1949-498X 4 1limax

See discussions, stats, and author profiles for this publication at: https://www.researchgate.
net/publication/295860390
Laboratory Observations on Biology of the Tawny Garden Slug Limax flavus

(Linnaeus) (Limacidae: Mollusca)
Article · January 2013
CITATIONS READS
0 621
1 author:
Reham Fathey
Cairo University
17 PUBLICATIONS 18 CITATIONS
SEE PROFILE
Some of the authors of this publication are also working on these related projects:
Biological aspects of the common terrestrial gastropods in Egypt View project
All content following this page was uploaded by Reham Fathey on 25 February 2016.
The user has requested enhancement of the downloaded file.

ANIMAL BIOLOGY JOURNAL
Volume 4, Issue 1, 2013
TABLE OF CONTENTS
Profiles of Carbohydrates During Osmoionic Regulation of the Stenohaline

Catfish, Heteropneustes fossilis (Bloch) 1
Fauzia Anwar Sherwani and Iqbal Parwez
Seasonal Changes in Condition Factor, Gonadossomatic and Hepatosomatic

Indices of the Protogynous Marbled Swamp Eel,
Synbranchus marmoratus 15
Nirlei Hirachy Costa Barros, Arrilton Araujo de Souza
and Sathyabama Chellappa
Histomorphology of Digestive Tract in Two Closely Related Mountain Newts

(Salamandridae: Neurergus kaiseri and N. microspilotus) 27
P. Parto, S. Vaissi and M. Sharifi
Anatomy and Histology of the Digestive Tract and Feeding Habits

of the Neotropical Fish Hypostomus pusarum (Starks, 1913)
(Osteichthyes: Loricariidae) 39
Emilly Kataline R. Pessoa, Naisandra Bezerra da Silva,
Naithirithi T. Chellappa, Arrilton Araújo de Souza
and Sathyabama Chellappa
Laboratory Observations on Biology of the Tawny Garden Slug Limax flavus

(Linnaeus) (Limacidae: Mollusca) 51
M. I. Mohamed and Reham F. Ali
A Brief Review of Noninvasive Methods of DNA Sampling for Wildlife

Conservation 63
Haseeb A. Khan and Ibrahim A. Arif
ii Contents
Anatomy and Histology of the Digestive Tract of a Rare Annual Fish

Hypsolebias antenori (Rivulidae) from Brazil 73
Wallace Silva do Nascimento, Naisandra Bezerra da Silva,
Maria Emília Yamamoto and Sathyabama Chellappa
New York
Animal Biology Journal
The Animal Biology Journal publishes original scientific articles that provide an international
readership with up-to-date ideas and studies surrounding Morphology, Physiology, and
Genetics from microorganisms, invertebrate, and vertebrate biological approaches.
The
Animal Biology Journal
is published quarterly by
Nova Science Publishers, Inc.

400 Oser Avenue, Suite 1600
Hauppauge, New York 11788-3619, U.S.A.
Telephone: (631) 231-7269
Fax: (631) 231-8175
E-mail: nova.main@novapublishers.com
Web: www.novapublishers.com
ISSN: 1949-498X
Institutional Subscription Rate per Volume

Print: $245 Electronic: $245 Combined Print + Electronic: $367
Additional color graphics might be available in the e-version of this journal.
Copyright © 2014 by Nova Science Publishers, Inc. All rights reserved. Printed in the United
States of America. No part of this Journal may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means: electronic, electrostatic, magnetic tape, mechanical,
photocopying, recording, or otherwise without permission from the Publisher. The Publisher
assumes no responsibility for any statements of fact or opinion expressed in the published
papers.
EDITOR-IN-CHIEF
José Rosa Gomes
Departamento de Biologia Estrutural
Molecular e Genética
Universidade Estadual de Ponta Grossa
Campus Uvaranas
Paraná, Brasil
E-mail: abiojournal@ig.com.br
EDITORIAL BOARD MEMBERS

André Carrara Morandini, NUPEM – UFRJ - RJ – Brasil
André Victor Lucci Freitas, UNICAMP - SP - Brasil
Ana Cristina Teixeira Bonecker, UFRJ - RJ - Brasil
Claudio Gustavo Barbeito, Univ. Nacional de La Plata, Buenos Aires, Argentina
Farhad F. Shadan, Scripps Research Institute and Scripps Clinic, La Jolla, USA
José Mauro Granjeiro, UFF - RJ - Brasil
Liya Regina Mikami, UNIBRASIL – PR - Brasil
Luciana Bolsoni Lourenço Morandini, UNICAMP - SP - Brasil
Maria Albertina de Miranda Soares, UEPG – PR - Brasil
Pedro Duarte Novaes, FOP/UNICAMP - SP - Brasil
Priscila A. Grohmann, UFRJ – RJ - Brasil
Ricardo Silva Absalão, UFRJ - RJ - Brasil
Sajal Ray, University of Calcutta - India
Sergio Potsch de Carvalho e Silva, UFRJ - RJ - Brasil
Sérgio Luiz Costa Bonecker, UFRJ – RJ - Brasil
Haseeb Ahmad Khan, King Saud University, College of Sciences, Saudi Arabia
Ajai Kumar Srivastav, Dept. of Zoology, D.D.U. Gorakhpur University, India
Luis Eduardo M. Quintas, UFRJ - RJ - Brasil
Lisheng Zhang, Beckman Research Institute, California, USA
Animal Biology Journal ISSN: 1949-498X
Volume 4, Issue 1 © Nova Science Publishers, Inc.
PROFILES OF CARBOHYDRATES DURING OSMOIONIC

REGULATION OF THE STENOHALINE CATFISH,
HETEROPNEUSTES FOSSILIS (BLOCH)
Fauzia Anwar Sherwani and Iqbal Parwez

Department of Zoology, Aligarh Muslim University, Aligarh, UP, India
ABSTRACT
The persistently high plasma osmolality values of the stenohaline catfish,
Heteropneustes fossilis following transfer from tap water (TW) to 30% and 35% sea
water (SW) suggest the lack of regulative phase in this species which further confirms
our earlier results. A significant increase in plasma glucose levels was observed within 3
h during transfer of the catfish from TW to 30% (p<0.05) and 35% (p<0.025) SW. It,
however, remained more or less constant throughout the duration of the experiment when
the catfish were transferred back to TW after being acclimated to 35% SW for 15 days.
This suggests that quantum of the stress was higher during transfer of the fish to higher
salinities. The elevated levels of plasma glucose during transfer of the catfish to higher
salinities and its sustained levels during reverse transfer to TW may possibly be due to
the process of glycogenolysis as is evident from decreased concentrations of glycogen in
liver and muscle during both types of transfers i.e., from TW to 30% and 35% SW and
the reverse transfer from 35% SW to TW. Thus, it may be concluded that the process of
osmotic adjustments in the catfish in different salinities has discernible effect on
carbohydrate metabolism as evident from the changes in the profiles of plasma glucose,
liver and muscle glycogen contents.
Keywords: Carbohydrate profiles, teleost fish, fresh water, osmoregulation
INTRODUCTION
Acclimation of teleosts to different salinities entails high energy demand for electrolyte
shift and other transport phenomenon (Chang et al., 2007; Febry and Lutz, 1987; Hegab and
Hanke, 1982; Hwang, et al., 2011; Kirstchner, 1993; Mehdi and Elham, 2011; Sangiao-
Alvarellos, et al. 2003, 2005; Tseng and Hwang, 2008). For this purpose they utilize different
energy reserves (such as, carbohydrates, proteins and fats) to cope with the osmotic stress.

Corresponding author: Iqbal Parwez, PhD, Tel.+91-571-272-0161, Fax. +91-571-270-6181, E-mail:
iparwez2002@yahoo.co, fasherwani@yahoo.com
2 Fauzia Anwar Sherwani and Iqbal Parwez
Therefore, studies on the relationship between energetic and osmoregulatory metabolism are
of prime importance. One of the approaches to study the energetic aspect of osmoregulation is
to analyze the alterations in the profiles of substrates of energy metabolism. Since
carbohydrates are utilized easily and have high ATP production rate, it is worthwhile to study
the changes in the carbohydrate metabolism during osmoionic adjustment of the fish. Such
studies have been conducted both on osmoreulatory and non-osmoregulatoru tissues. (Chang
et al., 2007; Hegab and Hanke, 1984; Hwang, et al., 2011; Mancera et al., 1993; Parwez et al.,
2001b; Prodocimo et al., 2008; Tseng and Hwang, 2008).Moreover, since liver and muscle
are directly associated with the formation of glucose through glycogenolysis or
gluconeogenesis, it is more logical to investigate the modifications of carbohydrates in the
above organs during the process of osmoregulatory adjustments (Saurez and
Mommsen, 1987).
The economically important airbreathing stenohaline catfish, Heteropneustes fossilis,
(Heteropneustidae) is widely distributed in freshwater ponds and reverine backwaters of
Indian subcontinent and constitutes an important component of culture and capture fishery.
Extensive work has been carried out at both osmoregulatory and endocrine levels (Goswami
et al., 1983; Parwez and Goswami, 1985; Parwez et al., 1979, 1984, 1994; Sherwani and
Parwez, 2000, 2008). However, no information is available regarding the energetic aspect of
osmoregulation of this fish. Therefore, in the present study, we have investigated the role of
readily oxidisable carbohydrate, glucose and carbohydrate reserves, mainly glycogen
following transfer of the catfish from tap water (TW) to 30% and 35% sea water (SW). We
have also analysedthe effect of reverse transfer of the catfish to TW after acclimation in 35%
SW for 15 days. Plasma osmolality profile has also been investigated under aforementioned
experimental conditions as plasma osmotic pressure/ionic concentration represents the
resultant factor of all adaptation mechanisms.
MATERIALS AND METHODS
Collection and Care of Fish
Adult specimen of catfish, H. fossilis, weighing 40-50g were obtained from local fish
market of Aligarh and acclimated to laboratory conditions (temperature 25+2ºC, photoperiod
12 L : 12 D) for 15 days in glass aquaria (60 X25 X 30 cm) containing stored dechlorinated
TW. During this period they were fed daily ad libitum with Hindlever laboratory animal feed
(Hindustan Lever Limited, Bombay, India) and the water in the aquaria was renewed daily by
siphoning off and replenishing simultaneously with TW adjusted to laboratory temperature.
The fish were also fed daily during the experiments.
Artificial Salt Water
Artificial SW was prepared in dechlorinated TW according to Goswami et al. (1983) and

30% and 35% SW were prepared by diluting full-strength artificial SW with dechlorinated
TW.
Profiles of Carbohydrates During Osmoionic Regulation of the Stenohaline Catfish … 3
Plasma Samples
Blood was drawn from caudal artery into heparinized glass van syringes fitted with 24-
gauge disposable needles. Immediately after collection, the blood was centrifuged for 10
minutes at 3000 rpm (Remi Ltd., India, model no. R8C) and plasma separated and stored at -
20C until analyzed.
Plasma Osmolality
Plasma osmolality was measured in 10 l sample with vapour pressure osmometer

(Wescor 5500, Utah, USA) and expressed as mOsmol/kg.
Plasma Glucose
Plasma glucose was assayed by the glucose-O-toluidine method (Hyvarinen and

