Newly emerging diseases of marine turtles, especially sea turtle egg fusariosis (SEFT), caused by species in the Fusarium solani complex (FSSC)

ABSTRACT Sea turtles are presently considered severely endangered species that are historically threatened by many environmental factors. Recently, additional threats to sea turtles from two pathogenic species of fungi in the Fusarium solani species complex (F. falciforme and F. keratoplasticum) have been identified. These species infect marine turtle eggs, causing sea turtle egg fusariosis, and kill their embryos, with recent reports of hatch-failure in seven globally distributed species of endangered sea turtles (Caretta caretta, Chelonia mydas, Dermochelys coriaceae, Eretmochelys imbricata, Lepidochelys olivacea, Lepidochelys kempi and Natator depressus). Mycelia and spores of pathogenic species of Fusarium are produced in disturbed terrestrial soils and are transported to the ocean in coastal run off. We propose that these fungi grow on floating particles of plant tissues (leaves and wood), animal tissues, silt and plastics, which are carried by wind and currents and the turtles themselves to the beaches where the turtles lay their eggs.


Introduction
In marine ecosystems the prevalence of infectious diseases caused by fungi has dramatically increased during the past two decades, likely due to the transmission of emerging pathogens into new environments and the rapid rate of global climate change (Altizer et al. 2003;Harvell et al. 2009;Groner et al. 2016). In fact, emerging infectious diseases are at present one of the main threats to global biodiversity (Altizer et al. 2003;Bax et al. 2003;Harvell et al. 2009;Hawkes et al. 2009;Lafferty 2009). Sea turtles are a well-known example of an endangered species whose populations are presently being significantly reduced in size by fungal infections (Sarmiento-Ramirez et al. 2014a;Reynolds et al. 2017). The general topics of turtle health and the effects of climate change on turtle populations were recently reviewed by Flint (2013) and by Hamann et al. (2013). The present review includes new information made available since these two reviews were published.
A large number of species of fungi infect both marine and freshwater species of juvenile and adult turtles and their eggs causing infectious diseases (Tables 1 and 2). In Table 1 we summarised reported fungal pathogens found recently in sea turtle species, to give an overview of how widely distributed fungal pathogens are within these marine animals and which species have been infected so far. The most abundant fungal orders are the Eurotiales and Hypocreales. The Fusarium species found in sea turtles and their nests belong to the order Hypocreales.
The primary focus of the present review is the ecology of pathogenic fungi in the Fusarium solani species complex (FSSC) and the relationship of these fungi to disease in marine turtles. A newly emerging fungal disease, sea turtle egg fusariosis (STEF), linked to sea turtle egg mortality in sea turtle nests worldwide, is caused by members of one lineage, the F. solani species complex (FSSC). The two common and cosmopolitan Fusarium species F. keratoplasticum and F. falicforme are known to be associated with STEF (Smyth et al. 2019). Fusarium solani Mart (Sacc) is defined on the basis of morphological characteristics but molecular investigations show that FSSC is actually a diverse complex of many phylogenetically distinct species (Short et al. 2013). The turtle-infecting strains of Fusarium have been placed by Zhang et al. (2006a) in the F. solani species complex group 2, which consists of many human, animal, and plant pathogens as well as environmental isolates (Sandoval-Denis et al. 2019).  (Sarmiento-Ramirez et al. 2014a. These two species of fungi infect the eggs of marine turtles in their nests resulting in low hatching success and subsequently lower population sizes in the oceans. F. falciforme and F. keratoplasticum have been implicated in hatch-failure in the seven globally distributed species of sea turtles (Caretta caretta, Chelonia mydas, Dermochelys coriaceae, Eretmochelys imbricata, Lepidochelys olivacea, Lepidochelys kempi and Natator depressus) (Sarmiento-Ramirez et al. 2014a).
The FSSC is thought to have a world-wide distribution (Sarmiento-Ramirez et al. 2017;Sandoval-Denis et al. 2019) and appears to include some of the world's most destructive pathogens, infecting both economically and ecologically important plant and animal hosts. These pathogens are found growing actively in multiple environments, including sink drains, human tissues, soil, silt, contact lenses and as saprotrophs in rich organic agricultural soils and in many other aquatic and terrestrial environments (environmental isolates) (Zhang et al. 2006b and2006c;Short et al. 2013;O'Donnell et al. 2015). A number of Fusarium species have been detected on sea turtles and their eggs. Eight different Fusarium species have been found associated with sea turtles and their nesting beaches (Table 2).
Fusarium is the major source of infection of sea turtle eggs and nests. Infections by Fusarium species (Figure 1) have been reported in nests and eggs of sea turtle for both wild and captive sea turtles. Recently reported Fusarium infections according to sea turtle species are summarised in Figure 2. The majority of reports come from loggerhead, green and hawksbill turtles and were from Australia ( Figure 3). Other parts of the world including Europe, North and South America and Asia have 3-4 reports, followed by Africa with only one reported case.
Plant diseases caused by species of Fusarium pose a significant threat to the human food supply and agricultural biosecurity in soil and in other terrestrial ecosystems (O'Donnell et al. 2015). Species of Fusarium can be a threat to marine animals in aquaculture as well, such as in commercial culture of the lobster (Homarus vulgaris) (Alderman 1981). The scope of the present review is restricted to diseases of marine turtle eggs caused by fungi in the FSSC.

