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WITHDRAWN: Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: Loricariidae) from Marikina River, Philippines
Accepted Manuscript Title: Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines Author: J.C. Jumawan A.A. Herrera PII: DOI: Reference:
S0040-8166(14)00030-5 http://dx.doi.org/doi:10.1016/j.tice.2014.05.001 YTICE 857
To appear in:
Tissue and Cell
Received date: Accepted date:
13-2-2014 2-5-2014
Please cite this article as: Jumawan, J.C., Herrera, A.A.,Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines, Tissue and Cell (2014), http://dx.doi.org/10.1016/j.tice.2014.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines
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J.C. Jumawana,b*, A.A. Herrerab Biology Department, Caraga State University, 8600 Butuan City, Philippines
Institute of Biology, University of the Philippines-Diliman, Quezon City, Philippines
Suggested Running Title:
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Testis characteristics of Pterygoplichthys disjunctivus
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Janitor fish, ultrastructure, reproduction
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Keywords:
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Acknowledgements:
Grants from the Commission on Higher Education- Science and Engineering Grants (CHED-
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SEGS) supported this study. The National Kidney Transplant Institute (NKTI) Transmission Electron Microscopy Center and the Philippine Kidney Transplant Institute (PKDF) HistoPathology laboratory are gratefully acknowledged for the TEM and histological preparations.
Correspondence author's details: Joycelyn C. Jumawan
Biology Department, Caraga State University, 8600 Butuan City, Philippines [email protected]; Tel: (6385) 342-3047, Fax: (6385) 342-1079 ORIGINAL SUBMISSION
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ABSTRACT
The histological and ultrastructural features of the testis of the invasive suckermouth sailfin
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catfish Pterygoplichthys disjunctivus rapidly proliferating in Marikina River, Philippines were characterized during the fish’s 2010-2011 reproductive season. The germinal
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compartment of the testes was composed of anastomosing tubules with cysts undergoing synchronous development. Spermatogenic cells were along the length of the testes indicate it
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to be of the unrestricted spermatogonial type. The spermatozoon is classified as type 1 ect
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aquasperm devoid of acrosome, has rounded nucleus, and a long flagellum—characteristics necessary for external fertilization. Male P. disjunctivus was reproductively active during half
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of the year-long study with peak spawning during the most rainy months (June to August) and prolonged recrudescence during the dry months (February to May). Results from this
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study form a histological baseline to describe the gonad dynamics and reproduction of this
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invasive fish species as well as provide possible means of mechanical control to curb the
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population of the fish in this river.
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1. Introduction
Pterygoplicthys disjunctivus Weber, 1991 is an invasive loricariid catfish that originally
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inhabited the Amazon River basin of Brazil and Peru (Weber, 2003) and Rio Madeira drainage of Brazil and Bolivia along with another species, Pterygoplichthys pardalis (Page
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and Robins, 2006). These two species are considered invasive in at least nine countries outside the neotropics (German et al., 2010), including the Philippines where they are
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popularly known as “janitor fish”. These species were first imported to the Philippines as
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ornamental aquarium fish. However, many propagules were believed to have accidentally escaped from aquarium fish breeding facilities into Laguna de Bay when typhoon Rosing
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caused flooding in 1995 (Guerrero, 2006). The two species rapidly proliferated in the Marikina River, a highly urbanized water system in Luzon (Chavez et al., 2006a). Individuals
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with intermediate abdominal pattern characteristics between the two species were observed
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with no defined species-specific DNA barcodes, suggesting possible introgressive hybridization between P. pardalis and P. disjunctivus in the river (Jumawan et al., 2011;
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Quilang and Yu, 2013).
Pterygoplichthys pardalis and P. disjunctivus have several features that account for their
rapid invasion in their non-native environments. These fishes were reported to be highly tolerant to poor water conditions (Chavez et al., 2006b; Lam and Su, 2009; Jumawan et al., 2010). Plasticity of the gastrointestinal tract and the broad diet of P. disjunctivus (German et al., 2010), its air-breathing capacity under hypoxic conditions, as well as the absence of natural predators of mature populations (Nico and Martin, 2001; Gibbs et al., 2008) contribute to their successful invasion in non-native environments. Knowledge of the reproductive biology of the two species and the family Loricariidae is quite limited but is important to enable a more accurate understanding of its natural history
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and possible reproductive plasticity. While it is known that female P. disjunctivus were capable of spawning at relatively smaller sizes (Gibbs et al., 2008) and some external features of the ovary were known, detailed studies on the testes have not been reported.
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This study will provide baseline information on the testicular morphology of P. disjunctivus in Marikina River, Philippines to provide insights into the knowledge about the
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reproduction of a neotropical teleost that has become invasive in a new environment. A description of the testicular maturity stages, as well as details on the ultrastructure of the
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gonad, are essential in creating management strategies and empirical models for the
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prediction of the fish’s invasive potential.
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2. Materials and Methods 2.1. Study area Pterygoplichthys disjunctivus were collected monthly from five sites (San Jose,
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Rodriguez Rizal; Banaba, San Mateo; Tumana, Malanday, River Park) along Marikina River,
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Philippines (Fig.1) from July 2010 to June 2011.
2.2. Sampling
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Fish were captured with a cast net (5 m long; mesh size of 3.8 cm). Due to the absence
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of defined sexual dimorphism in P. disjunctivus, females were inevitably collected and were excluded after sorting in the laboratory. Segregated males were sacrificed by immersing fish
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in ice 4 h prior to dissection. Standard length (SL) was measured to the nearest 0.1 cm. Body weight was measured with an electronic scale to the nearest 0.1 g. Testes were weighed using
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an analytical balance of 0.1 to 0.01 g accuracy. Gonado-somatic index (GSI) was calculated
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as: GSI = [(gonad weight)/ (body weight)] x 100.
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2.3 Visual characterization and Histological preparation Testis was characterized using visual-based macro scale of maturity based on testicular
size, color and swelling (Cailliet et al., 1986; Nunez and Duponchelle 2008; Brown-Peterson et al., 2011; Lowerre-Barbieri et al., 2011) with some modifications. To assess possible regionalization of germinal epithelium and glycoproteic region of secretion along the testes length, fragments from the cranial, medial and caudal portions were selected for histological characterization. A total of 360 testes samples were fixed in Bouin’s fluid for 12-24 h, dehydrated through a 75-90% ethanol series, embedded in paraffin, and sectioned at 5-6 µm. Cross and longitudinal sections were stained with haematoxylin-eosin (HE). The diameter of 100
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spermatogenic cells in each stage of development was measured in 5 to 10 histological sections a light microscope with a calibrated micrometer each month. Alcian blue (AB)Periodic Acid Schiff (PAS) pH 2.5 (ArtisanTM) was used to detect proteins and carbohydrates
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in the three testicular regions.
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2.4. Transmission and Scanning electron microscopy
Additional testicular tissue fragments from the cranial and medial regions of mature P.
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disjunctivus were immersed in 4% glutaraldehyde for 2 h followed by two changes of 0.2 M
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sodium cacodylate buffer at pH 7.4 for 10 min each and post-fixed in osmium tetroxide in the same buffer (0.2 M) for 15 min. After dehydration in an increasing alcohol series, the tissues
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were embedded in Epon resin mixture. Semi-thin sections (1 µm) were stained with toluidine blue for selection of adequate fields under light microscope. Ultra thin sections (70 nm) of
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the selected areas were counterstained with uranyl acetate and Reynold's lead citrate,
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analyzed and photo-documented using an energy filtering transmission electron microscope (EFTEM), Zeiss LIBRA 120™, Germany. Testicular maturity stages were then characterized
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based on the thickness of the tunica albuginea, the diameter of the tubule lumen and the predominant spermatogenic cells in each stage. Percentage (%) composition of the maturity stages derived from the histology of the testis was summarized for the 12 sampling months to establish reproductive seasonality in males. Spermatozoa were analyzed through scanning electron microscopy (SEM). Seminal
fluid was collected from adult males through maceration of the testes and dilution of the milt in phosphate buffer at 1:50 ratio suspension in a 1.5 ml microcentrifuge tube. Twenty microliters of the sperm suspension was transferred to a separate tube and centrifuged at low speed to allow sperm to settle at the bottom of the tube. Sperm samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 2 h at 40C. Primary washing of fixed
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samples (3 washes; 15 min. duration each) were done using 0.1 M cacodylate buffer pH 7.4. Osmium tetroxide (aqueous) pH 7.4 was used for secondary fixation. Samples were dehydrated in increasing ethanol series, mounted in a glass slide, and then dried overnight in
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a desiccator. The mounted samples were placed in a metal stub and then coated with a colloidal mixture of palladium gold (20 nm). Micrographs were obtained using Inspect ™
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Scanning Electron Microscope.
Size at first maturity (L50) was also determined from fishes classified as sexually
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mature and spawning capable through histological features and GSI of the testes using a
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logistic curve fitted by a weighted non-linear least squares regression. For all measured structures, the mean and standard deviation (SD) were calculated. Statistical analyses were
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conducted using Graphpad Prism5 software.
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3. Results
3.1. Internal morphology
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A total of 526 males were collected during the year-long study. Individuals ranged from 5 to 45 cm SL. Externally, sex in P. disjunctivus cannot be accurately distinguished due
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to lack of defined sexual dimorphism (Fig. 2 a, b). The testes of adults are paired, elongated organs joined together ventrally by the mesorchium and joined caudally by a common
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spermatic duct separated from the urethra through a thin sphincter. A resulting common duct
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extends anteriorly into a common urogenital opening. The terminal end of the rectum traverses across the testes midline and converge with the common urogenital tract separated
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by another muscular sphincter. Release of excreta and sperm is regulated by this sphincter as there is only a single aperture that serves both as a gonadal and anal orifice visible exteriorly.
3.2 Gonadosomatic Index
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An accessory seminal vesicle was absent at the caudal region of the testes.
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GSI values declined significantly from November to January (P<0.0001). The
minimum length at maturity for males is 18 cm SL. Males have longer spawning season extending until October (Fig. 3).