Nikkila 1962).
Glycogen
Glycogen, both in muscle and liver was estimated by Anthrone method (Roe and
Daily 1966).
Statistical Analysis
Data for all parameters were expressed as mean +standard error. Statistical comparisons
between experimental and control groups were made by student’s t-test (Snedecor and
Cochran, 1971).
EXPERIMENTAL PROTOCOL
Experiment 1
Groups of catfish were transferred from TW to aquaria containing 30% (320

mOsmol/Kg) and 35% (360 mOsmol/Kg) SW and to TW to serve as control. Fish in all
groups were subjected periodically to handling to eliminate stress factor during sampling.
Four to five fish from each group were sampled at 0, 3 and 24 h and 3, 6, 10 and 15 days
post-transfer.
Blood samples were collected in heparinized syringes from the caudal artery, centrifuged
immediately and plasma was separated for the measurement of osmolality and glucose. A
piece of muscle was excised from just beneath the dorsal fin and liver was dissected out to
estimate glycogen content. The data are presented in Figures 1-4.
Experiment 2
Catfish acclimated for 15 days in 35% SW were transferred to TW and 35% SW

(control). Four to five fish from each group were sampled at 0, 3 and 24 h and 3, 6, 10 and 15
days following transfer.
Blood was collected for the estimation of osmolality and glucose and glycogen contents
in liver as well as muscle were analysed according to the method described earlier. The data
are presented in Figures 5-8.
RESULTS
Experiment 1
Plasma Osmolality
Plasma osmolality increased significantly (p<0.001) 3 days after transfer of catfish in
both 30% (data of 30% SW already published, Sherwani and Parwez 2008) and 35% SW. The
elevated levels were maintained until 15 days post-transfer (Figure 1).
Figure 1. Changes in plasma osmolality of the catfish, Heteropneustes fossilis following

transfer from tap water (TW) to 30% and 35% sea water (SW).
Figure 2. Changes in plasma glucose concentration of the catfish, Heteropneustes fossilis

following transfer from tap water (TW) to 30% and 35% sea water (SW).
Plasma Glucose
Transfer of catfish to 30% and 35% SW significantly increased (p<0.05 and p<0.025
respectively) plasma glucose 3 h after transfer (Figure 2). Plasma glucose level decreased
significantly (p<0.05) in 35% and 30% SW at 24 h and on day 3 respectively after transfer.
Plasma glucose levels were almost similar in all treatments from day 10 onwards. The levels
on day 6 were lower but not significantly different from TW control.
Liver Glycogen
As shown in Figure 3, transfer of catfish to 30% and 35% SW significantly decreased
liver glycogen content from3 h (p<0.025) to 24 h post-transfer (p<0.001). On day 3, the levels
of liver glycogen in both groups exhibited a transient increase during which the values
became almost equivalent to TW control. However, the levels significantly decreased again
on day 6 (p<0.001) after which the parity with TW control was achieved in both groups
(Figure 3).
Muscle Glycogen
Muscle glycogen contents significantly increased (p<0.001) 3 h after transfer of the
catfish to 35% SW but not in those transferred to 30% SW. In both groups the levels declined
from 24 h to 6 days after transfer although they were statistically significant only in 30% SW
on day 3 (p<0.005) (Figure 4). From day 10 onwards muscle glycogen contents were not
significantly different in any group.
Figure 3. Changes in liver glycogen concentration of the catfish, Heteropneustes fossilis

following transfer from tap water (TW) to 30% and 35% sea water (SW).
Figure 4. Changes in muscle glycogen concentration of the catfish, Heteropneustes

fossilis following transfer from tap water (TW) to 30% and 35% sea water (SW).
Figure 5. Changes in plasma osmolality of the catfish, Heteropneustes fossilis following

reverse transfer to tap water (TW) after acclimation in 35% sea water (SW) for 15 days.
Figure 6. Changes in plasma glucose concentration of the catfish, Heteropneustes fossilis

following reverse transfer to tap water (TW) after acclimation in 35% sea water (SW) for
15 days.
Experiment 2
Plasma Osmolality
Plasma osmolality were significantly lower after reverse transfer of catfish from 35% SW
to TW at all sampling time (Figure 5).
Figure 7. Changes in liver glycogen concentration of the catfish, Heteropneustes fossilis

following reverse transfer to tap water (TW) after acclimation in 35% sea water (SW) for
15 days.
Figure 8. Changes in muscle glycogen concentration of the catfish, Heteropneustes

