The ability to steal functional chloroplasts from algae and retain them as endosymbionts is unique in the animal kingdom, found only in some sacoglossan sea slugs and a few species of rhabdocoel flatworms (Trench 1969, van Steenkiste et al., 2019). This ability has been termed functional kleptoplasty (Rumpho, et al., 2011). In sacoglossan slugs, kleptoplasts (stolen chloroplasts) are obtained directly from their algal food. Using a radular tooth, the slug pierces algal cells and sucks out the cytoplasm (Jensen, 1980). The chloroplasts are isolated from the rest of the algal cytoplasm and retained in digestive gland epithelial cells (Trench 1969).
Functional kleptoplasty could provide a slug with numerous benefits including camouflage, and/or energy and oxygen produced via photosynthesis, yet prolonged retention for (> 1 month) has only been observed in seven species, and likely originated independently in 4 + lineages (Christa et al., 2014). To date, only ~ 50 species have been investigated out of 400 + described sacoglossan species. Since sacoglossan diversity is likely underestimated and screening for kleptoplastic potential is rarely performed when new species are described, the diversity of kleptoplastic species may be much higher.
The recently described sacoglossan, Caliphylla yemanjae (Alves et al. 2022) feeds on the chlorophyte algae, Bryopsis plumosa (Hudson) C. Agardh, 1823 (Fig. 1A-B) and has thus far, only been observed in Brazilian waters. Since chloroplasts from B. plumosa can be retained by other sacoglossans for more than one month (Christa et al. 2014), we predicted that C. yemanjae may also demonstrate long-term kleptoplastic potential. To assess this, specimens were collected at 0-0.5m depth on Curacao in the Southern Caribbean, a locality where they have not been previously described. At the CARMABI field station, they were housed in 0.5L tanks containing continuously aerated seawater. Full spectrum light was provided for 12 hours each day via LED lamps (LEDVANCE GmbH, Germany) that provided 400 µmol m− 2s− 1 light intensity. Kleptoplast retention was assessed in three ways:
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Non-kleptoplastic slugs digest their chloroplasts rapidly and their coloration rapidly changes from green to whitish or transparent (bleached). Visual inspections were performed daily to examine the coloration of six C. yemanjae specimens in starvation. After five days, every individual was completely bleached (Fig. 1C-D). They also lost multiple cerata (dorsal projections of mantle tissue) and decreased in length. By the seventh day, a considerable proportion of individuals had died or were highly debilitated.
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To observe chloroplast density via autofluorescence (chlorophyll excitation = 350–450 nm, emission = 630–750 nm), several cerata were removed from unstarved and five-day-starved specimens and imaged using an epifluorescence microscope (Nikon e800 Eclipse, Nikon Worldwide). Unstarved specimens contained chloroplasts throughout their digestive gland tissues, but starved specimens no longer emitted detectable fluorescence (Fig. 1C-E).
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Specimens were also examined with a Pulse-Amplitude Modulated fluorometer (white light version, Walz GmBH, Germany) to determine the maximum quantum yield of photosystem II (Fv/Fm), which indicates the photosynthetic efficiency. Two specimens, unstarved and five days-starved, were measured three times in different regions along their body displaying a mean of Fv/Fm 0.640 ± 0.03 and 0.494 ± 0.037, respectively. High specimen mortality at later starvation time points prevented subsequent Fv/Fm measurements.
Both green coloration and chlorophyll autofluorescence disappeared after a few days in starvation, indicating that C. yemanjae is not able to retain functional chloroplasts for an extended time span (Fig. 1C-E). As Fv/Fm measurements are relative values and cannot be used to quantify chlorophyll, the moderate PAM values we recorded in five-day-starved specimens indicate that some degree of photosystem II activity is still possible. This likely stems from a small amount of residual chlorophyll, since intact kleptoplasts were not observed under the fluorescence microscope. These observations suggest C. yemanjae is capable of kleptoplast acquisition but incapable of long-term retention, and that kleptoplast acquisition fails to energetically sustain C. yemanjae during starvation. Since B. plumosa chloroplasts can be retained in other sacoglossans (Christa et al. 2014), the lack of kleptoplasty in this species is likely caused by the slug lacking the ability to retain what it acquired. Continued surveys of sacoglossan species for their kleptoplastic abilities remain necessary to understand the evolution of this remarkable ability and uncover why some sacoglossans are kleptoplastic, while others, like C. yemanjae, are not, especially when these species feed on the same algal food source.