3.3. Ultrastructural characterization Testes of a mature P. disjunctivus is covered by a thin tunica albuginea (Fig. 4 a),
with the germinal compartment composed of anastomosing tubules with cystic organization. Spermatogenic cells are observed to undergo synchronous development (Fig. 4 b) and are delimited by cytoplasmic extensions of the Sertoli cells at the base of the germinal compartment forming an anastomosed tubular system (Fig. 4 c). Spermatogonia are present
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along the length of each testis tubule, hence, could be described as unrestricted spermatogonial testicular type. There are no defined regions exclusive for secretion as evidenced by the absence of transition secretory prismatic cells and a negative reaction for
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carbohydrates and protein secretion in the lumen of the lobule through AB-PAS staining (Fig. 4 d). The germinal epithelium, supported by the basal lamina with the interstitial tissue is
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composed of myoid cells, blood vessels, Leydig cells and neighboring tubules (Fig. 5). Germ cell types were characterized based on morphology and size of the nucleus, organelles,
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complexes and pericentriolar features and are depicted using annotated photomicrographs.
3.3.1. Spermatogonia
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Primary spermatogonia (PSG) are the largest cells in the lineage (14.87 ± 0.22 µm), and attached nearest to the basal lamina of the tubule, surrounded by Sertoli cell extensions
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forming a cyst (Fig. 6 a). It has an extensive cytoplasm containing abundant mitochondria, a
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large spherical but faint nucleus (7.59 ± 0.11 µm) covered by an irregularly contoured nuclear membrane, and a distinct nucleolus (1.76± 0.08 µm) with darkly staining chromatin. Highly
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electron-dense particles or nuages are seen associated with several mitochondria (intermitochondrial cement) near the nuclear envelope or seen freely dispersed inside the cytoplasm. PSG dominate immature and regenerating testicular stages although these cells are seen in other testicular stages as they formed the reserve pool of germ cells. Secondary spermatogonia (SSG) are smaller than PSG (8.15±0.35µm) and form cysts of
two to four cells. The nucleus contains 2-3 nucleoli while the cytoplasm contains few organelles (Fig. 6 b).
3.3.2. Spermatocytes
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Primary spermatocytes (SC1) are formed after secondary spermatogonia (SSG) had undergone first meiotic division, resulting in an increased number of cysts and increased testes size during the developing (stage 2) and regenerating stages (stage 6). SC1 are smaller
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(7.16 ±0.60 µm) than SSG with very little cytoplasm and abundant mitochondria. The nucleus (6.51±0.24 µm) is slightly condensed with granular chromatin and had prominent
(Fig. 6 c). SC1 often occur at minimum of 6-12 cells in a cyst.
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synaptonemal complexes (chromosomic masses) appearing as parallel electron dense strips
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Secondary spermatocytes (SC2) are even smaller than SC1 (6.19 ±0.32 µm), with much
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reduced cytoplasm and abundant mitochondria. The pale-staining nucleus (4.8 ± 0.27 µm) contained chromatin material appearing as electron-dense clots (Fig. 6 d). These cells occur
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in large numbers ≥ 20 in a cyst nearest the lumen.
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3.3.3. Spermatids
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Spermatids (ST) are haploid cells formed after the second meiotic division of SC2. Spermatids exhibit several stages of chromatin condensation, shape remodeling and
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cytoplasm reduction while undergoing spermiogenesis. In the initial stages of ST differentiation, cells are closely attached to each other with a homogenously translucent chromatin in the nucleus. In the intermediate stages, the cytoplasm exhibited intense vacuolization and are often displaced caudally to the electron dense nucleus along with the abundant mitochondria (Fig 7 a). The nuclear fossa eventually deepens and forms a depression caudal to the nuclear envelope where the proximal and distal centrioles were located. The centrioles eventually move into the nuclear fossa bringing with them the basal body that forms the early flagellum segment and the plasma membrane (Fig. 7 b-e). The later stages of ST differentiation is characterized by a very dense nucleus (2.88 ±0.074 µm) covered by a thin perinuclear space and an elongated flagellum emerging from the nuclear
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fossa (Fig. 7 f). A large amount of cytoplasm is displaced to the nucleus and eliminated while still inside the cysts and then eventually absorbed by the cytoplasm of the Sertoli cells.
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3.3.4. Spermatozoa Spermatozoa (SZ) are the smallest cells of the lineage formed from STs upon completion
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of spermiogenesis. A mature spermatozoon has a round, opaque nucleus devoid of acrosome (2.46 ±0.105 µm) and has a long flagellum (21.94± 0.94 µm) devoid of flagellar fin (Fig. 7 h-
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i). Once differentiation of SZ is completed, cytoplasmic extensions of the Sertoli cells that
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enclose the cyst retract and SZ are released into the lumen.
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3.4. Histological characterization
Six stages of testicular maturity based on testes color, texture, weight, features of
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spermatogenic cells, thickness of the tunica albuginea, and diameter of the lumen were
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described (Table 1; Fig. 8). Minimum length of maturity in males with testes containing mature SZ and considerable swelling throughout the testicular surface were initially observed
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at 29 cm SL and onwards. Early stage recovering spent (stage 6) testes peaked in January along with increasing observations of mature testes until April (Fig. 9). Recurrence of early developing-spent (stage 5) testes overlapping with regressing
stages was seen during the hottest months of the study period (April- May 2011) when virtually no mature and late stage regenerating testes were observed. Testes of individuals sampled in May had a peculiar morphology, having a highly thickened tubular wall with excessive meiotic divisions and with SC2s and STs conspicuously lodged in the germinal epithelium. PSGs and SSGs were scanty or absent in the lobule lamina while the lumen are devoid of SZs.
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4. Discussion 4.1. Absence of sexual dimorphism P. disjunctivus exhibit no defined sexual dimorphism. However, it may be possible that
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during the actual spawning season, sex-related features may be prominent. Highly fecund and large female P. disjunctivus have bigger and swollen abdomen and can be distinguished from
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males during the peak of the spawning season. Catfishes do not reproduce spontaneously under culture conditions (Legendre et al., 1996), making it difficult to observe courtship
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frequencies, possible sexual signals in choosing mate and other male-female sexual
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interactions. However, sexual dimorphism has been reported in about half of all families of catfish (Friel and Vigliotta, 2006). For instance, in Hoplosternum littorale, apart from the
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sexual size dimorphism wherein males are larger than females, an elongate and recurved
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4.2. Testicular characterization
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pectoral spine was observed in males during the spawning season (Winemiller, 1987).
The testis of P. disjunctivus from Marikina River contain spermatogonia along the entire
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testis length and could be described as an “unrestricted” spermatogonial type (Grier, 1981) and an anastomosing tubular type, a common characteristic of fishes belonging to the basal taxa (Grier, 1993; Parenti and Grier 2004). Testis morphology and ultrastructure of the P. disjunctivus in this study appeared similar to those of relative species, Liposarcus anisitsi (Cruz et al., 2005) and R. aspera (Agostinho et al., 1987) both characterized by unrestricted lobular testes. Further, analyses of the gametic distribution show no exclusive spermatogenic, secretory or transition regions along the testes length. This observation is contrary to related catfishes L. lentiginosa, which has a characteristic cranial spermatogenic, spermatogenicsecretory transition and exclusively secretory caudal region (Guimaraes-Cruz et al., 2005),
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and R. aspera where secretory tubules in the caudal portion were observed (Melo et al., 2011). The absence of seminal vesicles in P. disjunctivus is also apparent in related species—L.
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alexandri and R. aspera, Iheringichthys labrosus, and P. maculates where the origin of secretion can be attributed to secretory cells located in the epithelium of the testes’ caudal
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region (Santos et al., 2001; Barros et al., 2007; Melo et al., 2011). Testicular morphology and ultrastructure of the gonads for hypostominae has not been explored fully.
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The spherical nucleus and the absence of the acrosome and the spherical head in P.
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disjunctivus spermatozoa classify it as having primitive type 1 aquasperm suitable for external fertilization (Jamieson, 1991; Mattei, 1991). This is similar with as is the case with
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other loricariids P. maculatus, P. corruscans, L. alexandri, R. aspera and C. conirostris (Grier, 1981; Santos et al., 2001; Martins et al., 2010; Melo et al., 2011). The absence of
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acrosome in the P. disjunctivus spermatozoa may be compensated by the approximate
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diameter of the sperm with the diameter of the micropyle in mature eggs (Jamieson, 1991) as
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opposed to the presence of the acrosome in agnathans and acipenciformes.
4.3. Gonadosomatic index and minimum length at maturity The minimum length to which male P. disjunctivus reach maturity is at 29 cm SL.
However, a number of males with mature spermatozoa at a still smaller size at 20 cm SL and onwards were observed especially during the peak of the spawning season. These size ranges of the fish capable of spawning have great impact on the increasing predominance of their population in the Marikina River where they can grow up to 55 cm TL. Mature males are apparent for 5 months (June to October). Percent distribution of maturity stages in the present study wherein mature and reproductively spawning males were observed for 5-6 months
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(three months of which as off the peak spawning) suggests that males have a long reproductive period spanning about half of the year.
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4.4. Reproductive phenology and testicular changes Histological characterization and establishing a defined criterion for testicular maturity
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stages in P. disjunctivus need the reference on the length of the fish as well as the defined establishment of the minimum length of maturity for males. The term “resting” stage,
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previously referring to stage 6, was no longer applied in this study following Brown-Peterson
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et al. (2002, 2011) since even in the stage where there seems to be no important changes after the spent stage, molecular and steroidal activities preparing the cysts for the next cycle of
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gamete development is dynamic. Hence, this stage is therefore is called “regenerating” or “developing-spent”.
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The long spawning season, the cystic synchronous spermatogenesis and the presence of
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fully spent and recrudescent testes in subsequent months suggest that P. disjunctivus males are batch spawners. Reproduction under culture conditions may have to be explored to
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determine the frequency of spawning within a reproductive season to verify this observation. Testis of P. disjunctivus seemed to have undergone a “prolonged spent” stage during the
hottest months of the study period (May) as the testes of mature individuals regained a flaccid state, devoid of milt while spermatogenesis seemed inhibited as conspicuous observations of excessive SC division in tubules devoid of spermatozoa were observed following three successive months where mature/spawning capable individuals were already apparent (February to April). Late recovering-spent stage and spawning-capable gonads were again observed during June in time for the rainy season. A very noticeable drop in the water level in the sampling stations was also observed in the sampling stations consequent to the minimal rainfall during the dry months (February to May).