fossilisfollowing reverse transfer to tap water (TW) after acclimation in 35% sea water
(SW) for 15 days.
Plasma Glucose
Reverse transfer of the catfish from 35% SW to TW did not significantly influenced
plasma glucose profile (Figure 6).
Liver glycogen
Liver glycogen content significantly declined (p< 0.001) within 3 h and remained
significantly lower for up to 24 h (p<0.05) compared to 35% SW control group (Figure 7).
From day 3 onwards, liver glycogen in both groups was not significantly different.
Muscle Glycogen
Reverse transfer of the catfish to TW showed consistently low muscle glycogen
concentration throughout the experimental period (Figure 8). The levels were significantly
low (p<0.005) at 24 h post reverse transfer.
DISCUSSION
The persistently high plasma osmolality values in catfish transferred to 30% and 35% SW
suggest the lack of a regulative phase in this species which further confirms the observation
of Goswami et al. (1983). A similar lack of regulative phase has also been found in another
air-breathing catfish, Clarias batrachus (Parwez et al., 2001a) when transferred to higher
salinities. The lack of regulative phase though in reverse direction has also been reported in
barred surfperch, Amphistichus argenteus, amarine teleost, which shows a continuous decline
in plasma osmolarity, plasma sodium and chloride concentration when transferred from 100%
SW to dilute SW (Homes and Lockwood,1970).
In the present study, when catfish acclimated to 35% SW for 15 days was transferred to
TW, there was a significant decline in plasma osmolality within 3 h but the fish was able to
regain its plasma osmotic pressure similar to TW fish within 24 h of the reverse transfer. This
is understandable since in the absence of any reversal of mechanism in higher salinities in the
catfish, the fall in the plasma osmolality is a consequence of simple decrease of ambient
salinity. The initial decrease in plasma osmolality within 3 h of transfer to TW suggested that
there is a continuous loss of ions and that fish is not able to immediately reactivate its salt
uptake mechanism in TW. Other reports also show decrease in plasma osmolality of fishes
with the decrease in external salinity (Evans, 1980; Mancera et al., 1993; 1994; Prodocimo et
al., 2008; Sangiao-Alvarellos et al., 2003, 2005; Sayer and Reader, 1996; Serrano, et al.,
2011;Woo and Fung, 1981).
The results also reveal a significant elevation of plasma glucose within 3 h of transfer of
the catfish both in 30% and 35% SW and are in accordance with earlier findings (Arjona et
al., 2007; Assem and Hanke, 1979; Fiess et al., 2007; Hegab and Hanke, 1984; Krayushkina,
1998; Parwez et al., 2001b; Sangio-Alvarellos et al., 2003, 2005). It is interesting to note that
the magnitude of increase in plasma glucose directly correlates with the magnitude of
gradient between plasma osmolality and the ambient salinity since it is more marked in 35%
SW (P<0.025) as compared to 30% SW (p<0.05). A similar pattern was observed in another
stenohaline catfish, Clasriasbatrachus (Parwez et al., 2001b) and also in the euryhaline
teleosts, Tilapia mossambica (Assem and Hanke, 1979) and Sparus aurata (Sangiao-
Alvarellos et al., 2003). However, in the catfish, H. fossilis, before attaining parity with TW
control, plasma glucose profile exhibited a declining pattern both in 30% and 35% SW up to
6 days post-transfer although the levels were significant (p<0.05) only at 24 h in 35% SW and
on day 3 in 30% SW. Similar situation was observed during brief aerial emersion in Limanda
limandawhere blood glucose concentration decreased after being hyperglycemic for a short
duration (Fletcher 1984). In stenohaline carp, Cyrinus carpio, plasma glucose levels remained
significantly elevated up to 30 days when it was transferred from fresh water (FW) to 1.5%
salt water (about 45% SW) (Hegab and Hanke, 1984).
The present study shows that there was no significant change in plasma glucose
concentrations during reverse transfer of H. fossilis from 35% SW to TW, which may suggest
that quantum of stress was not as high as that of higher salinity (Sangiao-Alvarellos et al.
2003). This is also corroborated by the fact that plasma osmotic pressure returns to normal
FW levels soon after reverse transfer to FW as against the constantly elevated levels
following transfer to higher salinity (see Figures 1 and 5). Reverse transfer of the stenohaline
fish, Cyprinus carpio from 1.5% salt water (about 45% SW) to FW also resulted in regulated
plasma glucose levels except an initial increase at 3h (Hegab and Hanke 1984). Plasma
glucose concentrations were also unaffected by salinity reduction in estuarine pufferfishes
Sphoeroides greeleyi and S. testudineus (Prodocimo et al., 2008). However other studies
show significantly high blood glucose concentrations during transfer of the fishes to lower
salinities (Assem and Hanke, 1979; Kelley and Woo, 1999; Mancera et al., 1993; Roche et
al., 1989; Sangiao-Alvarellos et al., 2005; Woo and Chung, 1995).
Our data show that change in ambient salinity is accompanied by a significant initial
decline in liver glycogen content both during transfer of H. fossilis to higher salinities and its
reverse transfer to TW. It appears that during transfer to higher salinities the catfish actively
osmoregulates during the initial phase which is evident from no appreciable increase in the
plasma osmolality. During this period, any increase in metabolic requirement may possibly be
met through liver glycogenolysis. Mobilization of liver glycogen was also observed when the
stenohaline fishes, Cyprinus carpio (De Boeck et al., 2000; Hanke, 1991) and Clarias
batrachus(Parwez et al., 2001b) were transferred to higher salinities. Glycogenolysis has also
been observed in rainbow trout, Salmo gairdneri (Soengas et al., 1993, 1995), tilapia,
Oreochromis mossambicus (Peters) (Chang et al., 2007; Hanke, 1991), red sea bream
Chryophyrus major L. (Woo and Murat, 1981), Japanese eel, Anguilla japonica (So and
Chan, 1985), gilthead sea bream, Sparus aurata (Sangiao-Alvarellos et al.,. 2003; 2005). In
the present study a significant increase in plasma osmolality beyond 24 h suggests that the
active osmoregulatory control breaks down and the survival in higher salinities is achieved by
passive tissue tolerance (Parwez et al., 1983). An apparent lack of any significant change in
liver glycogen content beyond 24 h (except on day 6) supports the above assumption.
Similarly during reverse transfer of the catfish from 35% SW to TW, it was able to reactivate
its salt uptake mechanism within 24 h during which plasma osmolality became more or less
similar to that of TW fish. The energy required to reactivate salt uptake mechanism during
reverse transfer may possibly be supplied through liver glycogen as is evident from
significant decline in liver glycogen content during the initial phase of 24 h. The liver
glycogen also decreased when the euryhaline teleost, Tilapia mossambicawas transferred to
FW after acclimation for 14 days in 2.5% of salt water (Assem and Hanke, 1979). Similarly,
liver glycogen declined when the carp, Cyprinus carpio (De Boeck et al., 2000); red sea
bream, Clarias major (Woo and Fung, 1981); black sea bream, Mylio macrocephalus ( Kelly
and Woo, 1999; Woo and Wu, 1982); rainbow trout, (Soengas et al., 1993; 1995); gilthead
sea bream, Sparus aurata (Soengas et al., 2003, 2005) were transferred to reduced salinities.
The present results also demonstrate a declining muscle glycogen profile from 24 h to 6
days following transfer of H. fossilis to higher salinities though the levels were significantly
low only on day 3 in 30% SW. The significant increase in muscle glycogen levels in 35% SW
at 3 h post-transfer was surprising and not clearly understood. Reverse transfer of 35% SW
adapted fish to TW also showed significant reduction at 24 h post-transfer and the parity with
control group is reached beyond that. A concomitant decline in liver and muscle glycogen
was also observed in euryhaline teleost, Tilapia mossambica, when transferred to higher
salinities (Bashamohideen and Parvatheswararao, 1972). Our data show that salinity induced
a significant and an early decline in glycogen in liver as compared to muscle, suggesting that
liver glycogen is the immediate source of energy during adaptation of this species of catfish
to different salinities.
The present study, therefore, suggests that ambient salinity has discernible effect on
carbohydrate metabolism as is evident from the changes in the profile of plasma glucose and
liver and muscle glycogen contents.
ACKNOWLEDGMENTS
The assistance of the Indian Council of Agriculture Research, New Delhi, India (Grant
no. 4(97)/2004-ASR-I) to one of the authors (IP) and Department of Science and Technology,
New Delhi, India (Grant no. SR/WOS-A/LS-1027/2003) to FAS is thankfully acknowledged.
REFERENCES
Arjona, F.J., Vargas-Chacoff, l., Ruiz-Jarabo, I., Martín Del Río, M.P. & Mancera, J.M.
(2007). Osmoregulatory response of Senegalese sole (Solea senegalensis) to changes in
environmental salinity. Comparative Biochemistry and Physiology, 148A: 413-421.
Assem, H. & Hanke, W. (1979). Concentrations of carbohydrates during osmotic adjustment
of the euryhaline teleost, Tilapia mossambica. Comparative Biochemistry and
Physiology, 64A: 5-16.
Bashamohideen, M. & Parvatheswararao, V. (1972). Adaptation to osmotic stress in the
freshwater euryhaline teleost Tilapia mossambica. IV. Changes in blood glucose, liver
glycogen and muscle glycogen levels. Marine Biology, 16: 68-74.
Chang, J.C., Wu, S., Tseng, Y., Lee, Y., Baba, O. & Hwang, P. (2007). Regulation of
glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis
mossambicus) during acclimation to seawater. The Journal of Experimental Biology, 210:
3494-3504.
De Boeck G., Vlaeminck, A., Van der Linden, A. & Blust, R. (2000). The energy metabolism
of common carp (Cyprinus carpio ) when exposed to salt stress: an increase in energy
expenditure or effects of starvation? Physiological and Biochemical Zoology, 73:102-
111.
Evans, D.H. (1980). Osmotic and ionic regulation by freshwater and marine fishes. In:
Environmental physiology of fishes (ed. M.A. Ali), pp. 93-121. Plenum Press, New York.
Febry, R. & Lutz, P. 1987. Energy partitioning in fish: the activity-related cost of
osmoregulation in a euryhaline cichlid. Journal of Experimental Biology, 128: 63-85.
Fiess, J.C., Kunkel-Pattersson, A., Mathias, L., Riley L.G., Yancey, P.H., Hirano, T. & Grau,
E.G. 2007. Effects of environmental salinity and temperature on osmoregulatory ability,
organic osmolytes, and plasma hormone profiles in the Mozambique tilapia
(Oreochromis moambicus). Comparative Biochemistry and Physiology, 146A: 252-262.
Fletcher, G.J. 1984. Plasma glucose and plasma fatty acid levels of Limanda limanda (L.) in
relation to season, stress, glucose load and nutritional state. Journal of Fish Biology, 25:
629-648.
Goswami, S.V., Parwez, I. & Sundararaj, B.I. 1983. Some aspects of osmoregulation in a
stenohaline freshwater catfish, Heteropneustes fossilis (Bloch), in different salinities.
Journal of Fish Biology, 23: 475-487.
Hanke, W. 1991. Mechanism of osmotic adaptation in fresh water teleost. Fischerei
Forschung, 29: 15 -19.
Hegab, S.A. & Hanke, W. 1982. Electrolyte changes and volume regulatory processes in the
carp (Cyprinus carpio) during osmotic stress. Comparative Biochemistry and Physiology,
71A: 157-164.
Hegab, S.A. & Hanke, W. 1984. The significance of cortisol for osmoregulation in carp
(Cyprinus carpio) and tilapia (Sarotherodon mossambicus). General and Comparative
Endocrinology, 54: 409-417.
Holmes, W.N. & Lockwood, L.N. 1970. Some aspects of osmoregulation in the barred
surfperch (Amphistichus argenteus). Journal of Zoology, London, 161: 1-14.
Hyvarinen, A. & Nikkila, E.A. 1962. Specific determination of blood glucose with O-
Toluidine. Clinical Chimica Acta, 7:140-143.
Kelly, S.P. & Woo, N.Y.S. 1999. The response of seabream following abrupt hypoosmotic
exposure. Journal of Experimental Biology, 55: 732-750.
Kirschner, L.B. 1993. The energetics of osmotic regulation in ureotelic and hypoosmotic
fishes. Journal of Experimental Zoology, 267: 19-26.
Krayushkina, L.S. 1998. Characteristics of osmotic and ionic regulation in marine
diadromous sturgeons Acipenser brevirostrum and A. oxyrhynchus (Acipenseridae).
Journal of Ichthyology, 38: 660-668.
Mancera, J.M., Perez-Figares, J.M. & Fernandez-Llebrez, P. 1993. Osmoregulatory response
to abrupt salinity changes in the euryhaline gilthead sea bream (Sparus aurata L.).
Comparative Biochemistry and Physiology, 95A: 171-175.
Mancera, J.M., Perez-Figares, J.M. & Fernandez-Llebrez, P. 1994. Effect of cortisol on
brackish water adaptation in the euryhaline gilthead sea bream (Sparus aurata L).
Parwez, I. & Goswami, S.V. 1985. Effects of prolactin, adrenocorticotropin,
neurohypophysial peptide, cortisol and androgens on some osmoregulatory parameters of
the hypophysectomized catfish, Heteropneustes fossilis (Bloch). General and
Comparative Endocrinology, 58: 51-68.
Parwez, I., Goswami, S.V. & Sundararaj, B.I. 1979. Salinity tolerance of the freshwater
catfish, Heteropneustes fossilis (Bloch). Indian Journal of Experimental Biology, 17:
810-811.
Parwez, I., Goswami, S.V. & Sundararaj, B.I. 1984. Effect of hypophysectomy on some
osmoregulatory parameters of the catfish, Heteropneustes fossilis (Bloch). Journal of
Experimental Zoology, 229: 375-381.
Parwez, I., Sherwani, F.A. & Goswami, S.V. 1994. Osmoregulation in the stenohaline
freshwater catfish, Heteropneustes fossilis (Bloch) in deionized water. Fish Physiology
Biochemistry, 13: 173-181.
Parwez, I., Nayyar, M., Sherwani F.A.& Parwez, H. 2001a. Salinity tolerance and the role of
cortisol during osmotic adjustments of an air-breathing catfish, Clarias batrachus.
Journal of Aquaculture, 9: 9-17.
Parwez, I., Nayyar, M., Sherwani F.A. & Parwez, H. 2001b. Changes in the profiles of
cortisol and carbohydrates during osmotic adjustments in an air-breathing catfish, Clarias
batrachus in higher salinities. Journal of Aquaculture, 9: 19-28.
Prodocimo,V., Souza, C.F., Pessini, C., Fernandes, L.C. & Freire, C. A. 2008. Metabolic
substrates are not metabolized from the osmoregulatory organs (gills and kidney) of the
estuarine pufferfishes Sphoeroides greeleyi and S. testudineus upon short-term salinity
reduction. Neotropical Ichthyology, 6: 613-620.
Roche, J.H., Chaar, K. & Pérés, G. 1989. The effect of a gradual decrease in salinity on the
significant constituents of tissue in the sea bass (Dicentrarchus labrax Pisces).
Roe, J.H. & Dailey, R. E. 1966. Determination of glycogen with the Anthron reagent .
Analytical Biochemistry, 15: 245-250.
Sangiao-Alvarellos, S., Laiz-Carrión, R, Guzmán, J.M., Martín del Río, M. P., Miguez, J.M.,
Mancera, J.M. & Soengas, J.L. 2003. Acclimation of S. aurata to various salinities alters
energy metabolism of osmoregulatory and nonosmoregulatory organs. The American
Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 285: 897-
907.
Sangiao-Alvarellos, S., Arjona, F.J., Martìn del Rìo, M. P., Mìguez, J.M., Mancera, J.M. &
Soengas, J.L. 2005. Time course of osmoregulatory and metabolic changes during
osmotic acclimation in Sparus auratus. The Journal of Experimental Biology, 208: 4291-
4304.
Saurez, R.K. & Mommsen, T.P. 1987. Gluconeogenesis in teleost fishes. Canadian Journal
of Zoology, 65: 1869-1882.
Sayer, M.D.J. & Reader, J.P. 1996. Exposure of goldspiny, rock cook and corkwing wrasse to
low temperature and low salinity: survival, blood physiology and seasonal variations.
Journal of Fish Biology, 49: 41-63.
Serrano, X., Serafy, J., Martin Grosell, M., 2011. Osmoregulatory capabilities of the gray
snapper, Lutjanus griseus: salinity challenges and field observations. Mar. Freshwat. Beh.
Physiol. 44, 185-196.
Sherwani, F.A. & Parwez, I. 2000. Effects of stress and food deprivation on the catfish,
Heteropneustes fossilis (Bloch). Indian Journal of Experimental Biology 38: 379-384.
Sherwani, F.A. and Parwez, I. 2008. Plasma thyroxine and cortisol profiles and gill and
kidney Na+/K+-ATP-ase and SDH activities during acclimation of the catfish,
Heteropneustes fossilis (Bloch) to higher salinity, with special reference to the effects of
exogenous cortisol on hypo-osmoregulatory ability of the catfish. Zoological Science, 25:
164-171.
Soengas, J.L., Barciela, P., Fuentes, J., Otero, J., Andres, M.D. & Aldegunde, M. 1993. The
effect of seawater transfer in liver carbohydrate metabolism of domesticated rainbow
trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology, 105B: 337-
343.
Soengas, J.L., Aldegunde, M. & Andres, M.D. 1995. Gradual transfer to sea water of rainbow
trout: effect on liver carbohydrate metabolism. Journal of Fish Biology, 47: 466-478.
Snedecor, G.W. & Cochran, W.G. 1971. Statistical methods. IOWA State University Press,
Ames, Iowa. 59 pp
So, S.T.C. & Chan, D.K.O. 1985. Metabolic changes during adaptation to seawater in the eel,
Anguilla japonica: role of hormones. In: Current trends in comparative endocrinology
(ed. B. Lofts and W.N. Holmes), pp. 922-927. Hong Kong University Press, Hong Kong.
Woo, N.Y.S. & Chung, K.C. 1995. Tolerance of Pomacanthus imperator to hypoosmotic
salinities: Changes in body composition and hepatic enzyme activities. Journal of Fish
Biology, 47: 70-81.
Woo, N.Y.S., & Fung, A.C.Y. 1981. Studies on the biology of the red sea bream Chrysophrys
major –II. Salinityadaptation. Comparative Biochemistry and Physiology, 69: 237-242.
Woo, N.Y.S. & Murat, J.C. 1981. Studies on the biology of the red sea bream Chrysophrys
major. III. Metabolic responses to starvation in different salinities. Marine Biology, 61:
255-260.
Woo, N.Y.S. & Wu, R.S.S. 1982. Metabolic and osmoregulatory changes in reduced salinities
in the red grouper, Epinephelus akaara (Temminck and Schlegel), and the black sea
bream, Mylio macrocephalus (Balsilewsky). Journal of Experimental Marine Biology
and Ecology, 65:139-161.
SEASONAL CHANGES IN CONDITION FACTOR,

