Methods

Twenty soil and eggshell samples were collected from the nesting sites of the green turtles at a depth of 25 cm within 24–48 hours of hatching. One gram of each sample was diluted by serial dilution method and plated onto petri dishes containing potato dextrose agar (PDA, Oxoid) and incubated at 29°C for 10 days. The mycoflora of the soil and eggshells samples were determined. Eggs were collected from the same site immediately after oviposition, incubated at 29°C at 95% relative humidity up to 70 days.

Aflatoxins were extracted from 20 A. flavus cultures, eggshells, and failed egg contents according to manufacturer instructions (Vicam). Five grams of NaCl and 100 mL of 80% high pressure liquid chromatography (HPLC) grade methanol (800 HPLC grade methanol plus 200 mL distilled water) were added to A. flavus cultures, eggshells, and failed egg contents, separately and were blended for 1 minute. The extracts were filtered through a filter paper and collected in a beaker. Ten milliliters of each filtered extract was diluted with 40 mL distilled water and filtered through a glass microfiber filter.

Ten milliliters of the filtrate was twice passed through aflatest B affinity column (Vicam) at a rate of 1–2 drops per second. Ten milliliters of distilled water was twice passed through the column at a rate of 2 drops per second. The column was eluted by 1 mL of HPLC grade methanol at a rate of 1–2 drops/second. The elute was collected in a glass cuvette. One milliliter of aflatest developer (Vicam) was added to the elute. Aflatoxins were quantified by Vicam fluorometer in parts per billion.

Eggs of green turtles were collected from the same area for other laboratory experiments investigating temperature and sex ratio determination. Three clutches of eggs (100 eggs/clutch) were collected directly from the ovipositor into plastic buckets containing sand from the nesting sites. Upon arrival at the laboratory, each egg was incubated separately in sterile polystyrene container (8 cm diameter, 11 cm deep) containing sterile vermiculite. Three control containers containing sterile vermiculite without eggs were also incubated. The bottom of the plastic container was padded with soft polystyrene. Holes were made on the side and top of the cover. Eggs were incubated at 24–31°C, 95% humidity for up to 70 days. Incubated eggs were checked for fungal growth. Mycelia and spores of fungi developing on failed and unfailed eggs were transferred by sterile needles to petri dishes containing PDA and were incubated at 29°C for 10 days. Slides were prepared from cultures of isolates and fungi were identified following Raper and Fennell (1965), Nelson et al. (1983), Ellis (1971, 1976), and Klich and Pitt (1994).

Fungi developing on and in eggs and eggshells were examined by light microscopy and scanning electron microscopy, during which small pieces of egg shell (1 cm²) were placed on top of a carbon background on aluminum stubs and allowed to air dry in a sterile petri dish, followed by gold coating (BIO-RAD, SEM Coating System, UK) for 10 minutes. The samples were placed in the SEM (JEOL 5600 LV- Low Vacuum, Japan) viewed with secondary electron detector (SE at 5 kV). Electronic images were captured.

Results

Eight genera and 14 species of fungi were isolated from soil and eggshells of C. mydas (Table 1). Most of the fungi found in soil were also present in the eggshells. The most commonly represented genus was Aspergillus, followed by Fusarium, Rhizopus stolonifer, and Penicillium species. Some fungi (Emericelula nidulans, Eurotium amystelodami, E. rubrum, and Trichoderma viridis) were found on eggshells but not in soil. Fungi isolated from nonnesting soil were A. flavus, A. niger, A. terreus, and Penicillium sp. Fungi were found growing on 29% and 4% of the failed and hatched eggs, respectively. Hyphae were found growing on the surface of failed eggs (Fig. 1), inside the eggshells (Fig. 2), and on the egg membranes (Fig. 3).

Table 1. The mycoflora of soil and eggshell samples at Ras Al-Jinz.

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Seventy-five percent of the A. flavus strains isolated from eggshells and grown in liquid media were found to be aflatoxigenic (Table 2), producing aflatoxins at concentrations ranging between 0.3 and 28 ppb. Aflatoxins were detected in 40% of eggshell samples at concentrations between 4.1 and 8.4 ppb (Table 3) and in the contents of 25% of failed eggs at concentrations of 0.14–2.0 ppb.

Table 2. Amount of aflatoxin produced by aflatoxigenic strains of Aspergillus flavus.

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Table 3. Aflatoxin content of eggshells.

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Discussion

Eight genera and 14 species of fungi were isolated from soil and eggshells. The genus Aspergillus was the most dominant. Solomon and Baird (1980) and Mo et. al. (1990) reported Aspergillus species as the most dominant species on green and olive ridley sea turtle eggs. A number of Aspergillus species isolated in this study are one known to be mycotoxin producers. Strains of A. flavus produce aflatoxins; A. niger produce malformic C and nigragillin; A. ochraceus produce ochratoxin; A. terreus produce citrinin and patulin; and Penicillium sp. produce citrinin and penicillic acid (Singh et al. 1991; Moss 2002). If these toxins are produced in and on eggs they may affect embryo development.