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Male Pterygoplichthys are known to excavate river banks to create burrows in which an attracted female will lay and guard her eggs (Nico et al., 2009), but, with the decline of water level and the rise in temperature during the dry months, males cannot effectively create
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burrows for females to lay their eggs, hence the tendency to suppress reproduction since the environmental conditions is not suitable for spawning. This suspension of reproduction was
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consistent with the frequent observations of a highly thickened tubular lamina with extreme growth of somatic cells instead of spermatogenic cells during these months.
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Majority of catfishes are considered seasonal breeders with the rate of testicular
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recrudescence being highly correlated with ambient photoperiod and optimum temperature to initiate spermatogenesis (Garg and Sundararaj 1985; Nayar and Sundararaj 1986; Legendre et
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al., 1996). Final stimulus for spawning may also be associated with rise in water level and flooding of marginal areas, as was needed for spawning in most clariid catfishes like Clarias
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batrachus and C. macrocephalus (Bruton 1979). Apart from these ideal conditions for
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reproduction, alternations in the dry and wet seasons in the tropics could lead to seasonal
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differences in water quality and food availability.
4.5. Possible mechanical control
In the Philippines, predators feeding on mature Pterygoplichthys are generally absent
apart from the chinese egret which have been seen attempting to pick on juveniles in many areas of the river. In temperate countries, extremely low temperatures during the winter season serves as natural means to control their population. Nico and Martin (2001) noted that P. anisitsi had high tolerance to low temperatures and apparently uses outflow from sewage treatment plants as thermal refugia during cold spells in Texas. In Palizada River, Mexico, the common snook Centropomus undecimalis preys on Pterygoplichthys pardalis (ToroRamirez et al., 2014).
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Freshwater systems in the tropics have relatively stable temperatures, and months exhibiting high temperatures and decline in water levels are opportunities for possible population control since spawning does not occur or is largely reduced during the dry
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seasons. Aggressive catching/harvest of these fishes during these months may curb their
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population especially in Marikina River.
5. Conclusion
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The primary objective of this study was to describe the histological and ultrastructural
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characteristics of the testis of P. disjunctivus which has rapidly dominated the ichthyofauna of Marikina River, Philippines and describe the testicular stages of development in the fish
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based on morphologic and histological observations on an annual reproductive season. Germinal compartment of the testis was composed of anastomosing tubules with cysts
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undergoing synchronous development. Spermatozoon is classified as type 1 ect aquasperm
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devoid of acrosome; has rounded nucleus and a long flagellum which are characteristics for external fertilization. Histological analyses and GSI data show that spawning in males peaked
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during the very rainy months (June- August) while recrudescence is very prominent during the dry months (February- May). Males attain reproductive capacity early on with gonad maturity observed in fishes starting 18 cm SL. The reproductive seasonality presented in this study has important implications in P.
disjunctivus’ nature of invasiveness, particularly in their capacity for rapid proliferation in a highly eutrophic, nutrient-rich, novel environment such as the Marikina River system. The apparent decline in water levels and increase in temperature during the dry months in the river were also noted to cause prolonged recrudescence in the testis. Dry months may serve as a good venue for massive mechanical control to curb the population of this invasive fish in the river.
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in an invasive exotic catfish Pterygoplichthys disjunctivus in Volusia Blue Spring, Florida, U.S.A. J. Fish. Biol. 73, 1562-1572.
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Guimaraes-Cruz, R.J., de los Santos, J.E., Santos, G.B., 2005. Gonadal structure and gametogenesis of Loricaria lentiginosa Isbrucker (Pisces, Teleostei, Siluriformes). Rev. Braz. de Zool. 22, 556-564. Grier, H.J., 1981. Cellular organization of the testis and spermatogenesis in fishes. Am. Zool. 21, 345–357.
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Grier, H.J., 1993. Comparative organization of sertoli cells including the Sertoli cell barrier. In: Russell, L.D., Griswold, M.D. (Eds), The Sertoli Cell. Cache River Press, Bolesta Clearwater. pp. 704–739.
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Jamieson, B.G.M., 1991. Fish evolution and systematics: evidence from spermatozoa. Cambridge: Cambridge University Press
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Jumawan, J.C., Catap, E.S., Salunga, T.C. 2010. Lipid peroxidation and patterns of cadmium and lead accumulation in vital organs of suckermouth armored catfish Pterygoplichthys
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pardalis Castelnau from Marikina River. J. Appl. Sci. Envi. Sanit. 5,375-390.
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Jumawan, J. C., Vallejo, B. M., Herrera, A. A., Buerano, C. C., Fontanilla, I. K. C., 2011. DNA barcodes of the suckermouth sailfin catfish Pterygoplichthys (Siluriformes:
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Loricariidae) in the Marikina River system, Philippines: molecular perspective of an invasive alien fish species. Phillip Sci Lett, 4, 103-113.
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Lam, J.C., Su, G.L., 2009. Arsenic and mercury concentrations of the waters and janitor fish
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(Pterygoplichthys spp.) in Marikina River, Philippines. J. Appl. Sci. Envl. Sanit., 4 187-
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Legendre, M., Linhart, O., Billard, R., 1996. Spawning and management of gametes, fertilized eggs and embryos in Siluroidei. Aquat. Liv. Resour. 9,59-80.
Lowerre-Barbieri, S.K., Brown-Peterson, N.J., Murua, H., Tomkiewicz, J., Wyanski, D.M., Saborido-Rey, F., 2011. Emerging issues and methodological advances in fisheries reproductive biology. Mar. Coast. Fish Dynam. Manag. Ecosys. Sci. 3(1), 32-51.
Martins, Y.S., de Moura, D.F., Santos, G.B., Rizzo, E., Bazzoli, N., 2010. Comparative folliculogenesis and spermatogenesis of four teleost fish from a Reservoir in southeastern Brazil. Acta Zool. 91, 466–473. Mattei, X., 1991. Spermatozoon ultrastructure and its systematic implications in fishes. Can. J. Zool. 69,3038-3055.
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Melo, R.M.C., Arantes, F.P., Sato, Y., dos Santos, J.E., Rizzo, E., Bazzoli, N., 2011. Comparative morphology of the gonadal structure related to reproductive strategies in six species of neotropical catfishes (Teleostei: Siluriformes). J. Morph. 272,525-535.
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Nayyar, S.K., Sundararaj, B.I., 1986. Seasonal reproductive activity in the testes and seminal vesicles of the Catfish, Heferopneustes fossilis (Bloch). J. Morph. 130,207-226.
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Nico, L.G., Martin, T., 2001. The South American suckermouth armored catfish, Pterygoplichthys anisitsi (Pisces: Loricaridae), in Texas, with comments on foreign fish
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introductions in the American Southwest. The Southwestern Natural. 46.98-104.
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Nico, G., Jelks, H.L., Tuten, T., 2009. Non-native suckermouth armoured catfishes in Florida: description of nest burrows and burrow colonies with assessment of shoreline
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conditions. Aquat NSRP Bull 9:1-30
Nunez, J., Duponchelle, F., 2008. Towards a universal scale to assess sexual maturation and
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related life history traits in oviparous teleost fishes. Fish Physiol. Biochem. 9242,340-
Page, L.M., Robins, R.H., 2006. Identification of sailfin catfishes (Teleostei: Loricariidae) in
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Southeastern Asia. Raffles Bull. Zool. 54,455-457.
Parenti, L.R., Grier, H.J., 2004. Evolution and phylogeny of gonad morphology in bony fishes. Integ. Comp. Biol. 44,333–348.
Quilang, J.P., Yu, S.C.S., 2013. DNA barcoding of commercially important catfishes in the Philippines.Mitochondria DNA. 0, 1-10.
Santos, J.E., Bazzoli, N., Rizzo, E., Santos, G.B., 2001.Morphofunctional organization of the male reproductive system of the catfish Iheringichthys labrosus (LuÈtken, 1874) (Siluriformes:Pimelodidae). Tissue & Cell. 33,533-540.
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Santos, J.E., Veloso-Junior, V.C.D., Oliveira, D.A., Hojo, R.E.S., 2010. Morphological characteristics of the testis of the catfish Pimelodella vittata (Lutken, 1874). J. Appl.
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Ichthyol. 26,942-945.
us
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Toro‐Ramírez, A., Wakida‐Kusunoki, A. T., Amador‐del Ángel, L. E., Cruz‐Sánchez, J. L.,
2014. Common snook (Centropomus undecimalis (Bloch, 1792) preys on the invasive
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Amazon sailfin catfish (Pterygoplichthys pardalis (Castelnau, 1855) in the Palizada River, Campeche, southeastern Mexico. J. Appl. Ichthyol. doi: 10.1111/jai.12391
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Weber, C., 2003. Loricariidae - Hypostominae (Armored catfishes). In: Reis, R.E., Kullander,
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S.O., Ferraris, C.J. Jr (Eds.), Checklist of the freshwater fishes of south and central
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America. Porto Alegre: EDIPUCRS, Brasil. p. 351-372. Winemiller, K.O., 1987. Feeding and reproductive biology of the currito, Hoplosternum
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littorale, in the Venezuelan llanos with comments on the possible function of the enlarged male pectoral spines. Environ. Biol. Fish. 20(3), 219-227
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Table
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Table 1. Macroscopic and microscopic characteristics used to assign stages of testes maturity in P. disjunctivus from the Marikina river system, Philippines.
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Figure captions
Fig. 1. Location map of sampling sites along the Marikina River system, Philippines.
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Fig. 2. P. disjunctivus collected from the Marikina River system, Philippines. (a) Dorsal view. (b) Ventral view of the abdomen showing a single anal orifice/ urogenital papilla. (c). Overview of the mature testes. i- small intestine; red arrows- testes; white arrows- spermatic ducts; black asterisk- cloaca; white asterisk-common anal orifice/ urogenital papilla.
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Fig. 3. The gonado-somatic index (GSI) of male P. disjunctivus in Marikina River, Philippines across sampling months. N: 526 males. Error bars represent standard error of the means.
