in Chl a cell⁻¹ in the high light acclimated B1-a, this lower absorption difference may have been due, in part, to the alga cell size which also declined during high light acclimation, thus chlorophyll cellular volume⁻¹ and pigment packaging may have remained similar across the two light levels.

Interestingly, acclimation to high light conditions reduced Fq′/Fm′ in both symbiont types, but only when excited with red spectrum (597–625 nm) wavelengths. Because photoprotective pigments such as xanthophylls do not absorb at these wavelengths, it is possible that the observed reductions in Fq′/Fm′ are indicative of decoupling between PSII reaction centers and light harvesting proteins. Apart from this reduction in Fq′/Fm′, the two holobionts showed marked photochemical differences in their patterns of photoacclimation, as the B1-a alga displayed a consistent down-regulation of functional PSII reaction centers (i.e., significant decline in Fv/Fm) (Figure 3B), while the A4-a symbiont reduced overall absorbance more and displayed a reduction in antennae-based non-photochemical quenching (Figure 4A) while Fv/Fm remained stable (Figure 3A). Under high light acclimation, microalgae tend to increase photo-protective mechanisms such as antennae-based non-photochemical quenching (Suggett et al., 2007). The reductions in non-photochemical quenching within the antennae bed observed for A4-a may possibly reflect a difference in optics associated with living inside the host anemone as opposed to free-living as is the case for most microalgal light acclimation studies. Coral tissues contain numerous fluorescent proteins which can substantially change the perceived light field for in situ symbionts (Dove, 2004). These host derived proteins may also differ across light and temperature gradients thus potentially also influencing symbiont response to elevated light conditions (Salih et al., 2000). Current efforts in understanding the miroenvironments in which Symbiodinium live (Wangpraseurt et al., 2012; Lichtenberg et al., 2016) are critical for improving our knowledge of coral/algal photoprotective mechanisms.

To ground-truth our fluorometer, we compared measurements of the same samples against a single excitation (peak 447 nm) Fluorescence Induction and Relaxation (FIRe) fluorometer (Satlantic Inc.). Both systems reported similar Fv/Fm values and overall trends in response to low and high light acclimation (Figure 6). Likewise, a similar response in functional absorption cross section was noted for both instruments, as high light acclimation led to elevated σ_PSII values. The slight reduction in Fv/Fm in the B1-a alga under high light acclimation noted with both systems is consistent with previously published work with most cultured Symbiodinium and reef corals (Lesser and Gorbunov, 2001; Hennige et al., 2009), however, the rise in σ_PSII under high light is not as common, and σ_PSII in cultured Symbiodinium tend to decrease slightly under high light (see Hennige et al., 2009 for one exception to this in a cultured F2 alga).

It is interesting to note that while the symbiont-anemone combinations used in this study contained a similar symbiont density and Chl. a algal cell⁻¹ (Table 2), multispectral active chlorophyll fluorescence revealed clear differences in symbiont photochemical efficiency that would be missed by a conventional fluorometer using a single wavelength band excitation light source. Importantly, symbiont density was normalized to the quantity of total soluble animal protein, and this does not account for possible structural differences between these two anemone populations, symbiont distributions across anemone location (e.g., tentacles vs. oral disc), or light absorbing animal pigments, all of which could significantly alter the photosynthetically usable radiation (PUR) in these animals further.

Different algal populations were separated by their light acclimated photochemical patterns (Figure 2C), but were not resolvable from dark acclimated samples alone (not shown). Spectrally dependent differences in algal photochemistry can not only provide more detail of photochemical differences within a single symbiosis, and also may potentially be a powerful tool for resolving genetically (and functionally) different Symbiodinium populations across diverse cnidarian hosts, including reef building corals. These results, while limited in scope, are similar to those of a more extensive study by Suggett et al. (2015) that examined 18 genetically distinct cultured Symbiodinium isolates with a single waveband excitation (peak excitation, 450 nm) fast repetition rate fluorometer. Specifically, that work noted that different phylotypes of Symbiodinium could be grouped into particular clusters based on the balance of how symbionts used the fraction of open PSII reaction centers (i.e., photochemical quenching or [1-C] vs. non-photochemical quenching [1-Q]).

There is a growing interest in the field of plant biology to better characterize genomic expression by characterizing specific phenotypic traits in a given environment, also known as phenomics (Furbank and Tester, 2011). Similar to the desire to link genomics to plant function and favorable agricultural traits, there is a growing need to characterize specific physiological traits of symbiotic reef building corals to their environment (Madin et al., 2016; Warner and Suggett, 2016). Active chlorophyll fluorescence is one such tool that has the potential to allow for rapid high throughput screening of photochemical response to different environmental conditions.

This work suggests that low-cost multispectral chlorophyll a fluorescence may prove useful in delineating functional traits as well as groups of different symbionts. Open source hardware and software platforms are becoming increasingly popular within the research community as their ease of use and low cost promotes high levels of customization for specific applications and a broad user base that reaches beyond traditional academic research. For example, the MultspeQ is a recently developed fluorometer designed to promote greater utilization across both scientific and community users interested in assessing terrestrial plant physiological responses via a phenomics-based approach (Kuhlgert et al., 2016). By utilizing chlorophyll fluorescence and absorbance based parameters, it is designed to provide a common platform to compare the photochemical traits of plant species worldwide. This device is relatively low cost, in part, due to the utilization and promotion of open access hardware and software platforms. The advantage of a greater user base also allows for higher level interpretation and analysis of the collected data. Although this current study does not offer a fully developed product for other users, it serves as an example of the type of research that can be achieved through the integration of open source platforms with specific research questions in mind and relatively inexpensive hardware. As more advanced hardware platforms adopt the open source concept, it is possible that even more sophisticated and low-cost instrumentation will become more accessible to a larger community of scientific and public community users interested in monitoring the physiological stability of algal-invertebrate symbioses as well.

In conclusion, spectrally dependent characteristics of symbiont photochemistry were effectively quantified using a multispectral chlorophyll-a fluorometer. It is likely that the spectrally dependent characteristics observed here correlate with differences in photopigment abundance across symbiont types as has been previously shown using immunoblot and HPLC analyses (Iglesias-Prieto et al., 1991; Iglesias-Prieto and Trench, 1997b; Hennige et al., 2009). With respect to symbiont types A4-a and B1-a, spectrally resolved Fv/Fm and σ_PSII values revealed additional changes in response to high-light acclimation that would not be possible if using more conventional active chlorophyll-a fluorometers with a single excitation wavelength centered at 450 nm. Importantly, these spectrally resolved fluorescence measurements were made using a custom built fluorometer using open source hardware and software platforms. Our work serves as an example of the potential for open source platforms to be utilized to construct purpose built and relatively low-cost instruments.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.