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1Biology Department, Caraga State University, Butuan City, Philippines

2Institute of Biology, University of the Philippines-Diliman, Diliman, Quezon City, Philippines

3Institute of Environmental Science and Meteorology, University of the Philippines-Diliman, Diliman,Quezon City, Philippines

*Corresponding author: [email protected]

Embryonic and larval development of thesuckermouth sailfin catfish Pterygoplichthys pardalisfrom Marikina River, Philippines

38

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

Siluriformes exhibit diverse reproductive

strategies and most studies are just focused on late

embryonic and larval development (Adriaens and

Vandewalle 2003). Nonetheless, knowledge of the

crucial phases of the life history of invasive species is

vital in order to understand their developmental

strategies and identify the advantageous

ontogenetic features for invasion and

establishment. The management of invasive alien

fish species with known high fecundity potential and

high success rate of establishment in invaded

environments requires knowledge of its early life

stages, such as the timing of embryogenesis and

organogenesis and the shift from endogenous to

exogenous feeding (Godinho et al. 2003).

The armoured suckermouth sailfin catfish

Pterygoplichthys pardalis

(Castelnau, 1855) is a

native of South America (Weber 2003), but has been

introduced to the Philippines through aquarium

trade. It has invaded many freshwater systems of

the country along with another hypostomine

loricariid, Ptergoplichthys disjunctivus. These two

species, popularly known in the country as janitor

fish, are not regarded as important commercial

fishes because of their hard body armour, very little

meat, propensity to compete for food resources,

and their potential to bioaccumulate heavy metals in

polluted environments (Chavez et al. 2006, Lam and

Su 2009, Jumawan et al. 2010a). Nonetheless, the

hardy nature of this genus, its capacity to down

Received: January 2014

Received in revised form: April 2014

Accepted: April 2014

Printed: May 2014

INTRODUCTION

AbstractBackground: There is little information about the early development of this invasive fish species inorder to understand its early life history and developmental strategies towards invasion.Material and Methods: Female Pterygoplichthys pardalis were induced to spawn using humanchorionic 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, theHCG-induced fertilized eggs had a low hatching success (49%), while from the free-living embryossuccessfully hatched, a high number (90%) survived to become juveniles. Embryonic developmentin P. pardalis was completed 168 h and 30 min after fertilization, with the total yolk resorptioncompleted on the 8th day post hatching, during which the suckermouth gradually shifted fromrostral to ventral position to commence the loricariid algae-scraping feeding mode.Conclusions:Pterygoplichthys pardalis does not undergo a true larval metamorphosis between thefree-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 thisinvasive species whose spawning and early developmental strategies were difficult to observe in thefield. Implications of some ontogenetic features in this species with regards to invasion are alsodiscussed.

Keywords: Development, embryogenesis, invasion, larvae, morphology, Pterygoplichthys pardalis.

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

http://dx.doi.org/10.5053/ejobios.2014.8.0.4

Joycelyn Cagatin Jumawan1*, Annabelle Aliga Herrera2, Benjamin Vallejo Jr3

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regulate metabolism during periods of scarcity of

food (German et al. 2010), its tolerance to poor

water conditions and its ability to breathe air under

hypoxic water conditions (Armbruster 1998) enabled

this fish to invade and successfully establish itself

even in disturbed freshwater systems.

The seasonality of reproduction and the gonad

features of the Pterygoplichthys

spp. population in

Marikina River has been previously described,

showing the females to be highly fecund and the

oocytes exhibiting features associated with parental

care (Jumawan et al. 2010b). However, the burrow-

spawning and nest-guarding nature involved during

the critical period of embryogenesis in these fishes

was found difficult to replicate under laboratory

conditions. To date, there are no available studies

describing the early development of P. pardalis and

P. disjunctivus

in their original habitat for comparison

with species thriving in non-native environments.

The present study will serve as a baseline reference

of the early embryogenesis, larval development and

organogenesis of the invasive P. pardalis

through in-

vitro fertilization.

Strip method for artificial fertilization

The artificial breeding protocol of P. pardalis

adapted the procedure by Tan-Fermin et al. (2008)

with some modifications. All experiments were

carried out in triplicates. Collection and

experimentation were performed during the peak

spawning months (July to September) of the

Pterygoplichthys

spp. population in Marikina River.