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Fig. 4. Anatomical overview and light micrographs of a mature P. disjunctivus testes. (a). Mature/ spawning-capable testes covered by a thin sheath of tunica albuginea. (b). Representative seminiferous tubule with cysts of the spermatogenic lineage cells in different stages development. HE. (c). Transverse section of the testes showing anastomosis in the seminiferous tubules. HE. (d). Representative section of the testes caudal region showing negative reaction of the lumen to AB-PAS stain (pH 2.5) test for presence of carbohydrates and glycoproteins. RBCs- red blood cells; White asterisk in b- Sertoli nucleus; White asterisk in d-lumen, White arrows-Sertoli cell cytoplasmic extensions; PSG-primary spermatogonia; SSG-secondary spermatogonia; SC2-secondary spermatocytes; ST-spermatids. Fig. 5. Ultrastructure of P. disjunctivus spermatogenic cells at the level of the interstitium. Interstitium , along with myoid cells, and red blood cells within blood vessels. Cysts close to the basement lamina consists of a primary spermatogonium and secondary spermatocytes. Iinterstitium; MC-myoid cells; RBCs- red blood cells; PSG-primary spermatogonium; SC1primary spermatocyte; L-lumen. Fig. 6. Ultrastructure of spermatogenic cells of P. disjunctivus from the Marikina river system. (a) Primary spermatogonia: Cytoplasm with mitochondria joined by intermitochondrial cement/nuages. (b). Secondary spermatogonia: Large nucleus with 2-3 nucleoli. (c). Primary spermatocyte: Nucleus with prominent synaptonemal complexes. (d).
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secondary spermatocyte: Nucleus with highly condensed chromatin forming electron-dense dots. C-cytoplasm; M-mitochondria; N- nucleus; Ns-nucleolus; CM-cell membrane; white asterisk- nuages; black arrowheads-synaptonemal complexes; black arrow-Sertoli cell cytoplasmic extensions.
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Fig. 7. Ultrastructure of spermiogenic cells in P. disjunctivus from the Marikina River system. (a-b). Intermediate phase spermatid: (a). heterochromatic nucleus with electronlucent vacoulations and covered by perinuclear space. Cytoplasm displaced caudally to the nucleus with electron-lucent vesicles. Conspicuous invagination of the nuclear fossa can already be observed. (b). Intermediate phase spermatid with cytoplasm largely eliminated. (ce). Migration of centrioles and formation of basal body in the region of the nuclear fossa. (c). Invagination in the nuclear fossa with undispersed centriole. (d). Basal body formed beneath the nucleus. (e). Movement of the proximal centriole and distal centriole near the basal body. (f). Late stage spermatid: Emergence of a flagellum from the once nuclear fossa. Nucleus is homogenously condensed with a defined nucleolus. (h-i). Scanning electron micrographs of the mature spermatozoon with oval head and long flagellum. (i). Closer view of the head. Nnucleus; ps-perinuclear space; bb- basal body; pc-proximal centriole; dc-distal centriole; pm-plasma membrane; F-flagellum; H-head; white arrow electron-lucent vesicles; black arrow/white asterisk-nuclear fossa.
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Fig. 8a-f Testicular maturity stages in P. disjunctivus. (a). Immature stage characterized by predominance of PSG among other spermatogenic cells. (b). Maturing stage: Increase in mitotic and meiotic division. SC1 and SC2 can be seen dominating the tubule. (c). Mature stage/ Spawning capable stage: Cytoplasmic extensions of Sertoli cells (black arrows) surrounding individual cysts retract resulting in anastomosis of the tubule filled with SZ. (d). Closer view of a single tubule: Lumen is compact with abundant mature SZ surrounded by cysts containing STs (e). Actively spawning stage. Massive anastomosis in the tubule. (f). Closer view of a single tubule in (e). Conspicuous space in the lumen dominated by SZ. PSGprimary spermatogonia; SSG-secondary spermatogonia; SC1-primary spermatocytes; SC2secondary spermatocytes; ST-spermatids; SZ- spermatozoa. Fig. 8 g-n Testicular maturity stages in P. disjunctivus continuation. (g) Spent stage: tubule lumen is composed of residual SZ. (h). Closer view of (g): Lining of the lumen is mainly composed of cysts containing PSG and SSG and occasional and scanty cysts of SC2. (i). Early stage developing spent stage: Lumen in each tubule is occluded. (j): closer view of (i): Thickened lining of the lobule with lining very little PSG. (k). Late developing spent stage (recrudescence): Normal spermatogenic progression during recrudescence. (l) Closer view of (k). (n). Mid-developing spent stage: Unusual thickening of the germinal epithelium with lining predominated by excessive SC2 division in the cyst. (n) closer view of (k): Excessive meiotic division of SC2 while absence of PSG,SSG and SC1 were seen during the dry months of study period. PSG-primary spermatogonia; SSG-secondary spermatogonia; SC1primary spermatocytes; SC2-secondary spermatocytes; ST-spermatids; SZ- spermatozoa.
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Fig. 9. Percentage (%) composition of maturity stages in P. disjunctivus testes from July 2010-June 2011. Stage 1-immature; Stage 2- maturing; Stage 3-mature/ spawning-capable; Stage 4-actively spawning; Stage 5- Spent/ regressing state; Stage 6- developing spent/ regenerating.
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Table 1. Macroscopic and microscopic characteristics used to assign stages of testes maturity in P. disjunctivus from the Marikina river system, Philippines. Macroscopic Features Small (≤ 0.1g), string-like and transparent, devoid of notable swelling and mesorchium in small males (22. 90± 7.88 mean, SD; range 6-27 cm SL). Difficulty in determination of sex.
Histologic features Testicular lining thin (Fig. 8 a) with cysts composed mostly of PSG and SSG held in cysts. SC2 hardly seen though cysts containing ST visible. Lumen of lobules small or occluded, devoid of SZ.
Maturing/ Developing (Stage 2)
Considerable increase in weight (mean 0.25 g ± 0. 25; range 0.1-0.98 g), with some degrees of swelling. Stage mostly observed in males of 26.81 ± 2.99 cm SL (range 19-32 cm). Color of testicular surface range from offwhite to pale pink.
Increased diameter of individual tubules due to increased number of cysts (Fig. 8 b). Lobules contain cysts of all germ cell types, though not consistently present together in all lobules. Lumen small, devoid of SZ.
Mature/ Spawning capable (Stage 3)
Testes swollen and attain maximum weight (mean 1.07 ± 0.609; range 0.03- 3.2 g). Initial swellings appear as small lobes during onset of maturity and even out as testes attain maximum weight. Surface has opaque pale-white color interspersed with fine pink/ brown spots.
The germinal epithelium thin and cysts containing SG1, SG2, and SC conspicuously reduced in number. Considerable degree of anastomosing lobules filled with SZ (Fig. 8 c, d).
Actively spawning (Stage 4)
Testicular weights comparable with Stage 3. External appearance overlaps from being overly swollen and opaque to being pinkish and spotted with varying degrees of twisting near the cranial area. Previous points of small swellings had evened out throughout testes length.
Heavy anastomosis of lobules; discontinuous epithelium with less compacted lumen filled with SZ (Fig. 8 e). Extensive retraction of the cytoplasmic extensions of the Sertoli cells result in a wider, irregularly shaped lumen filled with SZ (Fig. 8 f).
Spent/ regressing (Stage 5)
Testes thin and flaccid. Fully spent testes weights comparable to immature and developing stage (≤0.8 g) except for surface color that varies from being dark to light brown.
Residual SZ in the lumen or none at all; lobule boundary wall (LBW) thin with occasional latedeveloping STs (Fig. 8 g-h). Absence of SCs indicates cessation of spermatogenesis (Fig.
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Stage Immature (Stage 1)
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8j). Testes regain swelling and increase in size. Early stage 6 very similar with stage 2 except for the thick testicular lining and rough surface texture.
Repopulation of the germinal epithelium with SG1; SCs and STs continue to repopulate lobules in the mid recovering spent stage (Fig. 8 k, l). Late stage characterized by thickened tunica albuginea, abundant SCs and STs and empty lumen. PSG-primary spermatogonia; SSG-secondary spermatogonia; SC1-primary spermatocytes; SC2-secondary spermatocytes; ST-spermatids; SZ- spermatozoa.
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Recovering spent (Stage 6)
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S0040-8166(14)00030-5 http://dx.doi.org/doi:10.1016/j.tice.2014.05.001 YTICE 857
To appear in:
Tissue and Cell
Received date: Accepted date:
13-2-2014 2-5-2014
Please cite this article as: Jumawan, J.C., Herrera, A.A.,Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines, Tissue and Cell (2014), http://dx.doi.org/10.1016/j.tice.2014.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Histological and ultrastructural characteristics of the testis of the invasive suckermouth sailfin catfish Pterygoplichthys disjunctivus (Siluriformes: loricariidae) from Marikina River, Philippines
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J.C. Jumawana,b*, A.A. Herrerab Biology Department, Caraga State University, 8600 Butuan City, Philippines
Institute of Biology, University of the Philippines-Diliman, Quezon City, Philippines
Suggested Running Title:
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Testis characteristics of Pterygoplichthys disjunctivus
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Janitor fish, ultrastructure, reproduction
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Keywords:
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Acknowledgements:
Grants from the Commission on Higher Education- Science and Engineering Grants (CHED-
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SEGS) supported this study. The National Kidney Transplant Institute (NKTI) Transmission Electron Microscopy Center and the Philippine Kidney Transplant Institute (PKDF) HistoPathology laboratory are gratefully acknowledged for the TEM and histological preparations.
Correspondence author's details: Joycelyn C. Jumawan
Biology Department, Caraga State University, 8600 Butuan City, Philippines [email protected]; Tel: (6385) 342-3047, Fax: (6385) 342-1079 ORIGINAL SUBMISSION
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ABSTRACT
The histological and ultrastructural features of the testis of the invasive suckermouth sailfin
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catfish Pterygoplichthys disjunctivus rapidly proliferating in Marikina River, Philippines were characterized during the fish’s 2010-2011 reproductive season. The germinal
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compartment of the testes was composed of anastomosing tubules with cysts undergoing synchronous development. Spermatogenic cells were along the length of the testes indicate it
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to be of the unrestricted spermatogonial type. The spermatozoon is classified as type 1 ect
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aquasperm devoid of acrosome, has rounded nucleus, and a long flagellum—characteristics necessary for external fertilization. Male P. disjunctivus was reproductively active during half
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of the year-long study with peak spawning during the most rainy months (June to August) and prolonged recrudescence during the dry months (February to May). Results from this
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study form a histological baseline to describe the gonad dynamics and reproduction of this
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invasive fish species as well as provide possible means of mechanical control to curb the
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population of the fish in this river.