Classification
The genus Fusarium contains a very large number of species, one of which is the species F. solani. The

The aetiology of sea turtle egg fusariosis
Pathogenic fungi grow in the nests of marine turtles by initially forming mycelial networks on damaged eggs. These mycelial networks subsequently cover the surface of the eggs, produce enzymes and organic acids which degrade the shells by dissolving the organic substrates and calcium carbonate and finally penetrate the viable tissues inside. Infected eggs develop yellowish-blue infection zones that eventually become necrotic lesions and eventually kill the living embryos itself Sarmiento-Ramirez et al. 2010).
The eggs appear to become infected through contact with the sand around the nests and by gravid turtles and are less susceptible in dry sand than sand in intertidal zones or clay/silt soils (Abella-Pérez 2011; Phillott et al. 2002;Sarmiento-Ramirez et al. 2014b). Infected eggs within a clutch can serve as a source of inoculum for neighbouring eggs, with infection beginning on non-viable eggs (Phillott and Parmenter 2001).
Because of the presence of microorganisms in the egg chamber sand, it has been suggested that sea turtle cloacal fluid contains antimicrobial properties to protect the developing eggs (Keene et al. 2014). This clear, sometimes viscous, fluid contains glycoproteins and coats eggs as it is secreted from the cloaca during egg deposition into the nest chamber. As such, olive ridley turtles deposit 250-500 ml of fluid in a single nest, which could potentially provide protection if antimicrobials are present in the fluid (Keene et al. 2014). The warm, moist environment and presence of organic matter at the nesting site of sea turtles is ideal for the growth of soil fungi that contribute to the hatching failure of the eggs either by decomposing the eggs and/or secreting mycotoxins that negatively affect the developing embryos. Fungi implicated in egg failure have been isolated from soil at nesting sites; exterior and/or interior of unhatched eggs; and embryonic tissue of the eggs. Although sandy soil from the nesting sites of the sea turtles often lack an adequate amount of organic matter for the rapid growth of fungi. Large amounts of organic matter represented by egg shells of hatched and failed eggs are added annually to the nesting sites (Bézy et al. 2014). This creates an optimum medium for fungal growth and sporulation that subsequently contributes to egg contamination. (Abella-Pérez 2011; Phillott and Parmenter 2001;Sarmiento-Ramirez et al. 2014b).
The most common fungi reported from soil at the nesting sites and from failed eggs were species in the genera Aspergillus and Fusarium (Neocosmospora). These fungi were found growing on 29% and 4% of the failed and hatched eggs respectively. Fungal hyphae were found growing on the surface of failed eggs, inside the eggshells, and on the egg membranes (Elshafie et al. 2007). Although some isolates are known to be potential pathogens, the presence of these fungi does not necessarily lead to the development of disease. During embryonic, development, the eggs are incubated for a long period covered by sand under conditions of high humidity and constant warm temperature, which are known to favour the growth of soil-borne fungi such as F. solani. However, these conditions may not be the only factors determining disease development (Sarmiento-Ramirez et al. 2010). Sarmiento-Ramirez et al. (2010) have also examined and detected the presence of F. solani in nests with asymptomatic eggs. This suggests that other factors such as specific microclimatic conditions, sand composition and natural immunosuppression influence the development of disease. The developing immune system gains full maturity and competence only during and after embryonic development of embryos. In addition, immunosuppression, e.g. due to accumulation of toxic substances in turtles and their eggs, etc. may be determining the development of the disease.
