Coral reefs are vulnerable ecosystems impacted by a range of environmental disturbances across small and large spatial scales (Bellwood et al., 2004) and from natural and anthropogenic sources (Hughes et al., 2003; Burke et al., 2011). Intensifying anthropogenic impacts, including climate change, have contributed to recent declines in reef-building corals (Hoegh-Guldberg et al., 2007; De’Ath et al., 2012; Hughes et al., 2018). Declines in live coral cover can impact key population-level processes, including reproductive capacity and concomitant replenishment of corals via coral larval recruitment, a primary driver of recovery on degraded reefs (Harrison and Wallace, 1990; Mumby and Harborne, 2010; Gouezo et al., 2019). These early life stages often represent a bottleneck in the successful reproduction and recruitment process for marine invertebrates (Pineda, 2000). Therefore, negatively impacted stock-recruitment processes reduce the overall ability for coral communities and reefs to naturally recover (Hughes et al., 2019).

Reef restoration is a pathway to recover degraded reefs and is increasingly included in natural resource management (Edwards, 2010; Omori, 2019; Boström-Einarsson et al., 2020). Coral restoration has routinely relied on using asexually produced coral fragments to restore corals (Rinkevich, 2005), but the use of sexually derived coral larvae for coral and reef restoration is rapidly increasing (dela Cruz and Harrison, 2017; Doropoulos et al., 2019; dela Cruz and Harrison, 2020; Harrison et al., 2021). Using coral larvae for restoration relies on the high fecundity of broadcast spawning coral colonies (Harrison and Wallace, 1990), allowing collection of millions of gametes on reefs and in laboratory settings. Collected gametes enable the mass culture of larvae which allows restoration efforts to supply sufficiently high numbers of larvae to degraded reefs. Equally critical is ensuring settlement of coral larvae and metamorphosis into settled polyps, and survival and growth into juvenile corals. These early life stages are an essential step in recruitment to adult populations (Harrison and Wallace, 1990; Richmond, 1997) and dictate the effectiveness of restoration efforts (Boström-Einarsson et al., 2020).

Most wild-spawned coral larvae die or are lost from reef systems during their planktonic larval phase or in the first few weeks and months post-settlement (Harrison and Wallace, 1990; Wilson and Harrison, 2005; Doropoulos et al., 2016; dela Cruz and Harrison, 2017; Harrison et al., 2021), and there remain many questions around the biological determinants of settlement and recruitment processes for corals. For example, endogenous energy stores in non-feeding larvae are a limiting factor that dictate settlement outcomes (Strathmann, 1985; Marshall and Keough, 2003; Botello and Krug, 2006; Graham et al., 2013a). Egg provisioning impacts larval competency and the initial stages of settlement and post-settlement survival. Prolonged larval phases are thought to decrease settlement rates as energy stores are depleted, thereby compromising the potential to complete metamorphosis with increasing age (Wilson and Harrison, 1998; Graham et al., 2008). Therefore, the dynamic interplay between larval endogenous energy stores, settlement success and the latent effects on recruitment success highlight the importance of understanding the link between larval energetics and restoration efforts (Harrison and Wallace, 1990; Graham et al., 2013b).

While coral larvae are considered lecithotrophic, provisioned with sufficient energy needed during their larval phase, competency periods for broadcast spawning coral larvae are often longer than energetic models predict (Tranter et al., 1982; Wilson and Harrison, 1998; Nozawa and Harrison, 2002). As a result, some researchers suggest that certain species of coral larvae gain exogenous nutrients to bridge these gaps (Fadlallah, 1983; Arai et al., 1993; Zaslow and Benayahu, 1996; Graham et al., 2013a; Rivest et al., 2017). One important source of nutrients for coral larvae appears to be dissolved organic material (DOM) (Sorokin, 1973; Fadlallah, 1983; Graham et al., 2013a). Moreover, there is evidence that free amino acids contribute up to 11% of the metabolic needs of coral larvae (Zaslow and Benayahu, 2000) and may play a role in inducing settlement (Baird and Morse, 2004). Other nutrients may also be physiologically important, for example, Pocillopora damicornis larvae have been found to lack sufficient quantities of nitrogen and phosphorous necessary for metamorphosis (Titlyanov et al., 1998). Similarly, when deprived of all sources of exogenous nutrients, Porites porites larvae cannot settle successfully (Fadlallah, 1983).