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1. Introduction
Pterygoplicthys disjunctivus Weber, 1991 is an invasive loricariid catfish that originally
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inhabited the Amazon River basin of Brazil and Peru (Weber, 2003) and Rio Madeira drainage of Brazil and Bolivia along with another species, Pterygoplichthys pardalis (Page
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and Robins, 2006). These two species are considered invasive in at least nine countries outside the neotropics (German et al., 2010), including the Philippines where they are
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popularly known as “janitor fish”. These species were first imported to the Philippines as
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ornamental aquarium fish. However, many propagules were believed to have accidentally escaped from aquarium fish breeding facilities into Laguna de Bay when typhoon Rosing
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caused flooding in 1995 (Guerrero, 2006). The two species rapidly proliferated in the Marikina River, a highly urbanized water system in Luzon (Chavez et al., 2006a). Individuals
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with intermediate abdominal pattern characteristics between the two species were observed
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with no defined species-specific DNA barcodes, suggesting possible introgressive hybridization between P. pardalis and P. disjunctivus in the river (Jumawan et al., 2011;
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Quilang and Yu, 2013).
Pterygoplichthys pardalis and P. disjunctivus have several features that account for their
rapid invasion in their non-native environments. These fishes were reported to be highly tolerant to poor water conditions (Chavez et al., 2006b; Lam and Su, 2009; Jumawan et al., 2010). Plasticity of the gastrointestinal tract and the broad diet of P. disjunctivus (German et al., 2010), its air-breathing capacity under hypoxic conditions, as well as the absence of natural predators of mature populations (Nico and Martin, 2001; Gibbs et al., 2008) contribute to their successful invasion in non-native environments. Knowledge of the reproductive biology of the two species and the family Loricariidae is quite limited but is important to enable a more accurate understanding of its natural history
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and possible reproductive plasticity. While it is known that female P. disjunctivus were capable of spawning at relatively smaller sizes (Gibbs et al., 2008) and some external features of the ovary were known, detailed studies on the testes have not been reported.
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This study will provide baseline information on the testicular morphology of P. disjunctivus in Marikina River, Philippines to provide insights into the knowledge about the
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reproduction of a neotropical teleost that has become invasive in a new environment. A description of the testicular maturity stages, as well as details on the ultrastructure of the
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gonad, are essential in creating management strategies and empirical models for the
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prediction of the fish’s invasive potential.
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2. Materials and Methods 2.1. Study area Pterygoplichthys disjunctivus were collected monthly from five sites (San Jose,
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Rodriguez Rizal; Banaba, San Mateo; Tumana, Malanday, River Park) along Marikina River,
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Philippines (Fig.1) from July 2010 to June 2011.
2.2. Sampling
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Fish were captured with a cast net (5 m long; mesh size of 3.8 cm). Due to the absence
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of defined sexual dimorphism in P. disjunctivus, females were inevitably collected and were excluded after sorting in the laboratory. Segregated males were sacrificed by immersing fish
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in ice 4 h prior to dissection. Standard length (SL) was measured to the nearest 0.1 cm. Body weight was measured with an electronic scale to the nearest 0.1 g. Testes were weighed using
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an analytical balance of 0.1 to 0.01 g accuracy. Gonado-somatic index (GSI) was calculated
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as: GSI = [(gonad weight)/ (body weight)] x 100.
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2.3 Visual characterization and Histological preparation Testis was characterized using visual-based macro scale of maturity based on testicular
size, color and swelling (Cailliet et al., 1986; Nunez and Duponchelle 2008; Brown-Peterson et al., 2011; Lowerre-Barbieri et al., 2011) with some modifications. To assess possible regionalization of germinal epithelium and glycoproteic region of secretion along the testes length, fragments from the cranial, medial and caudal portions were selected for histological characterization. A total of 360 testes samples were fixed in Bouin’s fluid for 12-24 h, dehydrated through a 75-90% ethanol series, embedded in paraffin, and sectioned at 5-6 µm. Cross and longitudinal sections were stained with haematoxylin-eosin (HE). The diameter of 100
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spermatogenic cells in each stage of development was measured in 5 to 10 histological sections a light microscope with a calibrated micrometer each month. Alcian blue (AB)Periodic Acid Schiff (PAS) pH 2.5 (ArtisanTM) was used to detect proteins and carbohydrates
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in the three testicular regions.
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2.4. Transmission and Scanning electron microscopy
Additional testicular tissue fragments from the cranial and medial regions of mature P.
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disjunctivus were immersed in 4% glutaraldehyde for 2 h followed by two changes of 0.2 M
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sodium cacodylate buffer at pH 7.4 for 10 min each and post-fixed in osmium tetroxide in the same buffer (0.2 M) for 15 min. After dehydration in an increasing alcohol series, the tissues
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were embedded in Epon resin mixture. Semi-thin sections (1 µm) were stained with toluidine blue for selection of adequate fields under light microscope. Ultra thin sections (70 nm) of
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the selected areas were counterstained with uranyl acetate and Reynold's lead citrate,
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analyzed and photo-documented using an energy filtering transmission electron microscope (EFTEM), Zeiss LIBRA 120™, Germany. Testicular maturity stages were then characterized
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based on the thickness of the tunica albuginea, the diameter of the tubule lumen and the predominant spermatogenic cells in each stage. Percentage (%) composition of the maturity stages derived from the histology of the testis was summarized for the 12 sampling months to establish reproductive seasonality in males. Spermatozoa were analyzed through scanning electron microscopy (SEM). Seminal
fluid was collected from adult males through maceration of the testes and dilution of the milt in phosphate buffer at 1:50 ratio suspension in a 1.5 ml microcentrifuge tube. Twenty microliters of the sperm suspension was transferred to a separate tube and centrifuged at low speed to allow sperm to settle at the bottom of the tube. Sperm samples were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 2 h at 40C. Primary washing of fixed
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samples (3 washes; 15 min. duration each) were done using 0.1 M cacodylate buffer pH 7.4. Osmium tetroxide (aqueous) pH 7.4 was used for secondary fixation. Samples were dehydrated in increasing ethanol series, mounted in a glass slide, and then dried overnight in
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a desiccator. The mounted samples were placed in a metal stub and then coated with a colloidal mixture of palladium gold (20 nm). Micrographs were obtained using Inspect ™
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Scanning Electron Microscope.
Size at first maturity (L50) was also determined from fishes classified as sexually
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mature and spawning capable through histological features and GSI of the testes using a
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logistic curve fitted by a weighted non-linear least squares regression. For all measured structures, the mean and standard deviation (SD) were calculated. Statistical analyses were
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conducted using Graphpad Prism5 software.
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3. Results
3.1. Internal morphology
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A total of 526 males were collected during the year-long study. Individuals ranged from 5 to 45 cm SL. Externally, sex in P. disjunctivus cannot be accurately distinguished due
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to lack of defined sexual dimorphism (Fig. 2 a, b). The testes of adults are paired, elongated organs joined together ventrally by the mesorchium and joined caudally by a common
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spermatic duct separated from the urethra through a thin sphincter. A resulting common duct
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extends anteriorly into a common urogenital opening. The terminal end of the rectum traverses across the testes midline and converge with the common urogenital tract separated
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by another muscular sphincter. Release of excreta and sperm is regulated by this sphincter as there is only a single aperture that serves both as a gonadal and anal orifice visible exteriorly.
3.2 Gonadosomatic Index
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An accessory seminal vesicle was absent at the caudal region of the testes.
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GSI values declined significantly from November to January (P<0.0001). The
minimum length at maturity for males is 18 cm SL. Males have longer spawning season extending until October (Fig. 3).
3.3. Ultrastructural characterization Testes of a mature P. disjunctivus is covered by a thin tunica albuginea (Fig. 4 a),
with the germinal compartment composed of anastomosing tubules with cystic organization. Spermatogenic cells are observed to undergo synchronous development (Fig. 4 b) and are delimited by cytoplasmic extensions of the Sertoli cells at the base of the germinal compartment forming an anastomosed tubular system (Fig. 4 c). Spermatogonia are present
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along the length of each testis tubule, hence, could be described as unrestricted spermatogonial testicular type. There are no defined regions exclusive for secretion as evidenced by the absence of transition secretory prismatic cells and a negative reaction for
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carbohydrates and protein secretion in the lumen of the lobule through AB-PAS staining (Fig. 4 d). The germinal epithelium, supported by the basal lamina with the interstitial tissue is
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composed of myoid cells, blood vessels, Leydig cells and neighboring tubules (Fig. 5). Germ cell types were characterized based on morphology and size of the nucleus, organelles,
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complexes and pericentriolar features and are depicted using annotated photomicrographs.
3.3.1. Spermatogonia
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Primary spermatogonia (PSG) are the largest cells in the lineage (14.87 ± 0.22 µm), and attached nearest to the basal lamina of the tubule, surrounded by Sertoli cell extensions
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forming a cyst (Fig. 6 a). It has an extensive cytoplasm containing abundant mitochondria, a
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large spherical but faint nucleus (7.59 ± 0.11 µm) covered by an irregularly contoured nuclear membrane, and a distinct nucleolus (1.76± 0.08 µm) with darkly staining chromatin. Highly
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electron-dense particles or nuages are seen associated with several mitochondria (intermitochondrial cement) near the nuclear envelope or seen freely dispersed inside the cytoplasm. PSG dominate immature and regenerating testicular stages although these cells are seen in other testicular stages as they formed the reserve pool of germ cells. Secondary spermatogonia (SSG) are smaller than PSG (8.15±0.35µm) and form cysts of
two to four cells. The nucleus contains 2-3 nucleoli while the cytoplasm contains few organelles (Fig. 6 b).
