EurAsian Journal of Biosciences Eurasia J Biosci 8, 38-50 (2014) http://dx.doi.Org/10.5053/ejobios.2014.8.0.4

Embryonic and larval development of the suckermouth sailfin Pterygoplichthys partialis from Marikina River, Philippines

Joycelyn Cagatin Jumawan1*, Annabelle Aliga Herrera2, Benjamin Vallejojr3

1 Biology Department, Caraga State University, Butuan City, Philippines institute of Biology, University of the Philippines-Diliman, Diliman, Quezon City, Philippines institute of Environmental Science and Meteorology, University of the Philippines-Diliman, Diliman, Quezon City, Philippines Corresponding author: [email protected]

Abstract Background: There is little information about the early development of this invasive species in order to understand its early life history and developmental strategies towards invasion. Material and Methods: Female Pterygoplichthys pardalis were induced to using human chorionic gonadotropin (HCG) so as to study the developmental stages from fertilization until yolk resorption. Results: The females subjected to a single dose of HCG responded positively to treatment (97%) with higher fertilization success (88%) compared to the untreated females (21%). Nonetheless, the HCG-induced fertilized eggs had a low hatching success (49%), while from the free-living embryos successfully hatched, a high number (90%) survived to become juveniles. Embryonic development in P. pardalis was completed 168 h and 30 min after fertilization, with the total yolk resorption completed on the 8th day post hatching, during which the suckermouth gradually shifted from rostral to ventral position to commence the loricariid algae-scraping feeding mode. Conclusions: Pterygoplichthys pardalis does not undergo a true larval metamorphosis between the free-living embryo and the juvenile stage and a definitive adult phenotype is developed directly. These results provided basic, yet essential information on the early developmental features of this invasive species whose spawning and early developmental strategies were difficult to observe in the field. Implications of some ontogenetic features in this species with regards to invasion are also discussed. Keywords: Development, embryogenesis, invasion, larvae, morphology, Pterygoplichthys pardalis.

Jumawan JC, Herrera AA, VallejoJrB (2014) Embryonic and larval development of the suckermouth sailfin catfish Pterygoplichthys pardalis from Marikina River, Philippines. Eurasia J Biosci 8: 38-50.

http://dx.doi.Org/10.5053/ejobios.2014.8.0.4 ©EurAsian Journal of Biosciences

INTRODUCTION Pterygoplichthys pardalis (Castelnau, 1855) is a native of South America (Weber 2003), but has been Siluriformes exhibit diverse reproductive introduced to the Philippines through aquarium strategies and most studies are just focused on late trade. It has invaded many freshwater systems of embryonic and larval development (Adriaens and the country along with another hypostomine Vandewalle 2003). Nonetheless, knowledge of the loricariid, Ptergoplichthys disjunctivus. These two crucial phases of the life history of invasive species is species, popularly known in the country as "janitor vital in order to understand their developmental fish", are not regarded as important commercial strategies and identify the advantageous because of their hard body armour, very little ontogenetic features for invasion and meat, propensity to compete for food resources, establishment. The management of invasive alien and their potential to bioaccumulate heavy metals in fish species with known high fecundity potential and polluted environments (Chavez et al. 2006, Lam and high success rate of establishment in invaded Su 2009, Jumawan et al. 2010a). Nonetheless, the environments requires knowledge of its early life hardy nature of this genus, its capacity to down stages, such as the timing of embryogenesis and organogenesis and the shift from endogenous to Received: January 2014 exogenous feeding (Godinho et al. 2003). Received in revised form: April 2014 The armoured suckermouth sailfin catfish Accepted: April 2014 Printed: May 2014