GONADOSSOMATIC AND HEPATOSOMATIC INDICES
OF THE PROTOGYNOUS MARBLED SWAMP EEL,
SYNBRANCHUS MARMORATUS
Nirlei Hirachy Costa Barros1, Arrilton Araujo de Souza1

and Sathyabama Chellappa2
1
Post-Graduate Programme in Psychobiology, Center of Bioscience,
Universidade Federal do Rio Grande do Norte, Lagoa Nova,
Natal, Rio Grande do Norte, Brazil
2
Department of Oceanography and Limnology, Center of Bioscience,
Universidade Federal do Rio Grande do Norte,
Praia de Mãe Luiza, s/n, Natal, RN, Brazil
ABSTRACT
Seasonal changes in the condition factor, gonadossomatic and hepatosomatic indices
of the protogynous marbled swamp eel, Synbranchus marmoratus were investigated.
Individuals collected from a brazilian tropical reservoir were used to determine the total
length-weight relationships, changes in the condition factor, gonadosomatic and
hepatosomatic indices in relation to rainfall. Seasonal variations in temperature,
concentration of dissolved oxygen, pH, electrical conductivity of the water and rainfall
were related to the spawning period of S. marmoratus. The sampled population
constituted of primary males, females, transitional individuals and secondary males,
wherein the secondary males were bigger and heavier than all the other individuals. This
species shows a higher increase in total length than in body mass (negative allometry).
The reproductive period occurred in July as indicated by the gonadossomatic index.
Hepatossomatic index was high prior to the breeding season and was low after spawning.
Concentration of dissolved oxygen and rainfall were important factors for reproduction of
this species, which occurred at the end of the rainy season. S. marmoratus adapts a
strategy which optimizes its reproductive potential under the semiarid climatic
conditions.
Keywords: Synbranchus marmoratus; Synbranchiformes; length-weight relationships;

reproduction

Corresponding Author: Sathyabama Chellappa, E-mail: chellappa.sathyabama63@gmail.com
16 Nirlei Hirachy Costa Barros, Arrilton Araujo de Souza and Sathyabama Chellappa
INTRODUCTION
Changes in reproductive strategies of fish are caused by various factors, such as, biotic
aspects, changes in the environmental characteristics and overfishing (Lowe-McConnell,
1987; Wootton, 1984). The abiotic factors act as triggers of reproduction in fish, which can
affect and define the period of gonadal maturation. Through changes in the environmental
factors it is possible to delimit the spawning period and reproductive success in most teleost
fish (Bromage et al. 2001; Bayarri et al. 2004).
The marbled swamp eel, Synbranchus marmoratus Bloch, 1975 (Synbranchiformes:
Synbranchidae), is a native fish of the Brazilian semiarid region (Nelson, 1994; Nascimento
et al., 2011). The members of Synbranchiformes have a long serpentine body lacking pelvic
fins, with the branchial aperture located on the lower side of the body (Britski et al., 1999).
Hermaphroditism is a dominant characteristic of this species presenting a sex reversal
sequence, which is considered as a specialized capacity. S. marmoratus presents protogynic
hermaphroditism with diandric type of males (Lo Nostro and Guerrero, 1996; Barros et al.,
2013). Due to its abundance in freshwater environments, S. marmoratus is used as a food
supplement by the fishing communities of semiarid Brazil (Montenegro et al., 2012).
Studies on the reproductive dynamics of native fish species are important to provide the
necessary information for fisheries and aquaculture development programs which aim at the
rational exploitation and conservation of the fish fauna in the natural and artificial
environments. In this work the following questions were addressed: What are the seasonal
variations of the condition factor, gonadossomatic and hepatosomatic indices of S.
marmoratus? What are the total length-weight relationships for females and secondary males
of S. marmoratus? When do the marbled swamp eel breed?

Fish sampling was carried out on a monthly basis from July, 2010 to July, 2011 in the
Marechal Dutra Reservoir (6° 26’ 24’’S and 36° 38’ 00’’W), located in Northeastern Brazil.
Fish were captured with the help of local fishermen, who used artisanal fishery traps, locally
known as “covos”. Fish captured were numbered, measured, weighed and used for
morphometric measurements to confirm the taxonomical identification of the study species
(Britski et al., 1999).
Total length (TL ±1 cm), total body weight (W ± 1 g), gonad weight (Wg ±0.1 mg) and
liver weight (Wl 1 g) were recorded. The ratio of the different sex types was calculated using
the total number of individuals captured. Variations in the sex ratio were analyzed using the
chi-square (χ 2) test, at a significance level of p<0.05.
The total length-weight relationships for females and secondary males were estimated
from the equation log W = log a + b log L, where W is the total body weight, L is the total
length, a is the intercept and b is the slope of the linear regression (Jobling, 2002). Log-log
plots were generated to remove outliers (Froese, 2006), and 95% confidence limits for anti-
log a and b were calculated for females and secondary males. The t-test was performed to
confirm whether the b departed from the isometric value 3. A single weight-length equation
(W=aLb) was fitted to estimate the value of coefficient b, using the data obtained from
individuals collected (Froese, 2006).
Seasonal Changes in Synbranchus marmoratus 17
The sexes were separated based on macroscopic examinations of gonads (Barros et al.,
2013). The relative condition factor (K) was calculated using the equation K=Wt/Lb (Le Cren,
1951). The gonadosomatic index (GSI) which is the percentage of the gonads in relation to
the whole fish, was calculated as per Wootton et al. (1978). The hepatosomatic index (HSI)
was determined using the equation HSI = 100 (Wl / Wt), where Wl= weight of liver and Wt=
body weight of fish (Wootton et al., 1978).
To characterize the dry and rainy seasons of the study area, rainfall data of the region
during the study period was obtained from the Meteorological Department of EMPARN
(Agriculture Research Company of Rio Grande do Norte), Natal, Brazil. Water temperature
(ºC), concentration of dissolved oxygen (mgL-1), pH and electrical conductivity (μScm-1) of
the waters were recorded monthly in situ, using specific probes from the WTW multi 340i
multiparameter kit, which was calibrated before each recording. The limnological parameters
were recorded regularly between 09:00 and 10:00h.
The principal component analysis (PCA) was used in ordering the five environmental
variables (water temperature, concentration of dissolved oxygen, pH, electrical conductivity
and rainfall) in two factorial axes, with an objective to reduce the dimensionality of the data
and to describe the relationships between these environmental variables in relation to the
spawning period of S. marmoratus. The PCA was performed from the linear correlation
matrix of environmental variables after logarithmic transformation of data. The ordinations
were carried out in the software XLSTAT 7.5.
Distributions of raw data were checked for normality and transformed when necessary.
Pearson correlation tests were conducted to check the correlation between the total lengths
and weights. The non-parametric Spearman test was used to verify the correlations between
HSI and condition factor; GSI and rainfall. The chi-square (2) test was applied to check the
differences between the established sex ratio.
RESULTS
A total of 179 individuals of S. marmoratus were captured during the sampling period
and it was observed that the sampled population constituted of four different sex types:
primary males (born and function as males; n=12), functional females (n=126), transitional
individuals (with both male and female gonadal tissues; n=14) and secondary males (females
transformed as males; n=27). There was a predominance of females during the entire
sampling period (χ2=57.51; p = 0.0001) (Figure 1).
The total length-weight ratio was represented by the mean values of functional females
and secondary males of S. marmoratus. The value of angular coefficient indicated negative
allometry (θ = 2. 585), with a slimmer body showing more growth in total length than in
weight (Figure 2).
The primary males showed amplitude of total length varying from 22 to 32 (26.3±3.95)
cm, females from 22 to 56 (43.9± 6.85) cm, transitional individuals from 37 to 56 (49.3±6.7)
cm and secondary males from 45 to 68 (57±4.9) cm. The secondary males had the largest
total body lengths, whereas the primary males presented the smallest total lengths. The
highest frequency of females and transitional individuals were in the class range of 42 to 52
cm (Figure 3A).
Figure 1. Relative frequency of occurrence of different sex types of S. marmoratus during

July, 2011 to July, 2012.
Figure 2. Total length-body weight relationship for females and secondary males
(distributed in individual empirical points) of S. marmoratus.
The amplitude of total weight of the primary males of S. marmoratus varied from 6 to 50
g (21.61±16.94), females from 9 to 210g (101.4 ±44.5), transitional individuals from 55 to
222g (148.3±48.3) and secondary males from 32 a 327g (203±70.3). The secondary males
were the heaviest whereas the primary males had the lowest weights. The highest frequency
of females and transitional individuals were in the class range of 66 to 126g (Figure 3B).
(A)
(B)
Figure 3. Percentage frequency of total length (A) and body mass (B) of the four different
sex types of S. marmoratus.
The hepatosomatic index (HSI) of females and secondary males of S. marmoratus

showed an increase from July to September, 2011 followed by a declining trend and attaining
the lowest value in December. This was followed by an increasing trend attaining a peak in
February, 2012 (Figure 4A). The condition factor of females and secondary males varied
from July, 2011 attaining a lowest value in January, 2012 which gradually increased from
April and reached the highest value in June, 2012 (Figure 4A). Condition factor and the HSI
showed a weak negative correlation, although it was not statistically significant (r = -0.718;
p > 0. 05).
The GSI of females and secondary males declined from July, 2011 and reached low
values in March, 2012. The GSI increased gradually from April 2012, with peaks in July,
2011 and 2012 (Figure 4B). The rainfall in the study region showed two well defined periods,
a dry season extending from August to December (rainfall 14.71 mm±11.09), and a rainy
season from January to July (143.61 mm±48.90) (Figure 4B). The mean rainfall of the study
region was 84.12±69.31 mm, which varied from a minimum of 7.3 mm to a maximum of
270.2 mm in April. GSI and rainfall showed significant negative correlation (r = -0.7187; p
=0.006). Spawning of S. marmoratus occurred at the end of the rainy season (July) when the
GSI had high values.
Figure 4. (A) Condition factor (K) and Hepatosomatic Index (HSI) of females
and secondary males; (B) Gonadosomatic Index (GSI) of females and secondary
males of S. marmoratus and rainfall during July, 2011 to July, 2012.
Figure 5. Analysis of the Principal Component Analysis (PCA) of the environmental