3.3.2. Spermatocytes
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Primary spermatocytes (SC1) are formed after secondary spermatogonia (SSG) had undergone first meiotic division, resulting in an increased number of cysts and increased testes size during the developing (stage 2) and regenerating stages (stage 6). SC1 are smaller
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(7.16 ±0.60 µm) than SSG with very little cytoplasm and abundant mitochondria. The nucleus (6.51±0.24 µm) is slightly condensed with granular chromatin and had prominent
(Fig. 6 c). SC1 often occur at minimum of 6-12 cells in a cyst.
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synaptonemal complexes (chromosomic masses) appearing as parallel electron dense strips
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Secondary spermatocytes (SC2) are even smaller than SC1 (6.19 ±0.32 µm), with much
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reduced cytoplasm and abundant mitochondria. The pale-staining nucleus (4.8 ± 0.27 µm) contained chromatin material appearing as electron-dense clots (Fig. 6 d). These cells occur
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in large numbers ≥ 20 in a cyst nearest the lumen.
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3.3.3. Spermatids
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Spermatids (ST) are haploid cells formed after the second meiotic division of SC2. Spermatids exhibit several stages of chromatin condensation, shape remodeling and
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cytoplasm reduction while undergoing spermiogenesis. In the initial stages of ST differentiation, cells are closely attached to each other with a homogenously translucent chromatin in the nucleus. In the intermediate stages, the cytoplasm exhibited intense vacuolization and are often displaced caudally to the electron dense nucleus along with the abundant mitochondria (Fig 7 a). The nuclear fossa eventually deepens and forms a depression caudal to the nuclear envelope where the proximal and distal centrioles were located. The centrioles eventually move into the nuclear fossa bringing with them the basal body that forms the early flagellum segment and the plasma membrane (Fig. 7 b-e). The later stages of ST differentiation is characterized by a very dense nucleus (2.88 ±0.074 µm) covered by a thin perinuclear space and an elongated flagellum emerging from the nuclear
Page 10 of 36
fossa (Fig. 7 f). A large amount of cytoplasm is displaced to the nucleus and eliminated while still inside the cysts and then eventually absorbed by the cytoplasm of the Sertoli cells.
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3.3.4. Spermatozoa Spermatozoa (SZ) are the smallest cells of the lineage formed from STs upon completion
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of spermiogenesis. A mature spermatozoon has a round, opaque nucleus devoid of acrosome (2.46 ±0.105 µm) and has a long flagellum (21.94± 0.94 µm) devoid of flagellar fin (Fig. 7 h-
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i). Once differentiation of SZ is completed, cytoplasmic extensions of the Sertoli cells that
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enclose the cyst retract and SZ are released into the lumen.
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3.4. Histological characterization
Six stages of testicular maturity based on testes color, texture, weight, features of
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spermatogenic cells, thickness of the tunica albuginea, and diameter of the lumen were
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described (Table 1; Fig. 8). Minimum length of maturity in males with testes containing mature SZ and considerable swelling throughout the testicular surface were initially observed
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at 29 cm SL and onwards. Early stage recovering spent (stage 6) testes peaked in January along with increasing observations of mature testes until April (Fig. 9). Recurrence of early developing-spent (stage 5) testes overlapping with regressing
stages was seen during the hottest months of the study period (April- May 2011) when virtually no mature and late stage regenerating testes were observed. Testes of individuals sampled in May had a peculiar morphology, having a highly thickened tubular wall with excessive meiotic divisions and with SC2s and STs conspicuously lodged in the germinal epithelium. PSGs and SSGs were scanty or absent in the lobule lamina while the lumen are devoid of SZs.
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4. Discussion 4.1. Absence of sexual dimorphism P. disjunctivus exhibit no defined sexual dimorphism. However, it may be possible that
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during the actual spawning season, sex-related features may be prominent. Highly fecund and large female P. disjunctivus have bigger and swollen abdomen and can be distinguished from
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males during the peak of the spawning season. Catfishes do not reproduce spontaneously under culture conditions (Legendre et al., 1996), making it difficult to observe courtship
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frequencies, possible sexual signals in choosing mate and other male-female sexual
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interactions. However, sexual dimorphism has been reported in about half of all families of catfish (Friel and Vigliotta, 2006). For instance, in Hoplosternum littorale, apart from the
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sexual size dimorphism wherein males are larger than females, an elongate and recurved
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4.2. Testicular characterization
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pectoral spine was observed in males during the spawning season (Winemiller, 1987).
The testis of P. disjunctivus from Marikina River contain spermatogonia along the entire
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testis length and could be described as an “unrestricted” spermatogonial type (Grier, 1981) and an anastomosing tubular type, a common characteristic of fishes belonging to the basal taxa (Grier, 1993; Parenti and Grier 2004). Testis morphology and ultrastructure of the P. disjunctivus in this study appeared similar to those of relative species, Liposarcus anisitsi (Cruz et al., 2005) and R. aspera (Agostinho et al., 1987) both characterized by unrestricted lobular testes. Further, analyses of the gametic distribution show no exclusive spermatogenic, secretory or transition regions along the testes length. This observation is contrary to related catfishes L. lentiginosa, which has a characteristic cranial spermatogenic, spermatogenicsecretory transition and exclusively secretory caudal region (Guimaraes-Cruz et al., 2005),
Page 12 of 36
and R. aspera where secretory tubules in the caudal portion were observed (Melo et al., 2011). The absence of seminal vesicles in P. disjunctivus is also apparent in related species—L.
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alexandri and R. aspera, Iheringichthys labrosus, and P. maculates where the origin of secretion can be attributed to secretory cells located in the epithelium of the testes’ caudal
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region (Santos et al., 2001; Barros et al., 2007; Melo et al., 2011). Testicular morphology and ultrastructure of the gonads for hypostominae has not been explored fully.
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The spherical nucleus and the absence of the acrosome and the spherical head in P.
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disjunctivus spermatozoa classify it as having primitive type 1 aquasperm suitable for external fertilization (Jamieson, 1991; Mattei, 1991). This is similar with as is the case with
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other loricariids P. maculatus, P. corruscans, L. alexandri, R. aspera and C. conirostris (Grier, 1981; Santos et al., 2001; Martins et al., 2010; Melo et al., 2011). The absence of
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acrosome in the P. disjunctivus spermatozoa may be compensated by the approximate
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diameter of the sperm with the diameter of the micropyle in mature eggs (Jamieson, 1991) as
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opposed to the presence of the acrosome in agnathans and acipenciformes.
4.3. Gonadosomatic index and minimum length at maturity The minimum length to which male P. disjunctivus reach maturity is at 29 cm SL.
However, a number of males with mature spermatozoa at a still smaller size at 20 cm SL and onwards were observed especially during the peak of the spawning season. These size ranges of the fish capable of spawning have great impact on the increasing predominance of their population in the Marikina River where they can grow up to 55 cm TL. Mature males are apparent for 5 months (June to October). Percent distribution of maturity stages in the present study wherein mature and reproductively spawning males were observed for 5-6 months
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(three months of which as off the peak spawning) suggests that males have a long reproductive period spanning about half of the year.
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4.4. Reproductive phenology and testicular changes Histological characterization and establishing a defined criterion for testicular maturity
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stages in P. disjunctivus need the reference on the length of the fish as well as the defined establishment of the minimum length of maturity for males. The term “resting” stage,
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previously referring to stage 6, was no longer applied in this study following Brown-Peterson
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et al. (2002, 2011) since even in the stage where there seems to be no important changes after the spent stage, molecular and steroidal activities preparing the cysts for the next cycle of
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gamete development is dynamic. Hence, this stage is therefore is called “regenerating” or “developing-spent”.
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The long spawning season, the cystic synchronous spermatogenesis and the presence of
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fully spent and recrudescent testes in subsequent months suggest that P. disjunctivus males are batch spawners. Reproduction under culture conditions may have to be explored to
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determine the frequency of spawning within a reproductive season to verify this observation. Testis of P. disjunctivus seemed to have undergone a “prolonged spent” stage during the
hottest months of the study period (May) as the testes of mature individuals regained a flaccid state, devoid of milt while spermatogenesis seemed inhibited as conspicuous observations of excessive SC division in tubules devoid of spermatozoa were observed following three successive months where mature/spawning capable individuals were already apparent (February to April). Late recovering-spent stage and spawning-capable gonads were again observed during June in time for the rainy season. A very noticeable drop in the water level in the sampling stations was also observed in the sampling stations consequent to the minimal rainfall during the dry months (February to May).
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Male Pterygoplichthys are known to excavate river banks to create burrows in which an attracted female will lay and guard her eggs (Nico et al., 2009), but, with the decline of water level and the rise in temperature during the dry months, males cannot effectively create
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burrows for females to lay their eggs, hence the tendency to suppress reproduction since the environmental conditions is not suitable for spawning. This suspension of reproduction was
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consistent with the frequent observations of a highly thickened tubular lamina with extreme growth of somatic cells instead of spermatogenic cells during these months.
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Majority of catfishes are considered seasonal breeders with the rate of testicular
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recrudescence being highly correlated with ambient photoperiod and optimum temperature to initiate spermatogenesis (Garg and Sundararaj 1985; Nayar and Sundararaj 1986; Legendre et
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al., 1996). Final stimulus for spawning may also be associated with rise in water level and flooding of marginal areas, as was needed for spawning in most clariid catfishes like Clarias
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batrachus and C. macrocephalus (Bruton 1979). Apart from these ideal conditions for
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reproduction, alternations in the dry and wet seasons in the tropics could lead to seasonal
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differences in water quality and food availability.
4.5. Possible mechanical control
In the Philippines, predators feeding on mature Pterygoplichthys are generally absent
apart from the chinese egret which have been seen attempting to pick on juveniles in many areas of the river. In temperate countries, extremely low temperatures during the winter season serves as natural means to control their population. Nico and Martin (2001) noted that P. anisitsi had high tolerance to low temperatures and apparently uses outflow from sewage treatment plants as thermal refugia during cold spells in Texas. In Palizada River, Mexico, the common snook Centropomus undecimalis preys on Pterygoplichthys pardalis (ToroRamirez et al., 2014).