38 EurAsian Journal of Biosciences 8: 38-50 (2014) Jumawan et al. regulate metabolism during periods of scarcity of (Merck, Germany) bath for 4 min. The female P. food (German et al. 2010), its tolerance to poor pardalis received an intramuscular injection of water conditions and its ability to breathe air under human chorionic gonadotrophin (HCG) (Argent hypoxic water conditions (Armbruster 1998) enabled Chemicals, Philippines) at 4 lU/g body weight this fish to invade and successfully establish itself (injection volume: 1uL/g BW). The hormone-injected even in disturbed freshwater systems. females were then placed separately in 1 m x 0.5 m The seasonality of reproduction and the gonad plastic tanks containing de-chlorinated tap water to features of the Pterygoplichthys spp. population in a depth of 0.4 m each. Females untreated with HCG Marikina River has been previously described, served as controls and were placed in a separate showing the females to be highly fecund and the tank. oocytes exhibiting features associated with parental Approximately 14-18 h after HCG administration, care (Jumawan et al. 2010b). However, the burrow- the females were checked for ovulation by applying spawning and nest-guarding nature involved during pressure to the abdomen to confirm ovulation. Eggs the critical period of embryogenesis in these fishes from ovulated females were then stripped in a dry was found difficult to replicate under laboratory plastic basin. At about the same time, males were conditions. To date, there are no available studies anesthetized, sacrificed by a sharp blow to the head describing the early development of P. pardalis and and had their testes removed. were collected P. disjunctivusin their original habitat for comparison after maceration of the testes and then immediately with species thriving in non-native environments. diluted with 0.9% NaCl to obtain milt solution. The The present study will serve as a baseline reference milt solution was poured into a bowl containing the of the early embryogenesis, larval development and stripped eggs and mixed for 30-60 sec using a organogenesis of the invasive P. pardalis through in- feather. Approximately 5 mL of tap water was added vitro fertilization. to the bowl to ensure fertilization. After 2 min of gentle stirring, the fertilized eggs were transferred to a plastic strainer and rinsed with running water MATERIALS AND METHODS for about 1 min to remove excess milt. Fertilized Strip method for artificial fertilization eggs were immediately transferred to a 45x30x30 The artificial breeding protocol of P. pardalis cm aerated plastic aquarium for incubation. The adapted the procedure by Tan-Fermin et al. (2008) aquaria were provided with partial shade by using a with some modifications. All experiments were 1x1 m black, plastic polyethylene bag to simulate carried out in triplicates. Collection and the darkened burrows in the field. The eggs were experimentation were performed during the peak examined 10-15 min after gamete mixing to check spawning months (July to September) of the for blastodisc formation. Unfertilized eggs were Pterygoplichthys spp. population in Marikina River. carefully removed from the aquaria using fine Because of the absence of defined sexual forceps. dimorphism in this fish, large and mature P. pardalis Initial observations showed that the fertilized weighing 350-500 g were collected. Females were eggs had a low hatching success rate when selected based on their full and heavy body, gravid incubated in de-chlorinated tap water (10-15%) or abdomen and reddish, swollen vent. To identify the rain water (10-20%), but had an improved hatching sex of the fish, pressure on the abdomen to extrude rate when using a mix of natural river and tap water. oocytes and the use of cannula were also attempted Hence, the subsequent trials used a mix of aerated for most samples. Males were selected based on a Marikina River water and tap water as an incubation streamlined body and flat abdomen. A total of 18 medium at 1:1 ratio. Prior to mixing the water females and 18 males were utilized for this study. medium, river water and tap water were analyzed Prior to hormonal induction, all females were for hardness (CaCO /L), chloride (mg/L), calcium anesthetized in a 200 ppm 2-phenoxyethanol 3

39 EurAsian Journal of Biosciences 8: 38-50 (2014) Jumawan et al.

(mg/L) and magnesium (mg/L) levels. The physical RESULTS and chemical parameters of the mixed water in the Description of fertilized eggs aquaria, such as temperature (°C), pH, salinity and The adult female P. pardalis were administered dissolved oxygen (D.O.), were recorded throughout HCG a day after samples were obtained from the the experiment. field. Difficulty in distinguishing between males and Embryogenesis, larval ontogenesis and females was noted due to lack of defined sexual biometry dimorphism as well as difficulty in extruding oocytes For each of the 3 replicate plastic aquaria, twenty and milt from the vent after the application of developing eggs were observed at 10-30 min considerable abdominal pressure and cannulation. intervals until completion of cleavage, 3 times per Table 1 shows the physico-chemical parameters of day until hatching. Hatchlings were documented the water used for embryo incubation. Oocyte twice daily, until the yolk was fully resorbed. The extrusion was performed 18 h after the exposure to developmental stages were divided into embryo and HCG. Approximately 200-250 oocytes were extruded free-living embryo stages. The embryonic stage from the ovaries of a single female exposed to HCG occurs inside the chorion and ends in hatching. The due to difficulty of handling and applying pressure in free-living embryo stage is characterized by the the abdomen of the fish. The females injected once nutritive contribution of the yolk sac and the stage with HCG responded positively to the treatment ends when the free-living embryo becomes capable (97% success rate) and hydrated, producing nearly of exogenous feeding after the yolk has been uniform sized (2-3 mm), transparent-yellow oocytes consumed (Geerinckx et al. 2008). after the ovary was stripped. In contrast, the oocytes Three subsamples of fertilized eggs were from females not exposed to HCG were mostly collected daily until yolk resorption and were fixed opaque yellow with occasional pre-vitellogenic in Bouin's fluid for histological purposes. oocytes (<1 mm) along with larger vitellogenic Additionally, three more fertilized eggs and oocytes. Adhesion of oocytes was observed hatchlings were collected until yolk resorption and immediately after any excess milt was washed off. were directly fixed in 4% neutral formaldehyde for The HCG-injected females had a higher fertilization biometry using a digital caliper (accuracy 0.001 mm; success (88.3%) compared to the untreated females (20.9%). The fertilized eggs from HCG-injected Control Company, USA), following the parameters females had low hatching success (48.6%), with described in the study of Guimaraes-Cruz et al. mortalities during early stage somitogenesis. (2009) (Fig. 1). From the successfully hatched embryos, a high The fertilization rate (number of eggs with number survived to become free-living embryos up blastodisc/total oocytes x 100), hatching rate to the termination of experiments on the 8th day post (number of hatched eggs/number of fertilized eggs hatching (8 dph) (Table 2). The diameter of the eggs x 100) and survival rate (number of surviving did not differ significantly between the two juveniles/total number of hatched embryos x 100) treatment groups. No change in the diameter of the were recorded. Three replicate runs from fertilized oocytes (3.2±0.23 mm) was also observed fertilization to hatching were conducted. from the onset of fertilization until the pre-hatching Analysis of variance (ANOVA) was used to stage, although a very small perivitelline space was compare the mean values of the morphometric formed surrounding P. pardalis eggs a few minutes variables according to each stage of larval after fertilization. development, and between oocyte mean diameter Embryonic development values for HCG-induced and non-HCG-induced The observations of P. pardalis were divided into samples. All tests were conducted in a 0.05 two periods: (1) before hatching (embryo), and (2) significance level using Graphpad Prism 5®. between hatching and yolk sac depletion (free-living