Fusarium sp. are common cosmopolitan soil fungi that occur widely. In this study, we isolated F. moniliforme from soil and eggshells. Some strains of this fungus produce a number of mycotoxins including fumonisins (Nelson et al. 1983). Phillott et al. (2001) isolated F. oxysporum and F. solani from eggs of green and loggerhead sea turtles. A. niger and C. cladosporoides are dematiaceous hyphomycetes that on growing on eggs give them a black appearance. The black coloration on eggs of turtles usually reported by workers may be due to contamination by fungi. E. nidulans is the telemorph of A. nidulans, E. amstelodami is the telemorph of A. hollandicus, and E. rubrum is the telemorph of A. rubrobrunneus. These telemorphic fungi are common in tropical and subtropical regions (Klich and Pitt 1994) and are capable of growing and producing toxic metabolites when they find a rich medium. In soil they are usually found in their anamorphic state.

All the fungal taxa found in this study are saprophytic soil fungi that are cosmopolitan and common decomposers of organic matter present in soils. They are capable of decomposing turtle eggs, producing mycotoxins that may affect egg hatching. Peters et al. (1994) reported that egg failure of loggerhead sea turtle was caused by molds and bacteria. Eckert and Eckert (1990) believed that it is possible that lower hatch success of the eggs of leatherback sea turtle might be due to fungal and bacterial infection. Wyneken et al. (1988) reported that several embryos dissected from nonviable eggs of loggerhead sea turtle showed extensive fungal invasion, possibly by Mucor spp. Egg and eggshell samples were collected from Ras Al-Jins, which is a small nesting site (1 km by 500 m) for the green sea turtles. The number of females that laid eggs in this small area in 2003 was estimated to be 31,872 females (Ministry of Regional Municipalities, Environment and Water Resources, 2003). After so many years of nesting in this small area, a large amount of organic matter originating from hatched and failed eggs is present. In this study, nonnesting soil was found to contain fewer fungi with fewer colony forming units (CFU) per gram than nesting soils. Similar observations were reported by Mo et al. (1990) who found that the number of bacterial counts (CFU/g sand) in nesting area was 6 times higher than nonnesting beaches due to accumulation of organic matter.

In this study, hyphae of fungi were found in the eggshells and egg membranes (Figs. 2, 3). Fungi have also been shown to cause turtle egg spoilage and decomposition (Acuna-Mesen 1992). Solomon and Tippett (1987) reported that fungal infection has caused early and late embryonic death of leatherback sea turtles and it was not uncommon for complete nests to be destroyed. Fungi were found on the exterior of unhatched eggs of green and loggerhead turtles and in some cases the entire egg mass was infected which resulted in nil hatch success (Phillott and Parmenter 2001).

Table 2 shows that 75% of the strains of A. flavus tested produced aflatoxin ranging between 0.3 and 28 ppb. This is in agreement with research finding that although not all A. flavus strains are aflatoxigenic, a high incidence of aflatoxigenic strains (50%–100%) are usually found among A. flavus isolates (Heperkan et al. 1994; Elshafie et al. 1999). The high percentage (75%) of aflatoxigenic strains of A. flavus in soils at the nesting site could be a serious threat to embryo development and egg hatching.

Aflatoxins were found in 40% of the eggshells studied at a concentration ranging between 3.6 and 8.4 ppb and in 25% of egg's contents at a concentration (3.6–8.4 ppb). This level of aflatoxins in eggshells and egg contents is high enough to cause embryo mortality. One part per billion of aflatoxin B1 was reported as causing 25% embryo mortality of chicken embryo (Neldon-Ortiz and Qureshi 1992). The maximum amount of aflatoxins allowed in food and feed by most countries is 10 and 20 ppb, respectively.

The warm, moist microenvironment 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 success of the eggs either by decomposing the eggs and/or secreting mycotoxins that affect the developing embryos.

According to Solomon and Baird (1980) fungi developing on eggs of sea turtles affect embryo development by 1) impending gas exchange of embryos, 2) calcium depletion of the eggshells, thus affecting embryo development, and 3) transfer of fungal spores from allantois to the embryonic tissue. In addition to this possible scenario, we think that mycotoxigenic fungi may produce highly toxic compounds that impair embryonic development and contribute to egg failure, low hatching success, and high mortality during embryonic development. Further investigation on the impact of some mycotoxins and mycotoxigenic fungi on embryo development of turtle eggs is underway.