variables (temperature, concentration of dissolved oxygen, rainfall, pH and electrical
conductivity) in relation to the reproductive period of S. marmoratus.
The temperature varied from 25ºC to 31ºC with a mean of 26ºC ±1.5, and the
concentration of dissolved oxygen varied from 2.03 mgL-1 to 7.63 mgL-1 (in April) with a
mean of 5.85 mgL-1 ±1.95. The pH had a mean value of 7.81 ±0.50, which varied from a
minimum of 6.5 to a maximum of 9.7.
The electrical conductivity of water varied from 556 μScm-1 to 638 μScm-1 with a mean
of 558 μScm-1 ±21.11. The Principal Component Analysis (PCA) applied to the
environmental variables showed that the two first axes together explained 95.05% of the total
variance in environmental variables. The first factorial axis explains 80.87% of data variance,
while the second explains 14.18%.
The ordination diagram shows that the environmental variables, such as, concentration of
dissolved oxygen and rainfall were the influential factors during the rainy season (January to
July), when GSI was increasing leading to spawning. Whereas variables, such as, water
temperature, pH and electrical conductivity were influential factors during the dry season
(August to December) (Figure 5). Since, S. marmoratus breeds in July, at the end of the rainy
season, its spawning period could be related to the beginning of the increase in water
temperature, pH and electrical conductivity, as well as the lowering of dissolved oxygen in
the reservoir.
DISCUSSION
The present study has documented seasonal changes in the condition factor,
gonadossomatic and hepatosomatic indices of S. marmoratus during its annual life cycle.
Some commonly used indirect measures of energy status are the condition factor (CF) and the
hepatosomatic index (HSI), and high values of CF and HSI are net product of energy
deposition following feeding and mobilization of energy reserves during reproduction
(Chellappa et al., 1995).
Life-cycle characteristics such as reproductive seasonality of different fish species are
regulated by environmental cues, and in environments that are clearly defined seasonally,
reproductive activity of fish species is restricted to a specific annual period. In tropics,
rainfall, pH, water temperature and concentration of dissolved oxygen are the environmental
factors which influence the reproductive period and breeding of fish. Exposure to
environmental disturbance presents a challenge for tropical fish, where the synchrony
between the onset of reproduction and environmental cues depend on rainfall (Izquierdo et
al., 2001; Wu et al., 2003; Migaud et al., 2010; Chellappa et al., 2010).
An appreciable predominance of females was registered during this study, differing from
the study carried out in Argentina where males predominated (53.3%) followed by functional
females (36.9) of S. marmoratus (Lo Nostro and Guerrero, 1996). Probably this was due to
the sex ratio, which can alter due to differences in growth rate, selective fishing method, male
preference for different habitats or mortality in different regions (Nikolsky, 1963; Edwards,
1998; Vicentini & Araújo, 2003).
The length-weight relationships provide an index which is frequently used as an
important tool in fish biology and ecology. With the help of this relationship the type of
growth could also be analyzed through the coefficient of allometry (Leis, 1981). The type of
growth of the fish species could be determined through the angular coefficient, which is
isometric when  = 3, positively allometric when > 3 and negatively allometric when  <3.
Isometric growth indicates that the body increases in all dimensions in the same proportion
during growth, whereas positive allometry indicates that the body becomes more rotund as it
increases in length, and negative allometry indicates a slimmer body (Jobling, 2002). S.
marmoratus showed negative allometry, which is logically expected since this species
presents a serpentine form.
Individuals of S. marmoratus with mean length of 58.73 (± 50.47) cm were encountered
in the Brazilian hydrographic basin of River Tibagi (Shibatta, 2003). The maximum body
lengths of S. marmoratus encountered were of 100 cm (Britski et al. 1999). In the River Alto
Uruguai of Brazil, S. marmoratus showed lengths ranging from 32.6 to 49.0 cm (Oliveira &
Zaniboni-Filho, 2009). In the present study, the big individuals which were the secondary
males measured from 60 to 68 cm.
Condition factor can quantify the state of well-being of a fish, and is a useful indicator of
changes in growth during the different seasons of the year. It is frequently used as an
indicator of the spawning period, since the condition factor is high before spawning (Jones et
al., 1999; Anene, 2005). Condition factor suffers alterations due to internal factors (gonadal
development and size of individuals) and external factors (food availability, temperature etc).
The HSI could reflect the depletion of energy reserves which are mobilized for gonadal
developmental processes (Huntingford et al., 2001; Zwolinski et at., 2001; Fontoura et al.,
2010). In this study, HSI showed a weak negative correlation with the condition factor. This
relationship occurs since the condition factor reflects the physiological state and/or spending
of energy reserves for reproductive activities. The fish generally stores large amounts of lipid
and glycogen as energy reserves, and these variations are evidenced significantly through
hepatossomatic relationships (Hoar Randall, 1971; Chellappa et al., 1995; Huntingford et al.,
2001).
The GSI could be considered as one of the good indicators of the spawning period
(Bazzoli; Godinho, 1991). In the present study, the GSI of S. marmoratus was high during the
end of the rainy season, which coincided with the spawning period. Similar observations were
recorded when the males showed high values of GSI between spring and summer (Lo Nostro
and Guerrero, 1996). After spawning, S. marmoratus generally undergoes a period of
aestivation during the dry season, getting buried in tunnels. This occurs during the months of
August to December in the semiarid Northeastern Brazil (Montenegro et al., 2012).
The electrical conductivity values remained low throughout the study period, but it could
increase during prolonged periods of drought (Chellappa & Costa, 2003; Chellappa et al.,
2009). Concentration of dissolved oxygen and rainfall are two important environmental
factors which influence the spawning period of S. marmoratus in the semiarid region of
Brazil.
ACKNOWLEDGMENTS
This study was supported by the Post-Graduate Federal Agency CAPES/MEC, Brazil,
and the National Council for Scientific and Technological Development of the Ministry of
Science and Technology of Brazil (CNPq/MCT) in the form of Research grants.
REFERENCES
Anene, A., 2005. Condition factor of four cichlid species of a man-made lake in Imo State,
Southeastern Nigeria. Turkish, Journal of Fisheries and Aquatic Sciences, 5: 43-47.
Barros, N. H. C., Souza, A. A., Chellappa, S., 2012. Histological aspects of gonad
development in the Diandric Protogynous Sequential Hermaphrodite fish Synbranchus
marmoratus (Osteichthyes: Synbranchidae). Animal Biology Journal, 3, (4): 159-172.
Bayarri, M.J., Rodriguez, L., Zanuy, S., Madrid, J.A., Sanchez-Vazquez, F.J., Kagawa, H.,
Kuzawa, K.O., Carrillo, M. 2004. Effects of photoperiod manipulation on the daily
rhythms of melatonin and reproductive hormones in caged European sea bass
Dicentrachus labrax. General and Comparative Endocrinology, 136: 72-81.
Bazzoli, N., Godinho, H.P. 1991. Reproductive biology of Acestrorhynchus lacustris
(Reinhardt, 1874) (Pisces: Characidae) from Três Marias reservoir, Brazil. Journal
Zoologischer Anzeiger, 226: 285-299.
Britski, H.A., Silimom, K.Z.S., Lopes, B.S. 1999. Peixes do pantanal. Manual de
identificação, H. A. Britski, Ed, Brasília: EMBRAPA, SPI, p 184.
Bromage, N., Porter, M., Randall, C. 2001. The environmental regulation of maturation in
farmed finfish with special reference to the role of photoperiod and melatonin.
Aquaculture, 197: 63-98.
Chellappa, N.T., Costa, M.A.M. 2003. Dominant and co-existing species of

Cyanobacteria from a eutrophicated reservoir of Rio Grande Norte State, Brazil. Acta
Oecologia, 24, (3): 1-10.
Chellappa, S., Bueno, R.M.X., Chellappa, T., Chellappa, N.T., Almeida, V.M.F., Val, V.M.F.
2009. Reproductive seasonality of the fish fauna and limnoecology of semi-arid Brazilian
reservoirs. Limnologica. 39(4): 325–329.
Chellappa, S., Huntinford, F.A.R., Strang, H.C., Thomson, R.Y. 1995. Condition factor and
hepatosomatic index as estimates of energy status in male three-spined stickleback.
Journal of Fish Biology, 47, (5): 775-787.
Chellappa, S., Lima, J. T. A. X., Araújo, A., Chellappa, N.T. 2010. Ovarian development and
spawning of Serra Spanish mackerel in the coastal waters of northeastern Brazil.
Brazilian Journal of Biology, 70, (2): 451-456.
Edwards, A.W.F. 1998. Notes and Comments. Natural selection and sex ratio: fisher's
sources. The American Naturalist, Chicago, 151, (6): 564-569.
Fontoura, N. F., Jesus, A. S., Larre, G.G., Porto, J. R. 2010. Can weight/length relationship
predict size at first maturity? A case study with two species of Characidae. Neotropical
Ichthyology, 8: 835-840.
Froese, R. 2006. Cube law, condition factor and weight-length relationships: history, meta-
analysis and recommendations. Journal of Applied Ichthyology, 22: 241–253.
Hoar, W.S., Randall, D.J. 1971. Fish physiology. New York, Academic Press: 457p.
Huntingford, F.A., Chellappa, S., Taylor, A.C., Strang, R.H.C. 2001. Energy reserves and
reproductive investment in male three spined sticklebacks, Gasterosteus aculeatus.
Ecology of Freshwater Fish, 10, (2): 111-117.
Izquierdo, M.S., Fernandez-Palacios, H., Tacon, A.G.J. 2001. Effect of broodstock nutrition
on reproductive performance of fish. Aquaculture, 197: 25-42.
Jobling, M. 2002. Environmental factors and rates of development and growth. In: Handbook
of fish biology and fisheries. P. J. Hart, J. D. Reynolds, Eds., Blackwell Publishing Ltd,
Oxford. p. 97-122.
Jones, R.E., Petrell, R.J., Pauly, D. 1999. Using modified length-weight relationships to
assess the condition of fish. Aquacultural Engineering, 20: 261-276.
Le Cren, E.D. 1951. The length-weight relationship and seasonal cycle in gonad weight and
condition in the perch (Perca fluviatilis). Journal of Animal Ecology, 20, (2): 201-219.
Leis, J.M. 1981. Distribution of fish larvae around Lizard Island, Great Barrier Reef: coral
reef lagoon refuge?. Marine Sciences Center, University of the Philippines, Quezon city
In: Gomez, G. D. et al., Eds., Proc. 4th Symp. Coral reefs, 2: 472-477.
Lo Nostro, F.L., Guerrero, G.A. 1996. Presence of primary and secondary males in a
population of the protogynous fish Synbranchus marmoratus. Journal Fish Biology, 49:
788–800.
Lowe-McConell, R.H. 1987. Ecological studies in tropical fish communities. Cambridge:
Cambridge University Press, p, 382.
Migaud, H., Davie, A., Taylor, J.F. 2010. Current knowledge on the photoneuroendocrine
regulation of reproduction in temperate fish species. Journal of Fish Biology, 76: 27-68.
Montenegro, L.A., Silva, N.B., Nascimento, W.S., Chellappa, S. 2012. Anatomy and
Histology of the digestive tract and feeding habits of the marbled swamp eel Synbranchus
marmoratus. Animal Biology Journal, 3, (3): 127 - 143.
Nascimento, W. S., Araújo, A. S., Gurgel, L. L., Yamamoto, M. E., Chellappa, N. T., Rosa,
R. S., Chellappa, S. 2011. Endemic fish communities and environmental variables of the
Piranhas-Assu hydrographic basin in the Brazilian Caatinga Ecoregion. Animal Biology
Journal, 2, (3): 97-112.
Nelson, J. 1994. Fishes of the World. 3rd, Edition, Wiley, New York, p. 600.
Nikosky, G.V. 1963. The ecology of fishes. Nova York, Academic, p.352.
Oliveira, A.P.N., Zaniboni-Filho, E. 2009. Length–weight relationships of fish species caught
in the Upper Uruguay River. Brazil, Journal of Applied Ichthyology, 25: 362–364.
Shibatta, O.A. 2003. Family Pseudopimelodidae, In: R. E. Reis, S. O. Kullander & C. J.
Ferraris Jr (Eds). Check List of the Freshwater Fishes of South and Central America.
Edipucrs, Porto Alegre, p. 401-405.
Vicentini, R.N., Araújo, F.G. 2003. Sex ratio and size structure of Micropogonias furnieri
(Desmarest, 1823) (Perciformes, Sciaenidae) in Sepetiba Bay, Rio de Janeiro. Brazil.
Brazilian Journal of Biology, 63: 559–566.
Wootton, R.J. 1984. Introduction: strategies and tactics in fish reproduction. In: G.W. Poots
& R.J. Wootton (ed.), Fish Reproduction: Strategies and Tactics. Academic Press, New
York, p. 1–12.
Wootton, R.J., Evans, W., Mills, L. 1978. Annual cycle in female threespined Sticlebacks
(Gasteroteus aculeatus L) from an upland and lowland population. Journal of Fish
Biology, 12: 331-343.
Wu, R.S.S., Zhou, B.S., Randall, D.J., Woo, N.Y.S., Lam, P.K.S. 2003. Aquatic hypoxia is an
endocrine disruptor and impairs fish reproduction. Environmental Science Technology,
37: 1137-1147.
Zwolinski, J., Stratoudakis, Y., Soares, E. 2001. Intra-annual variation in the batch fecundity
of sardine off Portugal. Journal of Fish Biology, 58: 1633-164.
HISTOMORPHOLOGY OF DIGESTIVE TRACT