The close proximity of eggs within the sea turtle nest could also allow fungal growth to influence eggs without direct contact. For example, volatile mycotoxins or other metabolites, that originate from fungal growth on the exterior of one egg, could affect adjacent eggs, and could have a detrimental effect on the development and condition of hatchlings (Phillott and Parmenter 2014).
Marine turtles, while feeding in the ocean from the surface down to and including the benthos, frequently ingest a variety of solid particles including insoluble inorganic particles (such as silt), insoluble organic particles (such as plant leaves and wood and tissues of animal origin) and microplastic particles (Duncan et al. 2018;Matiddi et al. 2017). Species of Fusarium and Aspergillus which are pathogenic to animals including humans are known to colonise these substrates (Das and Kumar 2014;Oberbeckmann et al. 2015). If F. solani spores or hyphae are attached to these particles, viable fungal cells can enter the digestive system during feeding, go through the digestive system and be left in faeces in the turtle nests where they can grow on fried cellulose containing plant substrates (Keene et al. 2014).
The possible roles of mycotoxins and proteases excreted by Fusarium and protease inhibitors excreted by turtles in diseases of marine turtle eggs Species of fungi which are plant pathogens or which digest plant cell wall materials saprotrophically in the soil would be expected to excrete enzymes which demonstrate high effectiveness for natural lignocellulosic biomass degradation and utilisation. In particular, species of Fusarium are indeed robust cellulose and hemicellulose degraders and excrete extracellular cellulases and xylanases which are stable over the wide pH and temperature ranges normally found in the soil (Huang et al. 2015). These pathogens have optimal growth temperatures of 28-29.7°C, similar to the temperature requirements of sea turtle embryos. Successful incubation of sea turtle eggs requires a narrow thermal range of 25°C to 35°C with varying tolerances between species and populations of sea turtles (Mrosovsky et al. 1992;Montero et al. 2019).
In addition, many species of Ascomycota, including some species of Fusarium, are known to excrete mycotoxins. Mycotoxins can be toxic to many species of animals which are either directly infected by pathogenic species of Fusarium (or by other Ascomycota) or which eat food resources such as plant materials infected by one of the mycotoxin-producing species. This topic has recently been reviewed by Duan et al. (2016), Azliza et al. (2014) and Lemmens (2012).
It is possible that mycotoxins excreted by Fusarium solani and other fungi growing on the surface of egg shells might diffuse into tissues of the embryos ultimately causing death. In fact, some species of Fusarium are known to produce a number of mycotoxins in culture, including fumonisins. If these toxins are produced in and on the surface of eggs they can affect embryo development (Elshafie et al. 2007). Elshafie et al. (2007) found aflatoxins in 40% of the eggshells studied at a concentration ranging between 4.1 and 8.4 ppb and in 25% of failed egg's contents at concentrations of 0.14-2.0 ppb. This level of aflatoxins in eggshells and egg contents is high enough to cause embryo mortality (Elshafie et al. 2007).
Proteases excreted by F. solani and other fungi might digest embryo cells as well. Olivieri et al. (2002) characterise a serine protease excreted by F. solani. Lowe et al. (2015) found that F. graminearum excretes a complex suite of extracellular proteases including metalloprotease, cysteine, serine, threonine and aspartate proteases.
However, some turtles have other defensive strategies to prevent disease. For example, they can protect their embryos from damage by proteases because their egg whites contain protease inhibitors. Ray et al. (1982) purified and characterised an acidic trypsin/subtilisin inhibitor in turtle eggs.