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Freshwater systems in the tropics have relatively stable temperatures, and months exhibiting high temperatures and decline in water levels are opportunities for possible population control since spawning does not occur or is largely reduced during the dry
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seasons. Aggressive catching/harvest of these fishes during these months may curb their
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population especially in Marikina River.
5. Conclusion
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The primary objective of this study was to describe the histological and ultrastructural
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characteristics of the testis of P. disjunctivus which has rapidly dominated the ichthyofauna of Marikina River, Philippines and describe the testicular stages of development in the fish
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based on morphologic and histological observations on an annual reproductive season. Germinal compartment of the testis was composed of anastomosing tubules with cysts
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undergoing synchronous development. Spermatozoon is classified as type 1 ect aquasperm
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devoid of acrosome; has rounded nucleus and a long flagellum which are characteristics for external fertilization. Histological analyses and GSI data show that spawning in males peaked
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during the very rainy months (June- August) while recrudescence is very prominent during the dry months (February- May). Males attain reproductive capacity early on with gonad maturity observed in fishes starting 18 cm SL. The reproductive seasonality presented in this study has important implications in P.
disjunctivus’ nature of invasiveness, particularly in their capacity for rapid proliferation in a highly eutrophic, nutrient-rich, novel environment such as the Marikina River system. The apparent decline in water levels and increase in temperature during the dry months in the river were also noted to cause prolonged recrudescence in the testis. Dry months may serve as a good venue for massive mechanical control to curb the population of this invasive fish in the river.
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References Agostinho, A.A., Barbieri, M.C., Agostinho, C.S., Barbieri, G., 1987. Biologia reprodutiva de Rhinelepis aspera (Agassiz, 1829) (Teleostei, Loricariidae) no rio Paranapanema. I.
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Estrutura dos testículos e escala de maturidade. Rev. Bras. Biol. 47(3), 309-317. Barros, M.D.M.,Guimaraes-Cruz, R.J., Veloso-Junior, V.C., Santos, J.E., 2007. Reproductive
Teleostei, Siluriformes). Rev. Bras. Zool. 24,213–221.
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apparatus and gametogenesis of Lophiosilurus alexandri Steindachner (Pisces,
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Brown-Peterson, N., Grier, H.J., Overstreet, R., 2002. Annual changes in the germinal
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epithelium determine reproductive classes in male cobia, Rachycentron canadum. J. Fish Biol. 60,178-202.
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Brown-Peterson, N.J., Wyanski, D.M., Saborido-Rey, F., Macewicz, B.J., Lowerre-Barbieri, S.K., 2011. A standardized terminology for describing reproductive development in
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fishes. Mar. Coastal Fish. 3(1), 52-70.
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Bruton, M.N., 1979. The breeding biology and early development of Clarias gariepinus (Pisces; Clariidae) in Lake Sibaya, South Africa, with a review of breeding in species of
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the subgenus Clarias. Trans. Zool. Soc. Lund. 35, 1-45.
Cailliet, G.M., Love, M.S., Ebeling, A.W., 1986. Fishes: a field and laboratory manual on their structure, identification, and natural history. Belmont, CA: Wadsworth Publication.
Chavez, J.M., Paz, R.M.D.L., Manohar, S.K., Pagulayan, R.C., Carandang, J., 2006a. New Philippine record of south American sailfin catfishes (Pisces : Loricariidae). Zootaxa 1109,57-68.
Chavez, H.M., Casao, E.A., Villanueva, E.P., Paras, M.P., Guinto, M.C., Mosqueda, M.B., 2006b. Heavy metal and microbial analyses of janitor fish (Pterygoplichthys spp.) in Laguna de Bay, Philippines. JESAM. 9, 31-40.
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Cruz, C., Marcos, A., Simoes, K, Vincentini, C.A., 2005. Structural and ultrastructural characteristics of the spermatogenesis of the grey armored catfish Liposarcus anisitsi (Holmberg, 1893) (Teleostei, Siluriformes). Braz. J. vet. Res. Anim. Sci. 42,37-44.
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Friel, J.P., Vigliotta, T.R., 2006. Synodontis acanthoperca, a new species from the Ogôoué River system, Gabon with comments on spiny ornamentation and sexual dimorphism in
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mochokid catfishes (Siluriformes: Mochokidae). Zootaxa. 1125,45-56.
Garg, S.K., Sundararaj, B.I., 1985. Response of the testes and seminal vesicles of the catfish
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Heteropneustes fossilis (Bloch) to various combinations of photoperiod and temperature.
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Physiol. Zool. 58,616-627.
German, D.P., Neuberger, D.T., Callahan, M.N., Lizardo, N.R., Evans, D.H., 2010. Feast to
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famine: The effects of food quality and quantity on the gut structure and function of a detritivorous catfish (Teleostei: Loricariidae). Comp. Biochem. Physiol. 155,281–293.
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Gibbs, M.A., Shields, J.H., Lock, D.W., Talmadge, K.M., Farrell, T.M., 2008. Reproduction
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in an invasive exotic catfish Pterygoplichthys disjunctivus in Volusia Blue Spring, Florida, U.S.A. J. Fish. Biol. 73, 1562-1572.
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Guerrrero, R.D., 2006. Aquatic Invasive Alien species in the Philippines: their status, impacts and management. In Proceedings of the conference-workshop on Invasive Alien Species in the Philippines and their Impacts on Biodiversity. Compac Disc, pp 1-3. Marikina City; Philippine Department of Environment and Natural Resources
Guimaraes-Cruz, R.J., de los Santos, J.E., Santos, G.B., 2005. Gonadal structure and gametogenesis of Loricaria lentiginosa Isbrucker (Pisces, Teleostei, Siluriformes). Rev. Braz. de Zool. 22, 556-564. Grier, H.J., 1981. Cellular organization of the testis and spermatogenesis in fishes. Am. Zool. 21, 345–357.
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Grier, H.J., 1993. Comparative organization of sertoli cells including the Sertoli cell barrier. In: Russell, L.D., Griswold, M.D. (Eds), The Sertoli Cell. Cache River Press, Bolesta Clearwater. pp. 704–739.
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Jamieson, B.G.M., 1991. Fish evolution and systematics: evidence from spermatozoa. Cambridge: Cambridge University Press
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Jumawan, J.C., Catap, E.S., Salunga, T.C. 2010. Lipid peroxidation and patterns of cadmium and lead accumulation in vital organs of suckermouth armored catfish Pterygoplichthys
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pardalis Castelnau from Marikina River. J. Appl. Sci. Envi. Sanit. 5,375-390.
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Jumawan, J. C., Vallejo, B. M., Herrera, A. A., Buerano, C. C., Fontanilla, I. K. C., 2011. DNA barcodes of the suckermouth sailfin catfish Pterygoplichthys (Siluriformes:
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Loricariidae) in the Marikina River system, Philippines: molecular perspective of an invasive alien fish species. Phillip Sci Lett, 4, 103-113.
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Lam, J.C., Su, G.L., 2009. Arsenic and mercury concentrations of the waters and janitor fish
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(Pterygoplichthys spp.) in Marikina River, Philippines. J. Appl. Sci. Envl. Sanit., 4 187-
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Legendre, M., Linhart, O., Billard, R., 1996. Spawning and management of gametes, fertilized eggs and embryos in Siluroidei. Aquat. Liv. Resour. 9,59-80.
Lowerre-Barbieri, S.K., Brown-Peterson, N.J., Murua, H., Tomkiewicz, J., Wyanski, D.M., Saborido-Rey, F., 2011. Emerging issues and methodological advances in fisheries reproductive biology. Mar. Coast. Fish Dynam. Manag. Ecosys. Sci. 3(1), 32-51.
Martins, Y.S., de Moura, D.F., Santos, G.B., Rizzo, E., Bazzoli, N., 2010. Comparative folliculogenesis and spermatogenesis of four teleost fish from a Reservoir in southeastern Brazil. Acta Zool. 91, 466–473. Mattei, X., 1991. Spermatozoon ultrastructure and its systematic implications in fishes. Can. J. Zool. 69,3038-3055.
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Melo, R.M.C., Arantes, F.P., Sato, Y., dos Santos, J.E., Rizzo, E., Bazzoli, N., 2011. Comparative morphology of the gonadal structure related to reproductive strategies in six species of neotropical catfishes (Teleostei: Siluriformes). J. Morph. 272,525-535.
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Nayyar, S.K., Sundararaj, B.I., 1986. Seasonal reproductive activity in the testes and seminal vesicles of the Catfish, Heferopneustes fossilis (Bloch). J. Morph. 130,207-226.
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Nico, L.G., Martin, T., 2001. The South American suckermouth armored catfish, Pterygoplichthys anisitsi (Pisces: Loricaridae), in Texas, with comments on foreign fish
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introductions in the American Southwest. The Southwestern Natural. 46.98-104.
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Nico, G., Jelks, H.L., Tuten, T., 2009. Non-native suckermouth armoured catfishes in Florida: description of nest burrows and burrow colonies with assessment of shoreline
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conditions. Aquat NSRP Bull 9:1-30
Nunez, J., Duponchelle, F., 2008. Towards a universal scale to assess sexual maturation and
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related life history traits in oviparous teleost fishes. Fish Physiol. Biochem. 9242,340-
Page, L.M., Robins, R.H., 2006. Identification of sailfin catfishes (Teleostei: Loricariidae) in
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Southeastern Asia. Raffles Bull. Zool. 54,455-457.
Parenti, L.R., Grier, H.J., 2004. Evolution and phylogeny of gonad morphology in bony fishes. Integ. Comp. Biol. 44,333–348.
Quilang, J.P., Yu, S.C.S., 2013. DNA barcoding of commercially important catfishes in the Philippines.Mitochondria DNA. 0, 1-10.
Santos, J.E., Bazzoli, N., Rizzo, E., Santos, G.B., 2001.Morphofunctional organization of the male reproductive system of the catfish Iheringichthys labrosus (LuÈtken, 1874) (Siluriformes:Pimelodidae). Tissue & Cell. 33,533-540.
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Santos, J.E., Veloso-Junior, V.C.D., Oliveira, D.A., Hojo, R.E.S., 2010. Morphological characteristics of the testis of the catfish Pimelodella vittata (Lutken, 1874). J. Appl.