IN TWO CLOSELY RELATED MOUNTAIN NEWTS
(SALAMANDRIDAE: NEURERGUS KAISERI
AND N. MICROSPILOTUS)
P. Parto 1, S. Vaissi 2 and M. Sharifi 2

1
Department of Biology, Razi University, Baghabrisham, Kermanshah, Iran
2
Department of Biology, Razi University, Center for Environmental Studies,
Baghabrisham, Kermanshah, Iran
ABSTRACT
Anatomical, histological and histochemical examinations were made from pharynx,
esophagus, stomach, duodenum and large intestine in two closely related mountain newts
(Salamandridae: Neurergus microspilotus and Neurergus kaiseri). Sections were stained
with Hematoxylin and Eosin, Periodic Acid Schiff's (PAS) and Alcian Blue (AB). In both
species the pharynx and esophagus are covered by pseudostratified ciliated columnar
epithelium with goblet cell which is positive with PAS and AB. The stomach in N.
kaiseri and N. microspilotus is a straight, expanded conical tube, laying slightly to the left
side of the body cavity, and terminating at the pylorus. The stomach in both species is
divided into three distinct parts, the cardia, fundus and pylorus. Although in both species
the epithelium of the stomach surface and of the lining of the crypts consists of a single
layer of high columnar cell, but the apical portion of the cells in N.kaiseri consists of
homogeneous acidophilic granules while in N.microspilotus is foamy. The duodenum is
short and is sharply reflexed along the medial aspect of the stomach. Duodenum in N.
kaiseri and N. microspilotus shows villi which consists of the epithelial covering and a
core of connective tissue containing blood and lymph capillaries. The large intestine in
both N. kaiseri and N. microspilotus is located along the median line. The intestine is a
coiled tube of a regular diameter, larger than, that of the duodenum. Histologically, these
are no villi in large intestine and goblet cells rise to numerous. The epithelium is simple
columnar, and the lamina propria and submocosa are strongly reduced.
Keywords: Neurergus kaiseri, Neurergus microspilotus, digestive system, anatomy,

histology

Corresponding author. E-mail: sharifimozafar2012@gmail.com
P. Parto, S. Vaissi, and and M. Sharifi
INTRODUCTION
The digestive system of vertebrates presents various structural and functional adaptations
to their diverse feeding habits. The digestive tract also represents a functional link between
foraging activity and energy conservation through energy allocation for various activities
(Hammond et al., 2001; Secor, 2001; Secor, 2005; Romão et al., 2011). Over the last decades,
field observations and experimental laboratory studies have shown that digestive tract
anatomy and function of many species are flexible, and can change in response to variation in
environmental conditions (Piersma & Lindstrom, 1997; Starck, 1999; McWilliams &
Karasov, 2004). However, most of these studies have been conducted in birds, mammals and
reptiles, while research on histological characteristics in amphibians is scarce. However,
attempts have been made to study of the microscopic structure of the amphibians alimentary
tract organs. Of these, the histological studies of the gut epithelium of the Neotenic Cave
salamander Proteus anguinus (Amphibia, Caudata) has been conducted by Mali & Bulog
(2004). Similarly, Liquoria et al., (2005) studied histological and immunohistochemical
response of alimentary tract to ATPase mediate acid secretion in gastric glands of Triturus
carnifex (Amphibia, Caudata). More studies have been carried out on histomorphology of
digestive tract in various species of anura (Jorquera et al., 1962; Ferrri & Medeiros, 1976;
Senler & Yildiz, 2000; Sancar-Bas, et al., 2009).
In Iran, genus Neurergus has a relatively wide geographic distribution, ranging from
southern Zagros Mountains to mid-Zagros range and extending into Iraq and southern Turkey
(Cope, 1862; Baloutch & Kami, 1995). Sharifi & Assadian (2004) demonstrated that N.
microspilotus occurs in several highland streams in the mid Zagros Mountains. N. kaiseri is
endemic to Iran and occurs only in southern parts of Zagros Mountains (Sharifi et al., 2008).
N. microspilotus is slightly larger than N. kaiseri and live in different climatic regime.
Although both species of the genus Neurergus occur in highland first order streams but
macro-ecology of these two areas (mid-Zagros and southern Zagros) are distinctively
different. In southern Zagros Range where N. kaiseri occurs the climate is warm without
winter freezing while in western Zagros where N. microspilotus occurs the climate is cold
with pronounced seasonal variations including a prolonged winter freezing (Sharifi et al.,
2008). In both areas the mountain newts are top predators of the diverse benthic macro-
invertebrates (Sharifi & Assadian, 2004).
The objective of this study was to describe the digestive system of two critically
endangered mountain newts by means of anatomical and histological inferences. This
description should enable better understanding of the digestive processes in these animals,
contributing to physiological, pathological and phylogenetic studies and to the management
and conservation of amphibian, and to extend their preventive and therapeutic medicine.

Four adult N. kaiseri, two males and two females, were collected from Vejenab in south
western Iran (32˚56' N, 48˚28' E). Four N. microspilotus, two males and two females, were
collected from Kavat Stream in northern Kermanshah in western Iran (34˚53' N, 46˚31' E).
Permits for collections were issued on the ground of scientific use by regional office of
environment in Kermanshah Province for N. microspilotus and in Khoramabad Province for
N. kaiseri. All animals were in resting condition and each with a body length of about
173.91±17.75 mm for N. kaiseri and 192.35±10.20 mm for N. microspilotus. Specimens from
both species, male and female, were dissected. The animals were sacrificed under ether
anesthesia and their digestive tracts were quickly removed. The body length was measured as
the distance from the tip of the snout to the posterior border of the cloacal opening.
Measurements of the various parts of the digestive tube were taken by digital Vernier
Calliper. Body divided into five parts. The specimens were fixed by 10% formaldehyde and
dehydrated in a series of ethanol treatments, starting from the 70% storing solution, then were
cleared in xylene, embedded in paraffin, and serially sectioned at 7 µm with a rotary
microtome. Most sections from each individual were stained with Hematoxylin-Eosin (for
general histology), and others sections were treated with Periodic Acid and Schiff’s reagent
(PAS), for identifying neutral carbohydrates and Alcian Blue (AB) at pH 2.5 to for acidic
polysaccharide, according to the protocol of Luna (1968). The control sections for PAS were
treated with saliva at 37°C (Pearse, 1968). Sections were observed with an Olympus
microscope (Leica Galen III) and were photographed with a digital camera (Leica with
Dinocapture 2.) mounted to the microscope. Anatomical and histological descriptions follow
the morphological nomenclature of Sever (1994 a).
RESULTS
The digestive tract of the Neurergus kaiseri and Neurergus microspilotus are relatively
simple extends from the mouth to cloaca, consist of mouth cavity, pharynx, esophagus,
stomach, duodenum, large intestine, rectum (Figure 1). The alimentary tract in both species
are histologically simple and consist of: 1) Mucosa, a cellular connective tissue which consist
of an inner epithelium, a middle lamina propria and an outer muscularis mucosa, 2)
Submocusa, a less cellular connective tissue layer, 3) Muscular coat, divided into an inner
circular and outer longitudinal layer, and 4) Serosa, a connective tissue surrounded by the
simple squamus mesothelial epithelium.
Pharynx leads directly from the mouth as a wide tube. In both species the pharynx is
covered by pseudostratified ciliated columnar epithelium with goblet cell. Lamina propria
submucosa is loose connective tissue with large blood vessels. A single layer of smooth
muscle fibers is presented in tunica muscularis (Figure 2).
Esophagus in both species a wide esophagus follows an almost straight course from the
pharynx without any sharp demarcation line. It is situated slightly to the left of the median
line, passing dorsal to the heart as it approaches the stomach and narrowing posteriorly
(Figure 1). The external walls of both esophagus and stomach, and in fact of the whole gut
also, are smooth, but the internal walls are variously corrugated, each section of the gut
having its own particular pattern. The length of esophagus in N. kaiseri and N. microspilotus
is 4.5±1.3mm and 7.1±2.68mm respectively and their girth is 1.90±0.70 mm and 2.35±0.65
mm respectively.
Histologically the esophagus in N. kaiseri and N. microspilotus is covered with
pseudostratified ciliated columnar epithelium with scattered goblet cells, which release large
amount of the mucus to lubricate the epithelium. The mucosa is thrown to longitudinal
mucosal folds. These are much serous, mucus and mixed glands in lamina propria and a
sparse longitudinally oriented muscularis mucosa is present. The muscular coat consists of
inner circular and outer longitudinal layer. The serosa is covered by simple squamus
mesothelium. Positive reaction of goblet cells to PAS and AB indicate the presence of neutral
and acidic carbohydrate (Figure 3).
Figure 1. General view of digestive tract in N. microspilotus (A and C) and N. kaiseri

(B and D).
Figure 2. A.The pharynx is lined with pseudostratified ciliated epithelium (EP)

(Magnification with ×400, (H & E)). B. Unicellular gablet cells (G) are abundant and
scattered among the ordinary epithelial cells. The dense connective tissue of the lamina
propriasubmucosa (LPS) is thick and comprises homogeneous collagen fibers and some
fibrocytes (Magnification with ×2500, (H & E)).
Figure 3. Section of the esophagus (A, B in N. microspilotus and B, C in N. kaiseri).