Because of the absence of defined sexual

dimorphism in this fish, large and mature P. pardalis

weighing 350-500 g were collected. Females were

selected based on their full and heavy body, gravid

abdomen and reddish, swollen vent. To identify the

sex of the fish, pressure on the abdomen to extrude

oocytes and the use of cannula were also attempted

for most samples. Males were selected based on a

streamlined body and flat abdomen. A total of 18

females and 18 males were utilized for this study.

Prior to hormonal induction, all females were

anesthetized in a 200 ppm 2-phenoxyethanol

(Merck, Germany) bath for 4 min. The female P.

pardalis received an intramuscular injection of

human chorionic gonadotrophin (HCG) (Argent

Chemicals, Philippines) at 4 IU/g body weight(injection volume: 1L/g BW). The hormone-injected

females were then placed separately in 1 m x 0.5 m

plastic tanks containing de-chlorinated tap water to

a depth of 0.4 m each. Females untreated with HCG

served as controls and were placed in a separate

tank.

Approximately 14-18 h after HCG administration,

the females were checked for ovulation by applying

pressure to the abdomen to confirm ovulation. Eggs

from ovulated females were then stripped in a dry

plastic basin. At about the same time, males wereanesthetized, sacrificed by a sharp blow to the head

and had their testes removed. Milt were collected

after maceration of the testes and then immediately

diluted with 0.9% NaCl to obtain milt solution. The

milt solution was poured into a bowl containing the

stripped eggs and mixed for 30-60 sec using a

feather. Approximately 5 mL of tap water was added

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

for about 1 min to remove excess milt. Fertilizedeggs were immediately transferred to a 45x30x30

cm aerated plastic aquarium for incubation. The

aquaria were provided with partial shade by using a

1x1 m black, plastic polyethylene bag to simulate

the darkened burrows in the field. The eggs were

examined 10-15 min after gamete mixing to check

for blastodisc formation. Unfertilized eggs were

carefully removed from the aquaria using fine

forceps.

Initial observations showed that the fertilized

eggs had a low hatching success rate when

incubated in de-chlorinated tap water (10-15%) or

rain water (10-20%), but had an improved hatching

rate when using a mix of natural river and tap water.

Hence, the subsequent trials used a mix of aerated

Marikina River water and tap water as an incubation

medium at 1:1 ratio. Prior to mixing the water

medium, river water and tap water were analyzed

for hardness (CaCO3/L), chloride (mg/L), calcium

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EurAsian Journal of BioSciences 8: 38-50 (2014)

MATERIALS AND METHODS

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(mg/L) and magnesium (mg/L) levels. The physical

and chemical parameters of the mixed water in the

aquaria, such as temperature (C), pH, salinity and

dissolved oxygen (D.O.), were recorded throughout

the experiment.

Embryogenesis, larval ontogenesis and

biometry

For each of the 3 replicate plastic aquaria, twenty

developing eggs were observed at 10-30 min

intervals until completion of cleavage, 3 times per

day until hatching. Hatchlings were documented

twice daily, until the yolk was fully resorbed. The

developmental stages were divided into embryo and

free-living embryo stages. The embryonic stage

occurs inside the chorion and ends in hatching. The

free-living embryo stage is characterized by the

nutritive contribution of the yolk sac and the stage

ends when the free-living embryo becomes capable

of exogenous feeding after the yolk has been

consumed (Geerinckx et al. 2008).

Three subsamples of fertilized eggs were

collected daily until yolk resorption and were fixed

in Bouins fluid for histological purposes.

Additionally, three more fertilized eggs and

hatchlings were collected until yolk resorption and

were directly fixed in 4% neutral formaldehyde for

biometry using a digital caliper (accuracy 0.001 mm;

Control Company, USA), following the parameters

described in the study of Guimaraes-Cruz et al.

(2009) (Fig. 1).

The fertilization rate (number of eggs with

blastodisc/total oocytes x 100), hatching rate

(number of hatched eggs/number of fertilized eggs

x 100) and survival rate (number of surviving

juveniles/total number of hatched embryos x 100)

were recorded. Three replicate runs from

fertilization to hatching were conducted.