Other species of fungi infecting sea turtles
Species in the FSSC are not the only fungi found to penetrate sea turtle egg shells. For example, Candan (2018) also identified several species of Aspergillus, Emericella, Rhizopus, Actinomucor, and Apohysomyces in the nests of living green turtles using molecular methods. There are reports of fungal pathogens of sea turtles in many parts of the world, including Australia, North America, South America, Asia, Europe and Africa (Table 1). In Figure 4, we show the distribution of diseases caused by fungal pathogens of turtle species in the world. Fungal infections of the loggerhead (Caretta caretta) have been reported around the world. The reports of fungal infections in the green turtle (Chelonia mydas) have not been found in Africa and South America. No reports of the hawksbill turtle (Eretmochelys imbricata) are known from Europe, North America and Africa. Fungal infections of olive ridley (Lepidochelys olivacea) and the leatherback turtle (Dermatochelys coricea) have only been reported in South America, whereas reports of Kemp's ridley (Lepidochelys kempi) have come from North America and of the flatback turtle (Natator depressus) only from Australia.
Species of fungi which appear to be pathogenic to turtle eggs have been observed in the fossil record. For example, Jackson et al. (2009) observed a species of Penicillium penetrating the shells of fossilised eggs from a lower Cretaceous geological formation in China. Fungal skin diseases are thought to be a major concern to the health of captive marine turtles (Orós et al. 2004b). All fungi so far identified from turtle skin lesions are common saprophytes (Wiles and Rand 1987). Fusarium solani, Aspergillus sp., Geotrichum sp., Penicillium sp., Scolecobasidium sp., Fusarium sp., Drechslera sp. and several unidentified fungi have been isolated from skin lesions in sea turtles.
Turtles can also be infected by systemic diseases such as mycotic granulomas (Orós et al. 2004a). Although mycotic granulomas can be found in the liver and throughout the coelomic cavity of wild and captive sea turtles, systemic mycotic infections occur primarily in the lungs. In captivity water quality appears to be an important factor in the incidence of cutaneous diseases due to the crowded conditions in the husbandry tanks (Orós et al. 2004a). The increased amount of nutrients in the water favour the growth of fungi in general.

Discussion and conclusions
The spread of fungal diseases is a serious threat to biodiversity especially due to the increasing number of pathogenic fungi that threaten wildlife and domesticated species worldwide (Sidique et al. 2017). Most fungal infections in chelonias are considered opportunistic and secondary to underlying health conditions or accelerated by immunosuppression. Factors that may predispose an individual include suboptimal environmental temperatures and humidity, nutritional imbalances, infections by other pathogens, and trauma (Donnelly et al. 2015). But not only adult animals in the ocean are threatened by fungal pathogens, the nests of chelonians and the eggs are especially susceptible to fungi. According to some researchers, the hatching success depends upon the interaction of a number of factors such as salinity, humidity, temperature, gas flow, rainfall, tidal inundation, erosion and predation (Özdemir and Türkozan 2006). The recognition of the FSSC as a potential global threat to sea turtle eggs represents another instance of the importance of an emerging fungal infection impacting on wildlife (Bailey et al. 2018).
In the present review we propose mechanisms for the transport of conidia and hyphae of the pathogenic species of Fusarium produced in disturbed soils in terrestrial ecosystems to the turtle eggs at the beach. Pathogenic species of fungi can be transported from terrestrial ecosystems to the ocean primarily in run off. These fungi can grow on floating particles of plant tissues, silt and plastics in the ocean which are carried by wind and currents to the beaches where the turtles lay their eggs. These fungi are also carried by the turtles themselves in the digestive system (Clukey et al. 2018;Duncan et al. 2018).

Highlights
• Seven species of sea turtles are presently considered severely endangered species   Table 1.