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Ichthyol. 26,942-945.
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Toro‐Ramírez, A., Wakida‐Kusunoki, A. T., Amador‐del Ángel, L. E., Cruz‐Sánchez, J. L.,
2014. Common snook (Centropomus undecimalis (Bloch, 1792) preys on the invasive
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Amazon sailfin catfish (Pterygoplichthys pardalis (Castelnau, 1855) in the Palizada River, Campeche, southeastern Mexico. J. Appl. Ichthyol. doi: 10.1111/jai.12391
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Weber, C., 2003. Loricariidae - Hypostominae (Armored catfishes). In: Reis, R.E., Kullander,
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S.O., Ferraris, C.J. Jr (Eds.), Checklist of the freshwater fishes of south and central
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America. Porto Alegre: EDIPUCRS, Brasil. p. 351-372. Winemiller, K.O., 1987. Feeding and reproductive biology of the currito, Hoplosternum
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littorale, in the Venezuelan llanos with comments on the possible function of the enlarged male pectoral spines. Environ. Biol. Fish. 20(3), 219-227
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Table
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Table 1. Macroscopic and microscopic characteristics used to assign stages of testes maturity in P. disjunctivus from the Marikina river system, Philippines.
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Figure captions
Fig. 1. Location map of sampling sites along the Marikina River system, Philippines.
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Fig. 2. P. disjunctivus collected from the Marikina River system, Philippines. (a) Dorsal view. (b) Ventral view of the abdomen showing a single anal orifice/ urogenital papilla. (c). Overview of the mature testes. i- small intestine; red arrows- testes; white arrows- spermatic ducts; black asterisk- cloaca; white asterisk-common anal orifice/ urogenital papilla.
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Fig. 3. The gonado-somatic index (GSI) of male P. disjunctivus in Marikina River, Philippines across sampling months. N: 526 males. Error bars represent standard error of the means.
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Fig. 4. Anatomical overview and light micrographs of a mature P. disjunctivus testes. (a). Mature/ spawning-capable testes covered by a thin sheath of tunica albuginea. (b). Representative seminiferous tubule with cysts of the spermatogenic lineage cells in different stages development. HE. (c). Transverse section of the testes showing anastomosis in the seminiferous tubules. HE. (d). Representative section of the testes caudal region showing negative reaction of the lumen to AB-PAS stain (pH 2.5) test for presence of carbohydrates and glycoproteins. RBCs- red blood cells; White asterisk in b- Sertoli nucleus; White asterisk in d-lumen, White arrows-Sertoli cell cytoplasmic extensions; PSG-primary spermatogonia; SSG-secondary spermatogonia; SC2-secondary spermatocytes; ST-spermatids. Fig. 5. Ultrastructure of P. disjunctivus spermatogenic cells at the level of the interstitium. Interstitium , along with myoid cells, and red blood cells within blood vessels. Cysts close to the basement lamina consists of a primary spermatogonium and secondary spermatocytes. Iinterstitium; MC-myoid cells; RBCs- red blood cells; PSG-primary spermatogonium; SC1primary spermatocyte; L-lumen. Fig. 6. Ultrastructure of spermatogenic cells of P. disjunctivus from the Marikina river system. (a) Primary spermatogonia: Cytoplasm with mitochondria joined by intermitochondrial cement/nuages. (b). Secondary spermatogonia: Large nucleus with 2-3 nucleoli. (c). Primary spermatocyte: Nucleus with prominent synaptonemal complexes. (d).
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secondary spermatocyte: Nucleus with highly condensed chromatin forming electron-dense dots. C-cytoplasm; M-mitochondria; N- nucleus; Ns-nucleolus; CM-cell membrane; white asterisk- nuages; black arrowheads-synaptonemal complexes; black arrow-Sertoli cell cytoplasmic extensions.
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Fig. 7. Ultrastructure of spermiogenic cells in P. disjunctivus from the Marikina River system. (a-b). Intermediate phase spermatid: (a). heterochromatic nucleus with electronlucent vacoulations and covered by perinuclear space. Cytoplasm displaced caudally to the nucleus with electron-lucent vesicles. Conspicuous invagination of the nuclear fossa can already be observed. (b). Intermediate phase spermatid with cytoplasm largely eliminated. (ce). Migration of centrioles and formation of basal body in the region of the nuclear fossa. (c). Invagination in the nuclear fossa with undispersed centriole. (d). Basal body formed beneath the nucleus. (e). Movement of the proximal centriole and distal centriole near the basal body. (f). Late stage spermatid: Emergence of a flagellum from the once nuclear fossa. Nucleus is homogenously condensed with a defined nucleolus. (h-i). Scanning electron micrographs of the mature spermatozoon with oval head and long flagellum. (i). Closer view of the head. Nnucleus; ps-perinuclear space; bb- basal body; pc-proximal centriole; dc-distal centriole; pm-plasma membrane; F-flagellum; H-head; white arrow electron-lucent vesicles; black arrow/white asterisk-nuclear fossa.
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Fig. 8a-f Testicular maturity stages in P. disjunctivus. (a). Immature stage characterized by predominance of PSG among other spermatogenic cells. (b). Maturing stage: Increase in mitotic and meiotic division. SC1 and SC2 can be seen dominating the tubule. (c). Mature stage/ Spawning capable stage: Cytoplasmic extensions of Sertoli cells (black arrows) surrounding individual cysts retract resulting in anastomosis of the tubule filled with SZ. (d). Closer view of a single tubule: Lumen is compact with abundant mature SZ surrounded by cysts containing STs (e). Actively spawning stage. Massive anastomosis in the tubule. (f). Closer view of a single tubule in (e). Conspicuous space in the lumen dominated by SZ. PSGprimary spermatogonia; SSG-secondary spermatogonia; SC1-primary spermatocytes; SC2secondary spermatocytes; ST-spermatids; SZ- spermatozoa. Fig. 8 g-n Testicular maturity stages in P. disjunctivus continuation. (g) Spent stage: tubule lumen is composed of residual SZ. (h). Closer view of (g): Lining of the lumen is mainly composed of cysts containing PSG and SSG and occasional and scanty cysts of SC2. (i). Early stage developing spent stage: Lumen in each tubule is occluded. (j): closer view of (i): Thickened lining of the lobule with lining very little PSG. (k). Late developing spent stage (recrudescence): Normal spermatogenic progression during recrudescence. (l) Closer view of (k). (n). Mid-developing spent stage: Unusual thickening of the germinal epithelium with lining predominated by excessive SC2 division in the cyst. (n) closer view of (k): Excessive meiotic division of SC2 while absence of PSG,SSG and SC1 were seen during the dry months of study period. PSG-primary spermatogonia; SSG-secondary spermatogonia; SC1primary spermatocytes; SC2-secondary spermatocytes; ST-spermatids; SZ- spermatozoa.
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Fig. 9. Percentage (%) composition of maturity stages in P. disjunctivus testes from July 2010-June 2011. Stage 1-immature; Stage 2- maturing; Stage 3-mature/ spawning-capable; Stage 4-actively spawning; Stage 5- Spent/ regressing state; Stage 6- developing spent/ regenerating.
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Table 1. Macroscopic and microscopic characteristics used to assign stages of testes maturity in P. disjunctivus from the Marikina river system, Philippines. Macroscopic Features Small (≤ 0.1g), string-like and transparent, devoid of notable swelling and mesorchium in small males (22. 90± 7.88 mean, SD; range 6-27 cm SL). Difficulty in determination of sex.
Histologic features Testicular lining thin (Fig. 8 a) with cysts composed mostly of PSG and SSG held in cysts. SC2 hardly seen though cysts containing ST visible. Lumen of lobules small or occluded, devoid of SZ.
Maturing/ Developing (Stage 2)
Considerable increase in weight (mean 0.25 g ± 0. 25; range 0.1-0.98 g), with some degrees of swelling. Stage mostly observed in males of 26.81 ± 2.99 cm SL (range 19-32 cm). Color of testicular surface range from offwhite to pale pink.
Increased diameter of individual tubules due to increased number of cysts (Fig. 8 b). Lobules contain cysts of all germ cell types, though not consistently present together in all lobules. Lumen small, devoid of SZ.
Mature/ Spawning capable (Stage 3)
Testes swollen and attain maximum weight (mean 1.07 ± 0.609; range 0.03- 3.2 g). Initial swellings appear as small lobes during onset of maturity and even out as testes attain maximum weight. Surface has opaque pale-white color interspersed with fine pink/ brown spots.
The germinal epithelium thin and cysts containing SG1, SG2, and SC conspicuously reduced in number. Considerable degree of anastomosing lobules filled with SZ (Fig. 8 c, d).
Actively spawning (Stage 4)
Testicular weights comparable with Stage 3. External appearance overlaps from being overly swollen and opaque to being pinkish and spotted with varying degrees of twisting near the cranial area. Previous points of small swellings had evened out throughout testes length.
Heavy anastomosis of lobules; discontinuous epithelium with less compacted lumen filled with SZ (Fig. 8 e). Extensive retraction of the cytoplasmic extensions of the Sertoli cells result in a wider, irregularly shaped lumen filled with SZ (Fig. 8 f).
Spent/ regressing (Stage 5)
Testes thin and flaccid. Fully spent testes weights comparable to immature and developing stage (≤0.8 g) except for surface color that varies from being dark to light brown.
Residual SZ in the lumen or none at all; lobule boundary wall (LBW) thin with occasional latedeveloping STs (Fig. 8 g-h). Absence of SCs indicates cessation of spermatogenesis (Fig.
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Stage Immature (Stage 1)
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8j). Testes regain swelling and increase in size. Early stage 6 very similar with stage 2 except for the thick testicular lining and rough surface texture.
Repopulation of the germinal epithelium with SG1; SCs and STs continue to repopulate lobules in the mid recovering spent stage (Fig. 8 k, l). Late stage characterized by thickened tunica albuginea, abundant SCs and STs and empty lumen. PSG-primary spermatogonia; SSG-secondary spermatogonia; SC1-primary spermatocytes; SC2-secondary spermatocytes; ST-spermatids; SZ- spermatozoa.
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Recovering spent (Stage 6)
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