Analysis of variance (ANOVA) was used to

compare the mean values of the morphometric

variables according to each stage of larval

development, and between oocyte mean diameter

values for HCG-induced and non-HCG-induced

samples. All tests were conducted in a 0.05

significance level using Graphpad Prism 5.

Description of fertilized eggs

The adult female P. pardalis were administeredHCG a day after samples were obtained from the

field. Difficulty in distinguishing between males and

females was noted due to lack of defined sexual

dimorphism as well as difficulty in extruding oocytes

and milt from the vent after the application of

considerable abdominal pressure and cannulation.

Table 1 shows the physico-chemical parameters of

the water used for embryo incubation. Oocyte

extrusion was performed 18 h after the exposure to

HCG. Approximately 200-250 oocytes were extruded

from the ovaries of a single female exposed to HCGdue to difficulty of handling and applying pressure in

the abdomen of the fish. The females injected once

with HCG responded positively to the treatment

(97% success rate) and hydrated, producing nearly

uniform sized (2-3 mm), transparent-yellow oocytes

after the ovary was stripped. In contrast, the oocytes

from females not exposed to HCG were mostly

opaque yellow with occasional pre-vitellogenic

oocytes (

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embryo). The main events in embryogenesis and

their respective times of observation are

summarized in Table 3 and Figs. 2-4. The fertilized

eggs of P. pardalis show meroblastic cleavage in

which the blastoderm is restricted to a small area at

the animal pole. The first segmentation that divided

the blastodisc into two blastomeres occurred within

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

fertilization. The spread of the blastoderm was

evident, covering the yolk with the embryo body

becoming more elongated, while the head and tailends of the embryo can be clearly seen at 20 h.

Finally, full yolk invasion and closure of the

blastopore was observed at 24 h.

Differentiation of the embryo

Pre-hatching organogenesis in P. pardalis

commenced with the formation of the notochord

and observations of the cranial-caudal portions of

the embryo (29 h), as well as the first somites and

the optic vesicles (36 h). Somitogenesis was

observed starting 2 days post fertilization (2 dpf)

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Fig. 1. Lateral view of a P. pardalisfree-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. 2. Stages of embryonic development in P. pardalis.(a) Early stage fertilized egg with distinct invagination at the animalpole before blastodisc formation; (b) Blastodisc stage; (c) Cleavageat 2-cell stage; (d) Cleavage at 4-cell stage; (e) Cleavage at 8-cellstage; (f) Cleavage at 64 blastomeres; (g) High blastula stage; (h)Low blastula; (i) Early gastrula.

Scale bar: 1 mm.

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) Rostrallocation of suckermouth (*), Note egg sac removed; (f) P. pardalis at6 dpf; (g) Newly hatched embryo (7 pf); (h) Note the ventral

position of the suckermouth in (g).

Scale bar: 1 mm.

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and subsequent somite formation enabled tail

movement, although the embryo was largelyconfined within the perivitelline space. No

corresponding somite formation for each hour

during the onset of embryogenesis in P. pardalis was

noted because of the difficulty in turning the

strongly adhesive eggs to locate the somites

without causing mechanical injury to the developing

embryos. The time when the pericardial cavity was

formed was not determined; however, the pulsating

heart and blood circulation was visible as a very faint

stream of capillary network in the pericardial cavity

at 36 h after fertilization. Ectodermal thickening toform the lens of the eyes was observed starting at

36 h, with the eye lens fully formed at 49 to 50 h post

fertilization. The suckermouth was rostrally

positioned inside the membrane (Fig. 3d-e,Fig. 4a).

Tail movement (72 h post fertilization) was

observed before the formation of the vitelline

circulatory system. Movement in the gill cavity and

blood circulation in the gill arches was noted

beginning at 5 dpf. Hatching was observed 167-168

h post fertilization. The caudal tail region was

observed detaching from the yolk mass with asubsequent increase in body movement, causing the

rupture of the chorion and the emergence of the

embryo from the capsule.

Free-living embryo development

Details of development and average body

measurements are reflected in Tables 4 and 5.

Histologic sections of larvae at different days post

hatching are shown in Figs. 5-7.