In addition to the assimilation of DOM, there is evidence that some coral larvae actively feed on particulate matter via ciliary currents and mucus strings (Tranter et al., 1982; Harii et al., 2009). The use of ciliary currents to move particulate matter to the oral pore has been observed in the larvae of Stylophora pistillata (Rinkevich and Loya, 1979), Cyphastrea ocellina (Wright, 1986), Pocillopora damicornis (Richmond, 1985) and Fungia scutaria (Krupp, 1983), and is common in other single-banded ciliated invertebrates (Strathmann et al., 1972; Paulay et al., 1985). In conjunction with ciliary currents, larvae have been observed using mucus strings to capture food particles (Tranter et al., 1982; Schwarz et al., 1999; Harii et al., 2009) in a process known as mucociliary transport (Young, 1971; Brown and Bythell, 2005). In this process, nutritive particulate matter is captured in mucus and transported to the oral pore, where it is then ingested (Tranter et al., 1982; Schwarz et al., 1999; Harii et al., 2009). With evidence that certain species of coral larvae are capable of gaining exogenous nutrients, the capacity to access exogenous nutrients may confer advantages to larval survival, settlement and post-settlement success.

Therefore, a strict dichotomy of binary nutritional modes - feeding or non-feeding - does not suit the wide biological variation seen among coral larvae. Alternatively, researchers have proposed an intermediate nutritional mode - facultative planktotrophy - to account for similar variation in other non-coral marine invertebrates (Allen and Pernet, 2007; Collin, 2012; Pernet, 2018). This nutritional mode is characterised by larvae capable of feeding but which are not required to do so to complete metamorphosis (Boidron-Métairon, 1995). Furthermore, when fed, facultative planktotrophic larvae often metamorphose into larger juveniles and exhibit increased post-metamorphic survivorship (Boidron-Métairon, 1995; McEdward, 2000; Allen and Pernet, 2007). However, to date, there are limited data that examine the relationships between the supply of exogenous nutrients and settlement in coral larvae. Therefore, these critical aspects of coral larval biology and ecology warrant more detailed investigation. Hence, the present study aims to quantify the effect of feeding on coral larval settlement and early post-settlement survival. Specifically, we hypothesise that rearing larvae with access to exogenous nutrients will enhance settlement and potentially improve post-settlement survival.

This experiment was conducted on Bindal country at the Australian Institute of Marine Science’s (AIMS) National Sea Simulator (SeaSim) in Townsville, Australia, to assess the effect of supplying nutrients to coral larvae on their settlement and early post-settlement survival. Aposymbiotic larvae of two broadcast spawning corals were used - Acropora tenuis and Acropora millepora. Both species are widespread in the Indo-Pacific region and common on a range of reef sites (Wallace, 1999). Acropora tenuis has been previously used in ex-situ larval restoration studies (dela Cruz and Harrison, 2017; Cameron and Harrison, 2020; Harrison et al., 2021). Furthermore, both species have been successfully spawned and reared in aquaria with well-established culturing and settlement techniques (Heyward and Negri, 1999; Humanes et al., 2016; Conlan et al., 2017; Pollock et al., 2017).

The results of this study demonstrate that rearing larvae with homogenised Artemia as a source of nutritive particulate matter doubled settlement rates for Acropora tenuis larvae and were 2.5 times higher for Acropora millepora larvae. This result suggests that endogenous energy content may be a limiting factor affecting larval settlement patterns. It has been demonstrated that Acropora tenuis larvae can typically lose between 30% and 75% of their energy stores within the first week of development (Harii et al., 2007; Figueiredo et al., 2012; Graham et al., 2013a), a result which is reflected in lipid depletion across other Acropora species (Harii et al., 2007; Okubo et al., 2008; Harii et al., 2010; Figueiredo et al., 2012; Graham et al., 2013a). Previous studies have shown that some coral larvae have the ability to feed (Tranter et al., 1982; Titlyanov et al., 1998; Schwarz et al., 1999; Harii et al., 2009). Therefore, by providing access to exogenous nutrients, larvae could supplement their endogenous energy stores, extending their potential to settle successfully and survive. This advantage is clearly seen in corals that produce larvae with photo-symbionts, which use photosynthetically derived nutrition to enhance survival and settlement (Harii et al., 2010). Photosymbiotic larvae reared under light were more likely to survive until settlement than larvae reared in the dark, as light-reared larvae could supplement up to 40% of their metabolic demands with exogenous nutrients (Harii et al., 2010). The results of the present study are consistent with the results of previous studies examining enhanced settlement associated with increased nutritional potential (Richmond, 1985; Schwarz et al., 1999; Harii et al., 2010).