40 EurAsian Journal of Biosciences 8: 38-50 (2014) Jumawan et al.

TL

PAD

Fig. 1. Lateral view of a P. pardalis free-living embryo (4 dph; 14.2 mm TL). TL: Total length; SL: Standard length; PAD: Pre-anal distance; HL: Head length; HH: Head height; SNL: Snout length; ED: Eye diameter; YSL: Yolk sac length; YSH: Yolk sac height, BH: Body height.

Fig. 3. Stages of embryonic and larval development in P. pardalis. (a) Late gastrula: formation of the embryonic shield (es); (b) Somites development (black arrow); (c) Late Neurula stage; f: Forebrain; m: Midbrain; h: Hindbrain, oc: Otic capsule; (d-e) Rostral location of suckermouth (*), Note egg sac removed; (f) P. pardalis at 6 dpf; (g) Newly hatched embryo (7 pf); (h) Note the ventral position of the suckermouth in (g). Scale bar: 1 mm.

5 to 15 min after fertilization. Subsequent successive cleavage until 64 blastomeres was observed until it was completed after 5 h with blastomeres very much decreased in size. Blastomeres very fine in appearance eventually flattened at the animal pole at 14-15 h post Fig. 2. Stages of embryonic development in P. pardalis. fertilization. The spread of the blastoderm was (a) Early stage fertilized egg with distinct invagination at the animal pole before blastodisc formation; (b) Blastodisc stage; (c) Cleavage evident, covering the yolk with the embryo body at 2-cell stage; (d) Cleavage at 4-cell stage; (e) Cleavage at 8-cell becoming more elongated, while the head and tail stage; (f) Cleavage at 64 blastomeres; (g) High blastula stage; (h) Low blastula; (i) Early gastrula. ends of the embryo can be clearly seen at 20 h. Scale bar: 1 mm. Finally, full yolk invasion and closure of the blastopore was observed at 24 h. embryo). The main events in embryogenesis and Differentiation of the embryo their respective times of observation are Pre-hatching organogenesis in P. pardalis summarized in Table 3 and Figs. 2-4. The fertilized commenced with the formation of the notochord eggs of P. pardalis show meroblastic cleavage in and observations of the cranial-caudal portions of which the blastoderm is restricted to a small area at the embryo (29 h), as well as the first somites and the animal pole. The first segmentation that divided the optic vesicles (36 h). Somitogenesis was the blastodisc into two blastomeres occurred within observed starting 2 days post fertilization (2 dpf)

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Table 1. Some physico-chemical features of the water medium used in this study.

Parameters Marikina River water Tap water (IB) Mix

Chloride, mg/L* 3.75 3.82 - Calcium, mg/L* 31.0 15.1 - Magnesium, mg/L* 10.8 3.91 - Hardness, mg equiv, CaCCVL* 122 54 - Dissolved oxygen, mg/L 4.56±0.67 5.0±0.73 4.6±0.98 Salinity (ppm) 0.3±0.02 0 0.2±0.01 Temperature (°C) 24.0±0.03 24.0±0.04 24±0.01 PH 7.9±0.91 7.5±0.77 7.6±0.87

*Data analyzed only once, through the Research and Analyti cal Servi ces Laboratory (RASL), Natural Sciences Research Institute (NSRI); Remaining data monitored once in every 3 days for 15 days. Values are listed as means±S.E.