Hatchling

Newly hatched, free-living embryos or eleuthe-

rembryos (Balon 1986) have a mean total length of

7.860.12 mm, still containing a large amount of yolk

(Fig. 5a). The newly hatched larvae had already

ventrally located the suckermouth and maxillary

barbels and were able to attach to the sides of the

glass substrata with their suckermouth, with water

inflow for the sucking action passing through the

furrows of the maxillary barbels, and attachment

sometimes assisted by body and tail movements

(Fig. 3h). Embryos initially had a rostrally located

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

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

*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 meansS.E.

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

*P

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mouth, which gradually shifted into the ventral

location during hatching. Dorsal and caudal fins were

observed. The bodies of the newly hatched free-

living embryos were transparent, except for the

onset of dendritic pigmentations initially found

interspersed finely on the head and on the edges of

the snout. The eye diameter was small (0.510.02

mm). The dorsal and caudal fins were well

recognizable. Serial sections of the digestive system

during the day of hatching showed that the gut is

tubular and closed at both ends, while the intestine

is composed of simple cubic epithelium. The first

outlines of the gill arches supported by blood

vessels were observed in the gill cavity. A tubular

heart was observed.

3-d old free- living embryo

A continued reduction of yolk sac and an increase

in body length (TL 12.410.05 mm) were observed

along with increasing pigmentation in the retina and

increasing complexity of the gills. Increasing

dendritic chromatophore pigmentation was

observed in the outlines of the head and dorsal side

of the body. The digestive system was characterized

by a simple striated border in the intestine (Fig. 5f).

The cranial kidney was now well developed with

readily recognizable glomeruli tufts within the

network of reticular fibers (Fig. 5e). The spleen could

be observed on the left lateral-dorsal-right lateral

part of the borderline between the gut and the

kidney. The liver could be observed ventrally located

in the cranial region with the hepatic parenchyma

very homogenous in appearance.

5-d old free-living embryo

The average length of a 5-day old free-living

embryo was about 14.150.04 mm TL with the yolk

sac length becoming gradually reduced (3.220.03

mm). Increasing pigmentation all throughout the

body was observed with dendritic chromatophores

reaching the lateral region caudally to the pectoral

fin (Fig. 4c-d). The yolk was visible, although less

compact near the digestive system. The intestines

became looped and lengthened, with the intestinal

epithelium exhibiting a complex columnar

arrangement of mucous and goblet cells. Intestinal

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

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content (residue) was observed in this stage.

Nephric ducts in the cranial kidney were very visible;

the encephalon was compact with associated cells

(Fig. 6e). The heart was compartmentalized with a

very visible atrium (Fig. 6f).


The average length of 7-day old free-living

embryo was about 14.840.34 mm TL with the yolk

sac length becoming gradually reduced (2.320.09

mm). Increased pigmentation all throughout the

body including the ventral part previously occupied

by the yolk sac (Fig. 4e). Full pigmentation of the

eyes observed (Fig. 7c). Increased use of the

suckermouth to anchor body in the glass aquaria was

observed in larvae. The gills were more developed

with elongated filaments and gill lamellae with

intense vascularization. Gas bladder was observed

with simple squamous epithelium (Fig. 7d). The heart

presented two compartments. Small intestine

mucosa can be seen, including microvilli, columnar

epithelium and muscularis mucosa. The head kidney

had visible renal tubules and extensive hemato-

poetic tissue.


The average total length of free-living embryo

was 6.950.20 mm TL and the yolk was fully

resorbed. The body had become opaque with the

accumulation of pigments all throughout its surface

as chromatophores became more numerous and

darker, maintaining the same distribution pattern.

Undifferentiated gonad with primordial germ cells

was observed (Fig. 7e), while a true larval stage

(Balon 1986, 1999) was not observed. P. pardalis

underwent a direct transition from a free-living

embryo with large yolk into a juvenile without

undergoing a true larval stage when the yolk was

fully consumed at 8 dph. Except for the absence of

hardened armour covering the body and the

abdominal pattern distinct for P. pardalis, an adult-

like appearance was observed at the moment the

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Fig. 4. Free-swimming embryo development in P. pardalis.(a) Embryo with yolk sac removed. Note the rostral position of themouth; (b) Development at 3 dph; (c-d) Development at 5 dph; Notethe capacity of the ventrally positioned mouth for attachment inthe glass substrata in; (c) Body pigmentation in (d); (e)Pigmentation in the ventral position of the 7 dph free-living

embryo.