In addition to increased settlement of fed larvae, another key finding from the present study was the increased post-settlement survival in A. millepora and A. tenuis spat. Mortality in the first year after settlement can be extremely high (Babcock and Heyward, 1986; Babcock and Mundy, 1996; Wilson and Harrison, 2005; Davies et al., 2013; dela Cruz and Harrison, 2017; Suzuki et al., 2018) and is highest immediately following settlement, declining markedly as coral spat increase in size (Martinez and Abelson, 2013; Doropoulos et al., 2016; Harrison et al., 2021). High early mortality may be due to the energy investment required for larvae to complete metamorphosis, as this process represents a significant energy investment for corals (Harrison and Wallace, 1990; Graham et al., 2013a). Therefore, coral larvae with lower energy stores may deplete these energy stores during metamorphosis and die shortly thereafter. However, for larvae provisioned with ample energy stores, metamorphosis into a coral spat should allow sufficient energy reserves to meet metabolic demands until they acquire photosymbiotic Symbiodiniaceae and gain photosynthetically derived nutrition (Harii et al., 2007; Harii et al., 2009; Harrison et al., 2021). Once the coral spat gain symbionts and develop tentacles, they can begin to feed in the same manner as adults, namely through autotrophy and planktotrophy, respectively. The high early mortality of coral spat from the unfed treatments in this study supports the hypothesis that some larvae run out of energy shortly after metamorphosis. Furthermore, the increased early post-settlement survival of both A. millepora and A. tenuis spat derived from larvae that were supplied with homogenised Artemia in this study, highlights the potential for supplying exogenous food to supplement energy stores to enhance post-settlement survival.

Although there was an initial difference in survival between A. tenuis spat from fed and unfed treatments, by seven days post-settlement, survival was similar between treatments. Fed A. tenuis larvae settled in much higher numbers than unfed larvae, even with consistent settlement surface availability, resulting in a higher density of spat in the fed treatments than the unfed treatments. Therefore, similar survival rates among recruits from both the fed and unfed A. tenuis larvae ( Figure 4 ) may have been influenced by post-settlement competition for space rather than metabolic demands. Juvenile survival after settlement can be affected by various ecological processes such as predation, sedimentation, and density-dependent competition (Ritson-Williams et al., 2009; Ricardo et al., 2017; Cameron and Harrison, 2020; Randall et al., 2021) While accidental grazing and predation are large sources of mortality in in-situ environments (Baria et al., 2010; Gallagher and Doropoulos, 2017), those pressures were not present in the current laboratory experiment and therefore, unlikely to influence the observed survival rates. Alternatively, density-dependent processes are known to impact survival as spat compete for space (Martinez and Abelson, 2013; Cameron and Harrison, 2020; Randall et al., 2020). With overall higher settlement in the fed A. tenuis larval treatment compared with the unfed larvae ( Figure 2 ), mortality due to space competition could have been higher in the fed than in the unfed treatment, resulting in less spat from fed treatments surviving overall. Moderate densities reduce space competition among newly settled spat (Cameron and Harrison, 2020). Therefore, more research is needed to optimise settlement density to maximise recruitment success.

While A. tenuis and A. millepora larvae benefit from being reared with access to exogenous nutrients, the exact feeding mechanism could not be determined during this study. Whether larvae assimilate nutrients via osmotrophy or ingest particulate matter through direct planktotrophy is yet to be resolved. However, due to their ability to utilise nutrients, A. tenuis and A. millepora are likely facultative planktotrophic rather than strictly lecithotrophic. Regardless of the feeding mechanism, the ecological relevance of enhanced settlement is clear- as is the importance of this approach for developing new larval rearing protocols for mass culture of coral larvae in future. Species-specific differences in larval quality impact settlement success in marine invertebrates (Marshall and Keough, 2004) with egg size and lipid composition influencing post-metamorphic performance (Pechenik, 2006; Pechenik, 2018). Furthermore, egg size, lipid composition, and lipid depletion are known to vary across acroporid coral larvae (Richmond, 1987; Zaslow and Benayahu, 2000; Graham et al., 2013a). For example, A. tenuis has been shown to deplete energetic lipids at a faster rate than other Acropora coral larvae examined (Graham et al., 2013a). These differences in egg quality may have resulted in a differential benefit of exogenous nutrients between A. tenuis and A. millepora. Therefore, differential utilisation of exogenous nutrients may be contributing to the differences in the mean settlement between A. tenuis and A. millepora. However, direct uptake of nutrients between species was not measured in the present experiment.

Overcoming high post-settlement mortality bottlenecks is a high priority goal in coral restoration (Boström-Einarsson et al., 2020; Randall et al., 2020). Current larval restoration techniques supply large numbers (often millions) of cultured larvae to degraded reefs (dela Cruz and Harrison, 2017; dela Cruz and Harrison, 2020; Harrison et al., 2021). Once on the reef, cultured larvae face the same barriers to settlement, survival and recruitment as wild-spawned larvae. Therefore, rearing cultured larvae with access to exogenous nutrients could produce energetically enhanced larvae with an increased capacity for settlement and post-settlement survival, thereby significantly increasing the efficiency of larval restoration efforts.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

PH provided the overall rationale for the study. CR, SW, and PH conceived and designed the experiment. CR and CH conducted the experiments. CR analysed the data and wrote the draft manuscript. PH obtained the primary research funding. All authors edited and approved the final draft.

Funding was provided by a Paul G. Allen Family Foundation grant to PH, with additional funding and support from the Australian Institute of Marine Science, and an SCU postgraduate research grant to CR.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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