Ecology and Protein Composition of Polypedates leucomystax (Gravenhorst, 1829) (Anura: Rhacophoridae) Foam Nests from Peninsular Malaysia

The four-lined tree frog, Polypedates leucomystax, spawns its eggs in a moist structure called a foam nest. Four foam nests constructed by this species were collected from the Sungai Sedim Recreational Forest, Kedah, Peninsular Malaysia. Two foam nests were found deposited on the leaves of low vegetation hanging over a rock pool. One was attached inside a water tank, and one was found on grass near an ephemeral puddle. In the laboratory, the foam nests were freeze-dried and the protein concentrations quantified, fractionated, and analyzed using LC-MS/MS. Twenty-two Original Research Article Shahriza et al.; ARRB, 14(6): 1-10, 2017; Article no.ARRB.34211 2 proteins, including seven enzymes, six structural proteins, five regulatory proteins, three receptors, and one antimicrobial peptide (AMP) were found in the foam nests. The function of the AMP (brevinin-2 type) is believed to protect the frog eggs from pathogenic microorganisms.


INTRODUCTION
Polypedates leucomystax is a medium to largesized frog. The snout-vent length (SVL) of males and females reaches 37-50 and 57-75 mm, respectively [1]. This commensal species is widely distributed throughout Bangladesh, Brunei, Cambodia, China, India, Indonesia, Laos, Malaysia, Myanmar, Nepal, the Philippines, Singapore, Thailand, and Vietnam [2]. They occupy various habitats and are usually found around human habitations in urban and rural areas [3,4,2]. During the breeding period, the females produce eggs in a white moist structure called a foam nest. The foam nest is created from skin secretions, produced by both the male and female frogs, and wiped by hind limbs onto the posterior dorsum. Usually, this foam nest is deposited on tree branches, twigs, or leaves and hangs over stagnant water bodies such as ephemeral pools and puddles [3,4,1].
Amphibian foam nests perform various functions including: protection against predators and pathogens [12]; prevention of egg-mass dehydration [13,14]; provision of respiratory advantages to embryos [15]; gliding substrates for larvae [14]; and provision of nutrients for the development of embryos [16]. The composition of anuran foam nests has been studied by several researchers. Kabisch et al. [14] analyzed the foam nests of P. leucomystax, which contained more than 93% protein. The proteins consisted of 17 amino acids; the main proteins were asparagines, lysine, and glutamate. Cooper et al. [17] discovered unusual primary structures and surfactant activity in ranaspumins proteins in the foam nests of Physalaemus pustulosus.
McMahon et al. [18] described the crystal structure of a 13 kDa surfactant protein, ranasmurfin, which was isolated from the foam nests of P. leucomystax. Hissa et al. [16] detected a strong surfactant protein, Lvranaspumin, in Leptodactylus vastus. These findings indicate the variety of chemical substances, especially proteins, contained in amphibian foam nests. Therefore, this study was conducted to analyze the ecology and protein composition of P. leucomystax foam nests.

Foam Nest Collection
Four P. leucomystax foam nests were collected from the Sungai Sedim Recreational Forest, Kedah, Peninsular Malaysia (5°25'N, 100°46'E; elevation <150 m above sea level). The foam nests were collected by searching around stagnant water bodies such as rock pools and temporary puddles. The foam nests were collected by hand and placed into moist plastic bags. To confirm that the foam nests belonged to the P. leucomystax species, each mating pair was collected. Visual examination indicated that the morphology of each mating pair was in accordance with P. leucomystax [3].

Freeze Drying
Eggs were separated from the foam nests using forceps. The foam nests were then placed into 50 mL Falcon tubes and frozen at -35°C. After 24 hours, the frozen foam nests were transferred into a freeze-dry machine (Labconco) and processed for 24 hours. The optimal temperature and vacuum conditions were -47°C and 0.025 mbar, respectively. Fifteen milligrams of each freeze-dried foam nest was added to 1 mL of 40 mM Tris-HCl (pH 8.8) extraction buffer. The mixtures were left for 20 minutes and occasionally stirred. The mixtures were then centrifuged at 12,000 x g for 30 minutes. The supernatants were collected and stored at -35°C.