Fig. 5. Organogenesis in P. pardalis.(a) Newly hatched free-living embryo; medial portion; (b) 2dph,cranial portion; (c) 3 dph, Retina (*) and its layers; (d) 3 dph, cranialportion; (e) 3 dph, Digestive system; (f) 3 dph, intestine with simplestriated border. N: Nostrils; E: Encephalon; H: Heart; B: Barbells; CK:Cranial kidney; GB: Gas bladder; I: intestine; y: Yolk; G: Ganglionarcell; IN: Inner nuclear layer; ON: Outer nuclear layer; YG: ocular

globe; GA: gill arches.Scale bars: a,d,e: 200 m; c,f: 20 m; b: 40 m.

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Table 5. Body measurements* (mm) of P. pardalisfrom hatching until yolk resorption.

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 meansS.E.; n= 212

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

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last yolk was consumed at 8 dph.

Biometric parameters registered a gradual

increase in all values except for the decrease in the

yolk sac length and height as the free-living embryo

neared and completed the yolk resorption

externally (Table 5). A full yolk resorption in the

juvenile P. pardaliswas observed 336 h or 14 dpf at

24C. All the oocytes observed for fertilization untilyolk resorption in juveniles developed synchro-

nously.

Most invasive loricariids do not reproduce

spontaneously when reared under laboratory

conditions (Alfaro et al. 2008). The present study

provides baseline information regarding the early

stages development of P. pardalis, a highly invasive

loricariid, whose early life history has not been

studied so far.

The reproduction and spawning behavior of the

janitor fish P. pardalis is difficult to observe, as they

are known to spawn in burrows with males guarding

the fertilized clutch. What is surprising in this

experiment, however, is the tendency of the eggs to

hatch well in river water characterized to be hardy,

with higher calcium and magnesium levels compared

with tap water.

The water quality of key areas along Marikina

River where spawning colonies of P. pardalis are

abundant showed that the river is highly eutrophic

and turbid, and had overall high conductivity,

nitrate, ammonia and phosphate levels. Fertilized

clutches from the river also have a higher hatching

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Fig. 7. Organogenesis in P. pardalis.(a) 6 dph; gas bladder and kidney; (b) 6dph, intestine; (c) 7 dph,cranial portion; (d) 7 dph, digestive system; (e) 8 dph,undifferentiated gonad; (f) 8 dph, segmentation of the notochord.B: Barbels; GB: Gas bladder; CK: Cranial kidney; I: Intestine; Y: Yolk.G: Ganglionar cell; IN: Inner nuclear layer. ON: Outer nuclear layer;YG: Ocular globe; GA: Gill arches; H: Heart; PGC: Primordial germ

cell; N: Notochord; DF: Dorsal fin; M: Muscle.Scale bars: c,d,f: 200 m; b,e: 20 m.

DISCUSSION

Fig. 6. Organogenesis in P. pardalis.(a) 3 dph, cranial portion; (b) 3 dph, spleen and kidney; (c) 4 dph,dorsal nervous tube; (d) 4 dph, digestive system; (e) 5 dph,encephalon; (f) 5 dph, heart. E: Encephalon; H: Heart; B: Barbells; K:Kidney; I: Intestine; y: Yolk; G: Ganglionar cell; IN: Inner nuclear

layer; ON: Outer nuclear layer; YG: Ocular globe; GA: Gill arches; S:Stomach; SP: Spleen, N: Notochord; M: Myomeres; NC:Neurocranium.

Scale bars: a: 200 m; b,d: 40 m; c,e,f: 40 m.

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rate if incubated in river water. It is not clear how the

nest guarding nature of the males for this species

actually influences the successful hatching because

of the difficulty of observing this process in the field.

Makeshift and darkened plastic aquaria to replicate

burrows in the field in the preliminaries of this

experiment failed to encourage spawning under

laboratory conditions. The low success rate in

hatching for P. pardalis under artificially-induced

spawning conditions may be attributed to the

absence of parental care during the experiment or

to the preference of eggs for river water

environments.