Table 2. Percent (%) response of P. pardalis to HCG-injection at 24°C

Conditions Fertilization rate Hatching rate Survival rate

HCG injected 88.33±7.890* 48.56±11.13* 90.44±7.299* Control (Non-HCG 20.89±11.50 0 0 injected)

*P<0.001; Values are listed as means±S.E. and subsequent somite formation enabled tail h post fertilization. The caudal tail region was movement, although the embryo was largely observed detaching from the yolk mass with a confined within the perivitelline space. No subsequent increase in body movement, causing the corresponding somite formation for each hour rupture of the chorion and the emergence of the during the onset of embryogenesis in P. pardalis was embryo from the capsule. noted because of the difficulty in turning the Free-living embryo development strongly adhesive eggs to locate the somites Details of development and average body without causing mechanical injury to the developing measurements are reflected in Tables 4 and 5. embryos. The time when the pericardial cavity was Histologic sections of larvae at different days post formed was not determined; however, the pulsating hatching are shown in Figs. 5-7. heart and blood circulation was visible as a very faint Hatchling stream of capillary network in the pericardial cavity Newly hatched, free-living embryos or eleuthe- at 36 h after fertilization. Ectodermal thickening to rembryos (Balon 1986) have a mean total length of form the lens of the eyes was observed starting at 7.86±0.12 mm, still containing a large amount of yolk 36 h, with the eye lens fully formed at 49 to 50 h post (Fig. 5a). The newly hatched larvae had already fertilization. The suckermouth was rostrally ventrally located the suckermouth and maxillary positioned inside the membrane (Fig. 3d-e, Fig. 4a). barbels and were able to attach to the sides of the Tail movement (72 h post fertilization) was glass substrata with their suckermouth, with water observed before the formation of the vitelline inflow for the sucking action passing through the circulatory system. Movement in the gill cavity and furrows of the maxillary barbels, and attachment blood circulation in the gill arches was noted sometimes assisted by body and tail movements beginning at 5 dpf. Hatching was observed 167-168 (Fig. 3h). Embryos initially had a rostrally located

42 EurAsian Journal of Biosciences 8: 38-50 (2014) Jumawan et al.

Table 3. Main events of the embryonic development of P. pardalis and their respective times (mean) after HCG-induced fertilization at 24°C. n: 192 developing eggs/stage.

Time post Stage Main event fertilization (h)

Zygote 2 Formation of the blastodisc and defined animal and vegetal pole (Fig. 2a, b).

Rapid mitotic divisions of the blastodisc into two to 64 blastomeres in the Cleavage 5 animal pole (Fig. 2c-F)

High blastula 9 Maximum number oF Fine blastomeres; protrudes in the animal pole (Fig. 2g).

Blastomeres Flatten at the animal pole, returning to the spherical shape oF Low blastula 15 the egg (Fig. 2h).

Epiboly movement covering the yolk; Forming the epiblast and hypoblast Gastrula 20 (Fig. 20. End oF epiboly; Embryo body becoming more elongated; Yolk completely Blastopore closure 24 surrounded by the blastoderm. DiFferentiation of Higher cell diFFerentiation; Notochord Formation, very distinct cranial and germ 29 caudal portion (Fig. 3a). layers

Somitogenesis 36 Visible somites, onset oF heart beat, blood circulation (Fig. 3b, c) Visible tail movement/contractions, however remains curved and conFined 72 Tail release within the yolk sac.

168 h, Increased body movement, breakdown oF chorion, larvae Free swimming (Fig. Hatching 30 min 3g, h).

Yolk resorption 336 Visible absence oFyolk; increase in body length. mouth, which gradually shifted into the ventral of the body. The digestive system was characterized location during hatching. Dorsal and caudal fins were by a simple striated border in the intestine (Fig. 5f). observed. The bodies of the newly hatched free- The cranial kidney was now well developed with living embryos were transparent, except for the readily recognizable glomeruli tufts within the onset of dendritic pigmentations initially found network of reticular fibers (Fig. 5e). The spleen could interspersed finely on the head and on the edges of be observed on the left lateral-dorsal-right lateral the snout. The eye diameter was small (0.51±0.02 part of the borderline between the gut and the mm). The dorsal and caudal fins were well kidney. The liver could be observed ventrally located recognizable. Serial sections of the digestive system in the cranial region with the hepatic parenchyma during the day of hatching showed that the gut is very homogenous in appearance. tubular and closed at both ends, while the intestine 5-d old free-living embryo is composed of simple cubic epithelium. The first The average length of a 5-day old free-living outlines of the gill arches supported by blood embryo was about 14.15±0.04 mm TL with the yolk vessels were observed in the gill cavity. A tubular sac length becoming gradually reduced (3.22±0.03 heart was observed. mm). Increasing pigmentation all throughout the 3-d old free- living embryo body was observed with dendritic chromatophores A continued reduction of yolk sac and an increase reaching the lateral region caudally to the pectoral in body length (TL 12.41±0.05 mm) were observed fin (Fig. 4c-d). The yolk was visible, although less along with increasing pigmentation in the retina and compact near the digestive system. The intestines increasing complexity of the gills. Increasing became looped and lengthened, with the intestinal dendritic chromatophore pigmentation was epithelium exhibiting a complex columnar observed in the outlines of the head and dorsal side arrangement of mucous and goblet cells. Intestinal