A.In this general view, the mucosal epithelial folds are seen (Magnification with ×400,
(H & E)). B, C, D. Detail of the esophageal wall. The lumen (L) of the esophagus is lined
by a pseudostratified ciliated columnar epithelium (EP) containing a huge amount of
goblet cells (G) (mucous cells). The lamina propria (LPS) is filled with serous (SG),
mucousand mixed glands (MG) (Magnification with, B: ×1000 (H & E), C: ×1000
(H & E), D: ×2500, (H & E)). E. Oesophageal goblet cells are PAS positive and react
with Alcian blue.C, Cilia; M, Tunica muscularis; S, Tunica serosa (Magnification with
×2500, (AB)).
Stomach in N. kaiseri and N. microspilotus is a straight, expanded conical tube, laying

slightly to the left side of the body cavity, and terminating at the pylorus. The stomach lies
almost longitudinally along the left side of the body. It narrows considerably at its posterior
end, and there is always a well-defined constriction between the stomach and the duodenum.
The constriction is especially noticeable when the gut is full, and a slight thickening of the
wall is discernible in histological cross section (Figure 1). The length of stomach in N. kaiseri
and N. microspilotusis 15.65±3.1 mm and 20.25±1.55 mm respectively and their girth is
3.8±0.65mm and 5.65±0.95 mm respectively. In the stomach from the surface of the
epithelium, numerous gastric pits (Crypts) sink down into the mucosa with the gastric gland
opening at their bottom in fundus and pylorus in both species. The stomach in N. kaiseri and
N. microspilotus is divided into 3 distinct parts, the cardia, fundus and pylorus. The
epithelium of the stomach surface and of the lining of the crypts consists of a single layer of
high columnar cell and the apical portion of the cell in N. kaiseri acidophil and homogeneous
while in N. microspilotus is foamy with H-E staining. In the cardia tubular glands empty their
secretion into the bottom of the gastric pit. These glands are lined with a single type of
secretory cell and extend throughout the connective tissue of lamina propria.
Figure 4. Stomach in N. kaiseri (A and D) and N. microspilotus (B and C). A.Cardia is

lining with simple columnar epithelium (EP). Invagination of tunica mucosa from gastric
pits, in which multiple tubular glands (G) drain. The muscularis mucosa (MM) is obvious
in this image (Magnification with ×400, (H & E)). B, C. In the fundus the lining
epithelium is simple columnar (EP) and the fundic glands occupy the lamina propria
(LP). Glands (*) are constructed mostly of oxynticopeptic cells, which secret both
hydrochloric acid and pepsinogen. A few mucosa neck cells (arrow) also present
(Magnification with B: ×400 (H & E), C: ×1000(H & E)). D. Note the deep gastric pits in
pylorus. The tubular glands (G) in this region are noting convoluted and very few in
number. Tunica muscularis (M) become thick to from the pyloro-duodenal sphingter
(Magnification with ×1000 (H & E)). E, F. Columnar cell and mucus neck cell showed
strong affinity for PAS and AB. SM, Tunica submucosa (Magnification with
E: ×1000(PAS), F: ×1000(AB)).
Figure 5. Duodenum in N. kaiseri (A) and N. microspilotus (B), these micrographs

displays elongated villi, deep finger-like processes of the intestinal mucosa extending in
the duodenal lumen. These expansions are lined by a simple columnar epithelium (EP)
and mucus secreting or goblet cells (G). The brush border (B) of the enterocytes is visible
in B. The villous core is filled with connective tissue of the lamina propria (LP)
containing blood and lymph capillaries. Tunica muscularis (TM) is covered by the serosa
(S).C. PAS positive goblet cells and striated border(Magnification with A: ×1000
(H & E), B: ×2500 (H & E), C: ×400(PAS).
The fundus branched tubular glands possess 2 types of cells. Most of the cells are
acidophilic which produces both pepsin and hydrochloric acid (Oxyntopeptic cell) and a few
mucus cells are situated in the neck of the glands (Mucous neck cell) which secrete a
protecting mucous, these cells are fewer in N. microspilotus. The pyloric glands are separated
more widely from one another and consist of the shorter but less frequently branched tubules.
A muscularis mucosa is found and consists of smooth muscle cells disposed longitudinally.
The submucosa contains nerves, blood vessels and lymphatics. The muscular coat is
composed of the inner circular and outer longitudinal smooth muscle cells and in the pylorus;
additional oblique layer makes the sphincter between stomach and intestine. Mucines were
observed in the apical portion of columnar cell and mucosa-neck cells, and these parts gave
strong PAS and AB reaction. (Figure 4).
Duodenum is definite and readily observable pyloric sphincter occurs consistently in N.
kaiseri and N. microspilotus. The portion of the small intestine between the pylorus and the
entrance of the bile duct comprises the duodenum. The duodenum is short and is sharply
reflexed along the medial aspect of the stomach. It is indistinguishable from the rest of the
intestine except that it receives the pancreatic and bile ducts (Figure 1). The dorsal pancreatic
duct enters the proximal end of the duodenum some 2-3 mm or so from the pylorus. The
length of duodenum in N. kaiseri and N. microspilotus is 14.55±0.55 mm and 17.70±1.20 mm
respectively and their girth is2.1±0.10mm and 2.7±1.25mm respectively. Duodenum in N.
kaiseri and N. microspilotus shows villi.
Figure 6. A. Large intestine in N. microspilotus. The mucosa presents a flat surface and
villi are absent. Numerous goblet cells (G) in the lining epithelium (EP) are characteristic
of this part. L: Lumen, M: Tunica muscularis (Magnification with ×1000 (H & E)). B, C.
Intestinal goblet cells were PAS positive and react with AB pH. 2.5 (Magnification with
B: ×2500(AB), C: ×2500 (PAS)).
A villous is a finger-like process of the mucosa which consists of the epithelial covering
and a core of connective tissue containing blood and lymph capillaries. The duodenal
epithelium of simple columnar is made up of enterocytes possessing a well marked striated
border (microvilli) and goblet (mucus-secreting) cells. The simple tubular invagination from
the surface is lacking in these newts species, thus no true glands of the Liberkuhn are present
in lamina propria. The lamina propria and submocusa contain large numbers of wandering
eosinophilic granular cells. The muscularis mucosa consists of a thin layer of smooth muscle
cells, longitudinal in direction. The muscular coat here well developed to insure peristaltic
activity. Mucose secreted by goblet cells was strongly PAS positive. No staining was
observed with AB. The brush border corresponding to glycocalyx also reacted with PAS
(Figure 5).
Large intestine in both N. kaiseri and N. microspilotusis located along the median line,
except when filled by an abundance of food, or by eggs in gravid female, and it extends
straight to the cloaca. The intestine is a coiled tube of a regular diameter, larger than, that of
the duodenum (Figure 1).The rectum is an expanded straight flask shaped structure arising
quite suddenly from the posterior end of the intestine. It is much thinner walled than the rest
of the gut (Figure 1). The length of large intestine in N. kaiseri and N. microspilotus is
36.30±4.7 mm and 47.55±3.45 mm respectively and their girth is 2.3±0.20 mm and 2.4±0.70
mm respectively.
Figure 7. The rectum is lined with a simple columnar epithelium (EP) housing numerous
side by side goblet cells (G). The mucosal folds become more flat and the number of
goblet cells increases considerably. Lamina propriasubmucosa is much reduced. Tunica
muscularisconsists of usual inner circular (CL) and outer longitudinal layer (LL) of
smooth muscle fibers lined by the serosa (S) (Magnification with A: ×320 (H & E),
B: ×2500 (H & E)).
Histologically, these are no villi in large intestine and goblet cells rise to numerous. The
epithelium is simple columnar, and the lamina propria and submocosa is strongly reduced.
The muscular coat is developed for peristalsis activity. A single layer of mesothelium is
covered the outside of the intestine. Goblet cells contain large secretory vesicles that were
intensely PAS positive and reacted with AB pH 2.5 (Figure 6). In the rectum, like the large
intestine, the epithelium is simple columnar but the number of goblet cells increased. Lamina
propria submucosa becomes a very thin layer (Figure 7).
DISCUSSION
The pharynx of the N. kaiseri and N. microspilotus similar to other salamanders is richly
vascular and unmarked by any Eustachian tubes and passes undetectably into a wide
esophagus. The pharynx leads into the stomach without any sharp line of demarcation
(Francis, 1934). Studies conducted by Wonderly (1963) on digestive systems in several
species of North American salamanders showed that in Siren lacertian, Notophthalmus v.