Eggs of P. pardalis

are highly adhesive, allowing

the formation of tight clutches of eggs. Teleost eggs

can be non-adhesive, weakly adhesive or strongly

adhesive. Rizzo et al. (2002) point out that adhesive

eggs are often large, laid in smaller numbers and

associated with the sedentary nature of the species

or with parental care. The zona radiata of

Pterygoplichthys spp. in Marikina River was thin

(mean 4.56 m), while its granulosa layer was thick

(mean 2.81 m) (Jumawan and Herrera 2014). A thick

granulosa layer may provide better adhesion of

eggs, while the thin zona radiata may be

compensated by the nest guarding nature of the

males of this species, as was the nature of some nest

guarding loricariids (Suzuki et al. 2000).

It was apparent in the results that although

artificially fertilized eggs had a low hatching rate, all

hatched P. pardalisfree-living embryos successfully

survived and had resorbed their yolk at 8 dph. A

large endogenous supply of yolk nutrients enhances

survival during the period when feeding structures

are still developing (Geerinckx et al. 2008), while also

enabling the free-living embryo to avoid an

intermediate larval stage and the cost of

metamorphosis since a definitive adult phenotype

was developed directly (Balon 1986).

Although hardened armour covering the body

was not observed when the last yolk was consumed

at 8 dph, hardening of the head and dorsal structure

towards development of the armoured covering of

the body was eventually observed 30 days post yolk

resorption in juveniles (data not shown).

Types of feeding (exogenous, endogenous),

nutrient supply (altricial, precocial) and life history

models (indirect, direct development) were used as

basis in the description of embryos (Balon 1986,

1999). Embryos with indirect development are a

consequence of poor vitellogenesis and depend

entirely on an endogenous nutrient supply, as eggs

are altricial in nature (Balon 1986). The short embryo

period in this type of development appears to be

extended by the larva period that feeds

exogenously prior to the formation of the definitive

phenotype. Fish taxa characterized by direct

development have a prolonged embryo period due

to a large endogenous supply as eggs are precocial

(large amount of yolk) that ultimately enables the

embryo to develop directly into a definitive

phenotype (Balon 1986). Embryos develop for

longer but directly into juveniles that are able to

compete in the adult habitat (Balon 1999). Direct

development occurs more frequently in the

reproductive guilds of guarders and bearers as these

fish groups exhibit parental care such as site

selection, egg deposition and nest guarding (Balon

1986). A true larva requires some tissues and

structures very different to those in the definitive

organism, and so, has to be remodeled through the

process of metamorphosis (Balon 1999). Fish species

with direct development lack the necessary cost of

forming temporary organs through metamorphosis.

The large yolk (mean 3.3 mm) in P. pardalis

is an

advantage in this context, as it requires none or little

external nutrients to develop into a definitive

phenotype. It has been proposed that the larger and

more advanced an individual at the onset of

exogenous feeding, the better its chances of

surviving (Balon 1999). This scenario improves

competitiveness in P. pardalis even in its early stages

and could be an advantage for invasion (Balon 1986).

The absence of a true larval stage in P. pardalis is

similar to most loricariids (Geerinckx et al. 2008). It is

however important to note that a slight variation in

measurements may be caused by the fixative used in

this study (4% neutral formaldehyde), as fixatives

may have some dehydrating effects contrary to the

measurement of fresh samples.

The eggs of P. pardalis contain a large amount of

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evenly distributed yolk, hence, is classified as

telolecitic (Ribeiro et al. 1995, Marques et al. 2008).

P. pardalis

has a meroblastic cleavage pattern

restricted to the animal pole, which is common in

teleosts. Blastopore closure occurred 24 h after

fertilization, indicating fertilization success. The

embryonic development of P. pardalis lasted 7 days

(168 h, 30 min), which is long compared with the

duration of embryogenesis in other siluriforms: 45 h,

50 min at 24C in R. aspera(Perini et al. 2009), 21 h,

20 min at 23C in Pimelodus maculates

(Luz et al.

2001) and 18 h at 27C in P. corruscans

(Marques et

al. 2008).