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Fig. 4. Free-swimming embryo development in P. pardalis. Fig. 5. Organogenesis in P. pardalis. (a) Embryo with yolk sac removed. Note the rostral position of the mouth; (b) Development at 3 dph; (c-d) Development at 5 dph; Note (a) Newly hatched free-living embryo; medial portion; (b) 2dph, the capacity of the ventrally positioned mouth for attachment in cranial portion; (c) 3 dph, Retina (*) and its layers; (d) 3 dph, cranial the glass substrata in; (c) Body pigmentation in (d); (e) portion; (e) 3 dph, Digestive system; (f) 3 dph, intestine with simple Pigmentation in the ventral position of the 7 dph free-living striated border. N: Nostrils; E: Encephalon; H: Heart; B: Barbells; CK: embryo. Cranial kidney; GB: Gas bladder; I: intestine; y: Yolk; G: Ganglionar cell; IN: Inner nuclear layer; ON: Outer nuclear layer; YG: ocular globe; GA: gill arches. content (residue) was observed in this stage. Scale bars: a,d,e: 200 urn; c,f: 20 urn; b: 40 urn. Nephric ducts in the cranial kidney were very visible; the encephalon was compact with associated cells had visible renal tubules and extensive hemato- (Fig. 6e). The heart was compartmentalized with a poetic tissue. very visible atrium (Fig. 6f). 8-d old free-living embryo 7-d old free-living embryo The average total length of free-living embryo The average length of 7-day old free-living was 6.95±0.20 mm TL and the yolk was fully embryo was about 14.84±0.34 mm TL with the yolk resorbed. The body had become opaque with the sac length becoming gradually reduced (2.32±0.09 accumulation of pigments all throughout its surface mm). Increased pigmentation all throughout the as chromatophores became more numerous and body including the ventral part previously occupied darker, maintaining the same distribution pattern. by the yolk sac (Fig. 4e). Full pigmentation of the Undifferentiated gonad with primordial germ cells eyes observed (Fig. 7c). Increased use of the was observed (Fig. 7e), while a true larval stage suckermouth to anchor body in the glass aquaria was (Balon 1986, 1999) was not observed. P. pardalis observed in larvae. The gills were more developed underwent a direct transition from a free-living with elongated filaments and gill lamellae with embryo with large yolk into a juvenile without intense vascularization. Gas bladder was observed undergoing a true larval stage when the yolk was with simple squamous epithelium (Fig. 7d). The heart fully consumed at 8 dph. Except for the absence of presented two compartments. Small intestine hardened armour covering the body and the mucosa can be seen, including microvilli, columnar abdominal pattern distinct for P. pardalis, an adult¬ epithelium and muscularis mucosa. The head kidney like appearance was observed at the moment the

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Table 4. Main morphologic events occurring during the development of P. pardalis from hatching until yolk resorption (TL, mm).

Stage Main events

Mouth already open, larvae can be seen sucking in the solid surface and actively swimming, Day 1 yolk is full and globular (Fig. 5a); Maxillary barbels are already present; tiny spine-shaped projections interspersed in the head surface. Digestive tract closed. Eyes with unpigmented (7.86 ±0.12) retina; Gill cavity containing the first outlines of gill arches supported by blood vessels; early evidence of chondrogenesis of arches (Fig. 5b).

Retina is conspicuously layered and pigmented (Fig. 5c, d); Nostrils opening visible; maxillary barbells thin but elongated; Gill filament can be seen inside gill cavity (Fig. 5d); Day 2 Chromatophores appear on the lateral part of the yolk sac, Spine-shaped projections (11.13±0.12) intersperse the linings of the eyes, nostrils, moving towards the tail. Intestine is composed of prismatic epithelium with simple striated border, although lumen is spaced but devoid of any digested particle, cranial kidney simple in appearance; gas bladder is visible (Fig. 5e, f).

Day 3 Retina increasing in pigmentation and gills increasing complexity (Fig. 6a); Well-developed spleen and kidney (Fig. 6b); Continued reduction of yolk sac. (12.41 ±0.05) Evidence of chondrogenesis in the vertebrate neural arch (Fig. 6c). Stomach and intestine open; Initial observations of mucous cells (Fig. 6d). Increasing growth of gill filaments. Kidney Day 4 well-interspersed within the uppermost portion of the coelomic cavity nearest the gas bladder; Increased pigmentation across the body, initial pigmentation stripe-like in the tail.