Bradford Assay
The total protein concentration of the foam nests was quantified using a Bradford assay [19]. Five micro liters of each supernatant was mixed with 250 µL of Bradford reagent in a 96-well plate and incubated at ambient temperature for 15 minutes. A standard curve ranging from 0-2.0 mg/mL was constructed with absorbance set at 595 nm. The total protein concentration of each sample was determined and averaged by comparing the absorbance value against the standard curve.

Protein Fractionation
Protein fractionation was conducted using a Gel free 8100 fractionation system (Expedeon, CA, USA). The procedure was carried out according to the protocol of Witkowski and Harkins [20]. Two hundred micrograms of each sample was loaded into a 10% Tris-Acetate cartridge. Twelve fractions were collected and concentrated using a vacuum concentrator.

Protein Digestion
Protein digestion was carried out according to Kinter and Sherman [21]. The samples were resuspended in 100 µL of 6 M urea and 10 mg/mL of 100 mM Tris buffer. DTT (200 mM) was added to each sample and the mixture held at room temperature for one hour. Iodoacetamide (200 mM) was then added and incubated at room temperature for a further hour followed by addition of excess DTT to consume unreacted iodoacetamide. The concentration of urea in the samples was then reduced by adding 775 µL of water. For digestion purpose, 20 µg of trypsin solution (Promega, WI, USA) was added to each sample and incubated overnight at 37°C. The digestion was halted the next day by adjusting the pH of the buffer to pH < 6.

LC-MS/MS Analysis
Each sample was mixed with 100 µL of 0.1% formic acid in de-ionized water and filtered using a 0.45 µm regenerated cellulose (RC) membrane syringe filter (Sartorius AG, Goettingen, Germany). The analysis was conducted using a LTQ-Orbitrap Velos Pro mass spectrometer, coupled with an Easy-nLC II nano liquid chromatography system. A C18 Easy column (10 cm, 0.75 mm i.d., 3 µm) (Thermo Scientific, San Jose, CA, USA) was used as the analytical column and a C18 Easy column (2 cm, 0.1 mm i.d., 5 µm) (Thermo Scientific, San Jose, CA, USA) was used as the pre-column. The analytical column was equilibrated at a flow rate of 0.3 µL/min for 4 µL; the pre-column was equilibrated at 3 µL/min for 15 µL; and 5 µL of each sample was injected and chromatographically separated at a flow rate of 0.3 µL/min. Running buffers (A) 0.1% formic acid in de-ionized water and (B) 0.1% formic acid in acetonitrile were used. The samples were eluted using a gradient of 5% to 100% of buffer B for 80 minutes. The eluent was sprayed into the mass spectrometer at 2.1 kV (voltage source), and the capillary temperature was set at 220°C. Protein and peptides were detected using full-scan mass analysis from m/z 300-2,000 at a resolving power of 60,000 (at m/z 400, FWHM; 1-s acquisition). Data-dependent MS/MS analyses (ITMS) were triggered by the eight most abundant ions from a parent mass list of predicted peptides, with rejection or unassigned charge states. Collision-induced dissociation (CID) was used as a fragmentation technique with a collision energy of 35. Each sample was analyzed twice.

Protein and Peptide Identification (De Novo Sequencing)
PEAKS Studio Version 7 (Bioinformatics Solution, Waterloo Canada) was used to perform de novo sequencing and database matching. The National Centre for Biotechnology Information (NCBI) amphibian database (as of October 2014) was used for database matching. Parent mass and precursor mass tolerance were set at 0.1 Da. A false detection rate (FDR) of less than 0.1% and a significant score of -10lgP for proteins greater than 30 were used for protein acceptance. A minimum unique peptide was set at 1 and a maximum variable post-translational modification was set at 4.