Long embryonic periods are known to be

associated with non-migratory species having large

eggs and with those that display parental care

(Sargent et al. 1987), features that both fit the

natural history of P. pardalis. Invasive species

exhibiting parental care may be considered an

advantage in the context of ensuring egg survival

until the juvenile period. By females precisely

selecting a safe and protected site for egg

deposition and males guarding the nest, the

progress of the early developmental stages in P.

pardalis

becomes ensured. This protection makes

the early stages difficult to observe in the wild as

they are well hidden from predators and other

threats, emerging only when post-yolk sac juveniles

are capable of feeding and the definitive phenotype

ensures the adult form.

A pigmentation of the retina observed early

during hatching in P. pardalis could be associated

with the need to develop a functional visual system

before the first feeding, typical in some fishes (Hall

et al. 2004). The undifferentiated gonad located

between the cranial kidney and the digestive tract in

P. pardalis

already contains a cluster of primordial

germ cells (PGCs) at 8 dph. PGCs in the

undifferentiated gonad were also observed at 5 dph

in R. aspera and at 13 dpf in Pimephales promelas

(Uguz 2008).

The shift of the suckermouth from rostral to

ventral position long before the hatching stage is a

common feature for most loricariids. This shift is of

more advantage to P. pardalis as newly hatched free-

living embryos already had a ventrally flattened

mouth despite the large yolk sac during hatching. On

the contrary, A. cf triradiatus, a relative loricariid,

was noted to have a rostro-ventrally positioned

suckermouth during hatching, but had to wait for 2-

4 days for the yolk sac to resorb for the transition to

a ventrally flattened mouth (Geerinckx et al. 2008).

The newly hatched P. pardalis in this study was able

to attach immediately to the substrate with their

suckermouth. This observation was also reported for

Sturisoma aureum(Riehl and Patzner 1991) and A.cf.

triradiatus

(Geerinckx et al. 2008). This feature may

be essentially an advantage for loricariid species

where hatchlings leave the shelter immediately

(Suzuki et al. 2000). In the case of an invasive species

such as P. pardalis, accidental release of eggs and

juveniles may result in assured higher survival rates

in the wild.

The SL of P. pardalis

during the time of hatching

(7.860.12 mm) and the large yolk sac during the

same time fall within the size range for most

loricariids (6-8 mm) (Riehl and Patzner 1991,

Nakatani et al. 2001, Geerinckx et al. 2008, Perini et

al. 2009). The morphometric development in P.

pardalis, which reflected a gradual increase in all

parameters with decreasing yolk size, is also

observed in A. cf triradiatus (Geerinckx et al. 2008), R.

aspera

(Perini et al. 2009) and Lophiosilurus alexandri

(Guimaraes-Cruz et al. 2009), all of which exhibited a

high degree of allometric growth. Geerinckx et al.

(2008) hypothesized that loricariid hatchlings often

exhibit rapid allometric growth in the snout and

remarkable lip transformation because it is a

necessity and advantageous for the suckermouth to

attach to the substratum for scraping and sucking

food.

In conclusion, this study pointed out several

baseline features of the early life strategies in P.pardalisthat may be of advantage to its biotic spread

potential: (1). The propensity of the embryo to thrive

in polluted water; (2) the adhesiveness of the eggs

allowing for higher hatching success rate, further

contributing to the nest guarding feature of males;

(3) the already ventrally positioned suckermouth

and sucking capacity of the free living embryo,

allowing for higher survival potential once left out of

parental care due to its substratum scraping capacity

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Jumawan et al.

49


for food and attachment; and (4) the absence of true

larval metamorphosis between the free-living

embryo and the juvenile stage due to the large

supply of yolk. Information on the early life history

strategies of P. pardalis from its original habitat

would be essential for comparison with the non-

native counterparts in order to determine possible

developmental plasticity of the fish in invaded water

systems.

JC Jumawan is grateful to the Commission on

Higher Education- Science and Engineering Grants(CHED-SEGS) for the dissertation grant and to the

Philippine Kidney Transplant Institute (PKDF)

Histology laboratory for the histological

preparations. The authors thank Drs PJ Denusta and

LMB Garcia for the technical help in the in-vitro

experiment.

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