(13.24±o.15) Encephalon still open (Fig. 6e); well-developed heart (Fig. 6f), well-developed kidney and gas bladder (Fig. 7a). Digestive tract contains digested food; Intestinal epithelium exhibiting complex columnar arrangement with goblet cells and blood vessels (Fig. 7b). Day 5 Retina epithelium heavily pigmented, cones and rods observed (Fig. 7c); further elongation of the gill filaments; Intestine looped and lengthened with complex columnar epithelium; yolk (14.15±0.04) more reduced, digested particles evident (Fig. 7d); anus open. Conspicuous pigmentation of the entire body.

Day 6 Gills with more elongated filaments; features largely similar with day 6, except for the presence of lesser yolk and heavy pigmentation across the length of the body

(14.88±0.18) Indifferent gonad with primordial germ cells (Fig. 7e); chondrogenesis (Fig. 7f). Well- developed gills and digestive tract containing food; Yolk is resorbed. Day 7

(15.84±0.34) Table 5. Body measurements* (mm) of P. pardalis from hatching until yolk resorption. Day 8 •(16.95±0.20 ) Days post hatching* 1 2 3 4 5 6 7 8

TL 7.86± 0.12 11.13±0.12 12.41 ±0.05 12.24±0.15 14.15±0.04 14.88±0.18 14.84±0.34 6.95±0.20 SL 7.28± 0.07 9.55±0.11 10.16±0.06 10.17±0.14 11.46±0.11 11.62±0.34 11.81±0.23 12.63±0.05 SNL 0.58+ 0.03 1.19±0.04 1.30± 0.05 1.19±0.04 1.39+0.01 1.33+0.01 1.49±0.04 1.50± 0.01 ED 0.51 ±0.02 0.65± 0.03 0.76±0.01 0.75±0.01 0.80±0.01 0.77± 0.02 0.97± 0.02 0.95± 0.02 HL 1.85±0.04 2.88± 0.21 3.84± 0.04 3.76±0.06 3.40±0.06 4.05± 0.03 3.92± 0.05 3.59± 0.03 HH 1.07±0.02 1.78±0.04 1.77±0.04 1.68±0.05 1.94±0.03 1.99±0.02 2.04± 0.03 2.19± 0.02 BH 0.61 ±0.02 0.88±0.02 1.02±0.02 0.99±0.01 1.28±0.01 1.19± 0.04 1.62±0.04 1.72±0.02 PAD 3.99±0.10 5.80±0.08 5.75±0.08 6.0±0.09 5.57±0.12 6.86± 0.04 6.50± 0.09 6.95± 0.05 YSL 3.93± 0.04 3.96±0.18 3.93±0.01 3.8±0.03 3.22±0.03 3.33± 0.02 2.32± 0.09 - YSH 2.28±0.11 2.04±0.24 1.74±0.01 1.6±0.06 1.58±0.05 1.42±0.04 1.08±0.13 -

TL: Total length; SL: Standard length; PAD: Pre-anal distance; HL: Head length; HH: Head height; SNL: Snout length; ED: Eye diameter; YSH: Yolk sac height; YSL: Yolk sac length; BH: Body height. Values are listed as means±S.E.; n= 212

45 EurAsian Journal of BioSciences 8: 38-50 (2014) Jumawan et al.

. ' • DF

Fig. 7. Organogenesis in P. pardalis. (a) 6 dph; gas bladder and kidney; (b) 6dph, intestine; (c) 7 dph, Fig. 6. Organogenesis in P. pardalis. cranial portion; (d) 7 dph, digestive system; (e) 8 dph, (a) 3 dph, cranial portion; (b) 3 dph, spleen and kidney; (c) 4 dph, undifferentiated gonad; (f) 8 dph, segmentation of the notochord. dorsal nervous tube; (d) 4 dph, digestive system; (e) 5 dph, B: Barbels; GB: Gas bladder; CK: Cranial kidney; I: Intestine; Y: Yolk. encephalon; (f) 5 dph, heart. E: Encephalon; H: Heart; B: Barbells; K: G: Ganglionar cell; IN: Inner nuclear layer. ON: Outer nuclear layer; Kidney; I: Intestine; y: Yolk; G: Ganglionar cell; IN: Inner nuclear YG: Ocular globe; GA: Gill arches; H: Heart; PGC: Primordial germ layer; ON: Outer nuclear layer; YG: Ocular globe; GA: Gill arches; S: cell; N: Notochord; DF: Dorsal fin; M: Muscle. Stomach; SP: Spleen, N: Notochord; M: Myomeres; NC: Scale bars: c,d,f: 200 um; b,e: 20 um. Neurocranium. Scale bars: a: 200 urn; b,d: 40 urn; c,e,f: 40 urn. stages development of P. pardalis, a highly invasive last yolk was consumed at 8 dph. loricariid, whose early life history has not been Biometric parameters registered a gradual studied so far. increase in all values except for the decrease in the The reproduction and spawning behavior of the yolk sac length and height as the free-living embryo janitor fish P. pardalis is difficult to observe, as they neared and completed the yolk resorption are known to spawn in burrows with males guarding externally (Table 5). A full yolk resorption in the the fertilized clutch. What is surprising in this juvenile P. pardalis was observed 336 h or 14 dpf at experiment, however, is the tendency of the eggs to 24°C. All the oocytes observed for fertilization until hatch well in river water characterized to be hardy, yolk resorption in juveniles developed synchro¬ with higher calcium and magnesium levels compared nously. with tap water. The water quality of key areas along Marikina DISCUSSION River where spawning colonies of P. pardalis are abundant showed that the river is highly eutrophic Most invasive loricariids do not reproduce and turbid, and had overall high conductivity, spontaneously when reared under laboratory nitrate, ammonia and phosphate levels. Fertilized conditions (Alfaro et al. 2008). The present study clutches from the river also have a higher hatching provides baseline information regarding the early