RESULTS AND DISCUSSION
Mating pairs (Fig. 1) and foam nests (Fig. 2) of P. leucomystax were collected from the Sungai Sedim Recreational Forest. In Fig. 1, the smallsized frog is male (top) and the larger frog is female (bottom). Two foam nests were found deposited on the leaves of low vegetation approximately 1 m above a rock pool. The rock pool was intermediate-sized, being approximately 4 m in length, 2 m wide, and 5-35 cm deep; contained clear water; had a sandygravel bed; and was covered with leaf litter and twigs. It was bordered by low vegetation and was directly exposed to sunlight. Tadpoles of other frog species, Microhyla heymonsi and Fejervarya limnocharis, were also found living in the pool. A single foam nest was collected from the inner surface of a water tank, which was located near a toilet. The water tank was about 1.5 m long and 1 m wide and was filled with clean tap water. This foam nest was located approximately 22 cm above the water level. The fourth foam nest was found deposited on grass at the edge of an ephemeral puddle. This intermediate-sized puddle was approximately 2 m long, 1 m wide, 2-15 cm deep, and located 6-7 m from a main river. It contained cloudy water, had a silty bottom, and was covered with sediments. The puddle was bordered by small sized grass and exposed to sunlight.
Anuran species lives in different types of environment and reproduce by a variety of modes. To date, 39 types of reproduction modes have been recognized and described in anurans, including the formation of foam nests [22,23]. Various frog species from Malaysia, especially tree frogs such as Polypedates macrotis, P. otilophus, P. leucomystax, Rhacophorus nigropalmatus, and R. pardalis deposit their eggs in foam nests during their breeding seasons [24]. Usually, foam nests are located adjacent to stagnant water bodies. Inger [25] found the foam nests of P. leucomystax attached to vegetation and rocks at the edges of standing or slowly flowing water. Berry [3] discovered foam nests in tubs around houses, tanks, rainwater butts, or on leaves overhanging small pools of water. Sheridan [26] found foam nests belonging to P. leucomystax above water in emergent vegetation or other suitable substrates. It breeds in standing water bodies such as natural ponds, cattle tanks, cisterns, and flowerpots. In this study, the foam nests of P. leucomystax were found deposited on leaves hanging over a rock pool, among grass, and attached inside a water tank. The average total protein concentration of the foam nests in the current study was 1.10 mg/mL. The seven-point calibration curve, ranged from 0-2.0 mg/mL (y=0.3652x0.006; R 2 =0.9914) (Fig. 3). Twenty-two proteins, including seven enzymes (32%), six structural proteins (27%), five regulatory proteins (23%), three receptors (13%), and one AMP (5%) were detected in the P. leucomystax foam nests ( Table 1). The enzymes detected in the foam nests were prohormone convertase 1, duodenase-1, Ca2 + -ATPase, membrane protease subunit 2, exosome complex exonuclease RRP43, and NADH-ubiquinone oxidoreductase chain 2. The structural proteins were collagen alpha-2(1) chain, alpha 1 type 1 collagen, adult keratin RAK, larval type 1 keratin, and larval specific keratin 1. The regulatory proteins were pumilio-related protein, protein 90B, C10orf27, and tropomyosin-1 alpha chain. The receptors were ryanodine receptor alpha isoform, ryanodine receptor beta isoform, and progesterone receptor. The single AMP belongs to the brevenin-2 family. The proteins, based on their biological functions, are shown in Fig. 4.
Various chemical substances including proteins, peptides, steroids, and biogenic amines have been detected in the skin secretions of amphibians [27,28]. Previous reports have documented AMPs in the skin secretions of amphibian species belonging to the families of Pipidae, Hylidae, Hyperoliidae, and Ranidae [29,30,31]. These AMP molecules are 10-50 amino acid residues in length, with remarkably diverse structures [31,32]. From our results, a single AMP belonging to the brevinin-2 family was detected in the foam nest of P. leucomystax.   [7].
The current study's findings notably include the first time that brevinin peptides coming from the foam nests of anuran species have been reported. Almost all peptides that have been extracted, isolated, and documented have come from amphibian skin secretions. In amphibians, these peptides have specific functions including protection from pathogenic microbes, defense from potential predators, wound healing, and oxidant scavenging [27,31,35]. In addition, peptides can also exert multiple functions such as chemotactic and immunomodulating activities, endotoxin neutralization, induction of angiogenesis, and wound repair [36].

CONCLUSION
Polypedates leucomystax deposits its eggs in a moist structure called a foam nest. Twenty-two proteins including seven enzymes, six structural proteins, five regulatory proteins, three receptors, and a single AMP (brevinin-2 type) were detected in the foam nests of this species in this study. The importance and functions of these proteins needs to be further investigated. The AMP (brevinin-2 type) is believed to play a vital role in protecting P. leucomystax eggs from pathogenic microorganisms, which also requires further study. The peptide should be isolated and its activities against several bacterial and fungal species tested.