46 EurAsian Journal of BioSciences 8: 38-50 (2014) Jumawan et al. rate if incubated in river water. It is not clear how the Types of feeding (exogenous, endogenous), nest guarding nature of the males for this species nutrient supply (altricial, precocial) and life history actually influences the successful hatching because models (indirect, direct development) were used as of the difficulty of observing this process in the field. basis in the description of embryos (Balon 1986, Makeshift and darkened plastic aquaria to replicate 1999). Embryos with indirect development are a consequence of poor vitellogenesis and depend burrows in the field in the preliminaries of this entirely on an endogenous nutrient supply, as eggs experiment failed to encourage spawning under are altricial in nature (Balon 1986). The short embryo laboratory conditions. The low success rate in period in this type of development appears to be hatching for P. pardalis under artificially-induced extended by the larva period that feeds spawning conditions may be attributed to the exogenously prior to the formation of the definitive absence of parental care during the experiment or phenotype. Fish taxa characterized by direct to the preference of eggs for river water development have a prolonged embryo period due environments. to a large endogenous supply as eggs are precocial Eggs of P. pardalis are highly adhesive, allowing (large amount of yolk) that ultimately enables the the formation of tight clutches of eggs. eggs embryo to develop directly into a definitive can be non-adhesive, weakly adhesive or strongly phenotype (Balon 1986). Embryos develop for adhesive. Rizzo et al. (2002) point out that adhesive longer but directly into juveniles that are able to eggs are often large, laid in smaller numbers and compete in the adult habitat (Balon 1999). Direct associated with the sedentary nature of the species development occurs more frequently in the or with parental care. The zona radiata of reproductive guilds of guarders and bearers as these Pterygoplichthys spp. in Marikina River was thin fish groups exhibit parental care such as site (mean 4.56 urn), while its granulosa layer was thick selection, egg deposition and nest guarding (Balon (mean 2.81 um) (Jumawan and Herrera 2014). A thick 1986). A true larva requires some tissues and granulosa layer may provide better adhesion of structures very different to those in the definitive eggs, while the thin zona radiata may be organism, and so, has to be remodeled through the compensated by the nest guarding nature of the process of metamorphosis (Balon 1999). Fish species males of this species, as was the nature of some nest with direct development lack the necessary cost of guarding loricariids (Suzuki et al. 2000). forming temporary organs through metamorphosis. It was apparent in the results that although The large yolk (mean 3.3 mm) in P. pardalis is an artificially fertilized eggs had a low hatching rate, all advantage in this context, as it requires none or little hatched P. pardalis free-living embryos successfully external nutrients to develop into a definitive survived and had resorbed their yolk at 8 dph. A phenotype. It has been proposed that the larger and large endogenous supply of yolk nutrients enhances more advanced an individual at the onset of survival during the period when feeding structures exogenous feeding, the better its chances of are still developing (Geerinckx et al. 2008), while also surviving (Balon 1999). This scenario improves enabling the free-living embryo to avoid an competitiveness in P. pardalis even in its early stages intermediate larval stage and the cost of and could be an advantage for invasion (Balon 1986). metamorphosis since a definitive adult phenotype The absence of a true larval stage in P. pardalis is was developed directly (Balon 1986). similar to most loricariids (Geerinckx et al. 2008). It is Although hardened armour covering the body however important to note that a slight variation in was not observed when the last yolk was consumed measurements may be caused by the fixative used in at 8 dph, hardening of the head and dorsal structure this study (4% neutral formaldehyde), as fixatives towards development of the armoured covering of may have some dehydrating effects contrary to the the body was eventually observed 30 days post yolk measurement of fresh samples. resorption in juveniles (data not shown). The eggs of P. pardalis contain a large amount of

47 EurAsian Journal of BioSciences 8: 38-50 (2014) Jumawan et al. evenly distributed yolk, hence, is classified as mouth despite the large yolk sac during hatching. On telolecitic (Ribeiro et al. 1995, Marques et al. 2008). the contrary, A. cf triradiatus, a relative loricariid, P. pardalis has a meroblastic cleavage pattern was noted to have a rostro-ventrally positioned restricted to the animal pole, which is common in suckermouth during hatching, but had to wait for 2¬ . Blastopore closure occurred 24 h after 4 days for the yolk sac to resorb for the transition to fertilization, indicating fertilization success. The a ventrally flattened mouth (Geerinckx et al. 2008). embryonic development of P. pardalis lasted 7 days The newly hatched P. pardalis in this study was able (168 h, 30 min), which is long compared with the to attach immediately to the substrate with their duration of embryogenesis in other siluriforms: 45 h, suckermouth. This observation was also reported for 50 min at 24°C in R. aspera (Perini et al. 2009), 21 h, Sturisoma aureum (Riehl and Patzner 1991) and A. cf. 20 min at 23°C in Pimelodus maculates (Luz et al. triradiatus (Geerinckx et al. 2008). This feature may 2001) and 18 h at 27°C in P. corruscans (Marques et be essentially an advantage for loricariid species al. 2008). where hatchlings leave the shelter immediately Long embryonic periods are known to be (Suzuki et al. 2000). In the case of an invasive species associated with non-migratory species having large such as P. pardalis, accidental release of eggs and eggs and with those that display parental care juveniles may result in assured higher survival rates (Sargent et al. 1987), features that both fit the in the wild. natural history of P. pardalis. Invasive species The SL of P. pardalis during the time of hatching exhibiting parental care may be considered an (7.86±0.12 mm) and the large yolk sac during the advantage in the context of ensuring egg survival same time fall within the size range for most until the juvenile period. By females precisely loricariids (6-8 mm) (Riehl and Patzner 1991, selecting a safe and protected site for egg Nakatani et al. 2001, Geerinckx et al. 2008, Perini et deposition and males guarding the nest, the al. 2009). The morphometric development in P. progress of the early developmental stages in P. pardalis, which reflected a gradual increase in all pardalis becomes ensured. This protection makes parameters with decreasing yolk size, is also the early stages difficult to observe in the wild as observed in A. cf triradiatus (Geerinckx et al. 2008), R. they are well hidden from predators and other aspera (Perini et al. 2009) and Lophiosilurus alexandri threats, emerging only when post-yolk sac juveniles (Guimaraes-Cruz et al. 2009), all of which exhibited a are capable of feeding and the definitive phenotype high degree of allometric growth. Geerinckx et al. ensures the adult form. (2008) hypothesized that loricariid hatchlings often A pigmentation of the retina observed early exhibit rapid allometric growth in the snout and during hatching in P. pardalis could be associated remarkable lip transformation because it is a with the need to develop a functional visual system necessity and advantageous for the suckermouth to before the first feeding, typical in some fishes (Hall attach to the substratum for scraping and sucking et al. 2004). The undifferentiated gonad located food. between the cranial kidney and the digestive tract in In conclusion, this study pointed out several P. pardalis already contains a cluster of primordial baseline features of the early life strategies in P. germ cells (PGCs) at 8 dph. PGCs in the pardalis that may be of advantage to its biotic spread undifferentiated gonad were also observed at 5 dph potential: (1). The propensity of the embryo to thrive in R. aspera and at 13 dpf in Pimephales promelas in polluted water; (2) the adhesiveness of the eggs (Uguz 2008). allowing for higher hatching success rate, further The shift of the suckermouth from rostral to contributing to the nest guarding feature of males; ventral position long before the hatching stage is a (3) the already ventrally positioned suckermouth common feature for most loricariids. This shift is of and sucking capacity of the free living embryo, more advantage to P. pardalis as newly hatched free- allowing for higher survival potential once left out of living embryos already had a ventrally flattened parental care due to its substratum scraping capacity

48 EurAsian Journal of BioSciences 8: 38-50 (2014) Jumawan et al.

for food and attachment; and (4) the absence of true ACKNOWLEDGEMENTS larval metamorphosis between the free-living embryo and the juvenile stage due to the large JC Jumawan is grateful to the Commission on supply of yolk. Information on the early life history Higher Education- Science and Engineering Grants strategies of P. pardalis from its original habitat (CHED-SEGS) for the dissertation grant and to the would be essential for comparison with the non¬ Philippine Kidney Transplant Institute (PKDF) native counterparts in order to determine possible Histology laboratory for the histological developmental plasticity of the fish in invaded water preparations. The authors thank Drs PJ Denusta and systems. LMB Garcia for the technical help in the in-vitro experiment.

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