viridescens, Desmognathus o. ochrophaeus, Plethodon g. glutinosus, P. c. cinereus, P. r.
richmondi and Gyrinophilus p. porphyriticus, there is a definite, external point of demarcation
between the esophagus and stomach. But this is absent in Cryptobranchus a. alleganiensis,
Necturus maculosus, Amphiuma tridactylum, Desmognathus quadramaculatus, and the genus
Ambystoma (except for a moderately definite constriction in one specimen of A.
jeffersonianum). In our study there is no clear demarcation line between esophagus and
stomach in N. kaiseri and N. microspilotus. In a histological study conducted on Triturus
carnifex (Liquori et al., 2005) it was found that the esophagus was lined by a columnar
ciliated epithelium with widespread mucous goblet cells. The mucosa appeared to be folded
and no esophageal glands were observed. Goblet cells contained large secretory vesicles that
were PAS-positive and reacted with AB at pH 2.5 (Liquori et al., 2005). But in N. kaiseri and
N. microspilotus the internal covering of esophagus is pseudostratified ciliated columnar with
large amount of goblet cells.
In N. kaiseri and N. microspilotus the stomach is divided into 3 parts, the cardia, the
fundus and the pylorus and each of them has its own microscopic characteristics. Also, the
epithelium of the stomach surface and of the lining of the crypts consists of a single layer of
high columnar cell. However, the apical portion of the cell in N. kaiseri is acidophil and
homogeneous while in N. microspilotus is foamy. Similar results have been obtained in other
species of Salamandra. The stomach of Triturus carnifex (Liquori et al., 2005), Bufo viridis
(Liquori et al., 2002) and Bombina variegata (Bani et al., 1992) appeared to be histologically
subdivided into a corpus, fundus and a wide pars pylorica. In these species the stomach lumen
waslined with a single layer of mucus-secreting cells. The mucosa was arranged in a few
longitudinal folds. The mucous neck cells were observed only in the oral fundus and were
PAS-positive, but they did not react with AB at pH 2.5 (Liquori et al., 2005). A definite and
readily observable pyloric sphincter occurs consistently in Cryptobranchus a. alleganiensis,
Necturus maculosus, Siren lacertina, and Amphiuma tridactylum which is the same of the
present study. But in the remaining species studied, very little pyloric constriction is evident,
and no appreciable thickening of the wall of the digestive tube at the junction of the stomach
and duodenum can be observed (Wonderly, 1936).
In N. kaiseri and N. microspilotus similar to many salamanders (Francis, 1934), the
duodenum is short and is sharply reflexed along the medial aspect of the stomach, but follows
a more transverse direction relative to the body than does that organ. It is indistinguishable
from the rest of the intestine except that it receives the pancreatic and bile ducts. The dorsal
pancreatic duct or rather the duct of the dorsal pancreas, enters the proximal end of the
duodenum some 2 mm or so from the pylorus in both species. The ventral pancreatic ducts,
which belong to the two ventral pancrease, discharge into the common bile duct which, in
turn, enters the distal end of the duodenum, some 4- 5 mm from the dorsal pancreatic duct. In
genus Salamandrina, the intestine is a coiled tube of a regular diameter, approximately equal
to, or slightly smaller than, that of the duodenum. Its length is about one-half that of the
whole gut, measured from the pharynx to the cloaca (Francis, 1934). The pattern of the
internal relief of the duodenum and intestine, considered as a whole, consists of a series of
sinuous longitudinal ridges. The ridges are thick proximally that is, at the duodenal end but
tend to become thinner and straighter towards the hinder end of the gut, until they are almost
knife-like. If the mucous epithelium is scraped or brushed off it is seen that the underlying
vascular network is also raised into ridges, and that the capillary loops follow the same wavy
outline. The network is much richer at the anterior end of the gut (Francis, 1934). In both N.
kaiseri and N. microspilotus the diameter of large intestine is larger that of the small intestine
and occupies approximately one third of the gut.
The findings of this study demonstrate that the morphological description of the digestive
tract of N. kaiseri and N. microspilotus are very similar and can be extended to the other
newts. Results obtained from current study are important for understanding the digestive
processes, underpinning not only physiological, pathological and Phylogenetic studies but
also the management and conservation, and also the preventive and therapeutic medicine of
these animals.
REFERENCES
Balouch, M. & kami, H. G. . (1995). Amphibians of Iran. Tehran University Publications,
177, 91-99.
Bani, G. L. F, Cecchi, R. (1992). Morphological observations on the glands of the oesophagus
and stomach of adult Rana esculenta and Bombina variegata. Ital. J. Embryo. 97, 75 – 87.
Cope, E. D. (1862). On Neurergus crocatus from Iran and Iraq, Proc. Acad. Natur. Sci . 1862,
343.
Ferri, S., & Medeiros, L. Q. (1976). Morphological study of the esophagus of Xenodon
merremii Wagler, 1924 (Ophidia). J. Herptol. 9 (3), 299-302.
Francis, E. T. B. (1934). The anatomy of the salamander. pp. 262 - 288.
Hammond, K. A., S Szewczak, J., & Krol E. (2001). Effects of altitude and temperature on
organ phenotypic plasticity along an altitudinal gradient. J. Exp. Biol. 20, 1991 – 2000.
Jorquera, B., Garrido, O., & Pugin, E. (1962). Comparative study of the digestive tract
development between Rhinoderma darwinii and R. rufum. J. Herptol. 16 (3), 204- 214.
Karasov, W.H., Pinshow, B., Starck, J. M., & Afik, D. 2004. Anatomical and histological
changes in the alimentary tract of migrating blackcaps (Sylvia atricapilla): a comparison
among fed, fasted, food-restricted, and refed birds. Physiol. Biochem. Zool. 77,
149 – 160.
Liquoria, G. E., Zizzaa, S., Mastrodonatoa, M., Scillitania, G., Calamitab, G., & Ferria, D.
(2005). Pepsinogen and H, K- ATPase mediate acid secretion in gastric glands of
Triturus carnifex (Amphibia, Caudata). Acta. Histochem. 107, 133- 141.
Pirsma, T., & Lindstrom, A. (1997). Rapid reversible changes in organ size as a component of
adaptative behaviour. Tren. Ecol. Evol. 12, 134 – 138.
Romao, M. F., Santos, A. L. Q., C. Lima, F., Desimone, S. S., Silva, J. M. M., Hirano, L. Q.,
Viera, L. G., & Pinto, J. G. S. (2011). Anatomical and topographical description of the
digestive system of Caiman crocodilus (Linnaeus 1758), Melanosuchus niger (Spix
1825) and Paleosuchus palpebrosus (Cuvier 1807). J. Morph. 29(1), 94-99.
Sanncar-bas, S., Kaptan, E., Sengezer, M. (2009). Glycoconjugate Histochemistry in the
Fundic Stomach and Small Intestine of the Frog (Rana ridibunda). IUFS J. Biol. 68(2),
93-104.
Secor, S. M. (2005). Evolutionary and cellular mechanisms regulating intestinal performance
of amphibians and reptiles. Integr. Comp. Biol. 45, 282 – 94.
Secor, S. M. (2001). Regulation of digestive performance: a proposed adaptive response.
Comp. Biochem. Physiol. 128, 565 – 577.
Sharifi, M. & Assadian, S. (2004). Distribution and conservation of Neurergus microspilotus.

Asia. Herpetol. Res. 10, 224-229.
Sharifi, M., Rastegar- Puyani, N., Akmali, V., & Assadian, S. (2008). On distribution and
conservation status of Neurergus kaiseri (caudata: salamandridae). Rus. J. Herpetol.
15(3), 169 – 172.
Sharifi, M., Papenfuse, T. Rastegar- Puyani, N., Assadian, S., &. Kuzmin, S. 2008. Neurergus
kaiseri. In: IUCN 2009. IUCN Red List of Threaten Species. Version 2009. 1.
http://www.iucnredlist.org. Downloaded on 22 October 2009.
Sinier, N. G & Yilgiz, I. (2000). The Ciliate Fauna in the Digestive System of Rana ridibunda
(Amphibia: Anura)-II Nyctotherus (Nyctotheridae: Heterotrichida). Turk. J. Zool. 24,
245-252.
Syarck, M. (1999). Structural flexibility of the gastro-intestinal tract of vertebrate’s
implications for evolutionary morphology. Zool. Anz. 238, 87– 101.
Williams, S. R., & Karasov, W.H.. (2001). Phenotypic flexibility in digestive system structure
and function in migratory birds and its ecological significance. Comp. Biochem. Physiol.
128, 579 – 593.
Wonderly, D. E. (1936). A comparative study of the cross anatomy of the digestive system of
some North American salamanders. Herpetol Soc. 4 (1/2), 31- 48.
ANATOMY AND HISTOLOGY OF THE DIGESTIVE

TRACT AND FEEDING HABITS OF THE NEOTROPICAL
FISH HYPOSTOMUS PUSARUM (STARKS, 1913)
(OSTEICHTHYES: LORICARIIDAE)
Emilly Kataline R. Pessoa1, Naisandra Bezerra da Silva2,

Naithirithi T. Chellappa3, Arrilton Araújo de Souza1
and Sathyabama Chellappa1
1
Programa de Pós-Graduação em Psicobiologia, Centro de Biociências,
Universidade Federal do Rio Grande do Norte (UFRN), Lagoa Nova,
Natal, Rio Grande do Norte, Brazil
2
Departamento de Morfologia, Centro de Biociências, Universidade Federal do Rio
Grande do Norte (UFRN), Lagoa Nova, Natal, Rio Grande do Norte, Brazil
3
Departamento de Oceanografia e Limnologia, Centro de Biociências,
Universidade Federal do Rio Grande do Norte (UFRN), Praia Mãe Luíza,
s/n, Natal, Rio Grande do Norte, Brazil
ABSTRACT
Hypostomus pusarum is a Neotropical fish with ecological and commercial
importance, and has a geographical distribution in almost all the hydrographic basins of
Central and South America. This study investigated the anatomy and histology of the
digestive tract besides the feeding habits of H. pusarum captured from a tropical reservoir
in Northeastern Brazil. The stomach contents were analyzed using the frequency of
occurrence method besides the dietary importance index. The digestive tract of H.
pusarum shows a small tubular ventral mouth with ability for suction, many small teeth,
and two barbells which help in selection of food. All four branchial arches are devoid of
visible gill rakers. The esophagus is in the form of a short tube, and the stomach has a U
shape, with the cardiac, fundic and pyloric regions. The intestine is very long and is
composed of double spiral loops which open out through the anus. The intestinal
coefficient is 10.8±0.7. The dietary importance index indicates that H. pusarum feeds
preferentially on organic matter in decomposition (88.7%) and filamentous microalgae
and diatoms (11.3%).
Keywords: Hypostomus pusarum, anatomy, histology, feeding habits

E-mail of the corresponding author: chellappa.sathyabama63@gmail.com
40 E. K. R. Pessoa, N. B. da Silva, Naithirithi T. Chellappa et al.
INTRODUCTION
The freshwater fish fauna of the Neotropical region is characterized by high species
richness which contributes to almost 31% of fish diversity of the planet (Reis et al., 2003).
Though morphology and histology of the fish digestive tract related to diet has been an
interesting topic of investigation, such information pertaining to the Neotropical freshwater
fish is still incipient (Albrecht et al., 2001; Canan et al., 2011; 2012; Montenegro et al., 2012).
Anatomical and histological characterization of the digestive tract coupled with analysis of
stomach contents, have aided in interpreting the dynamics of feeding, habitat occupation,
nutritional status and adaptations of the digestive tract in relation to the feeding habits of fish
(Wootton, 1999; Novakowski et al., 2004; Pessoa et al., 2013).
Morphological variations reflect the differential use of ecological resources, exhibiting a
relationship between morphological and ecological similarities (Norton et al., 1995; Pianka,
2000). Morphological characteristics indicate adjustments to the ecological niche dimension,
with a possibility of predicting the distribution of species in the environment or the
delimitation of trophic groups. As such, morphological analyses are used as interesting
measures in ecological studies (Hugueny; Pouilly, 1999; Pouilly et al., 2003).
Hypostomus is one of the genera of the family Loricariidae, with approximately 120
species, with a wide distribution in the freshwaters of Central and South America (Montoya
Burgos, 2003; Weber, 2003; Nascimento et al., 2011). Hypostomus species are encountered in
the hydrographic basins of Northeastern Brazil (Chellappa et al., 2009; Chellappa et al.,
2011). It is of commercial importance and is also used by the fishing communities as a food
supplement, due to the abundance of this species in freshwater ecosystems of the semiarid
region of Northeastern Brazil. The loricarideans generally play an important role in energy
recycling in Neotropical aquatic ecosystems (Delariva; Agostinho, 2001). This study
describes the feeding habits besides anatomical and histological characterization of the
digestive tract of H. pusarum.
Study Site and Fish Sampling
Fish sampling was carried out on a monthly basis from July, 2011 to June, 2012, in the
Marechal Dutra Reservoir, State of Rio Grande do Norte, located in the Caatinga ecoregion of
Northeastern Brazil (6°26’11’’S and 36°36’17’’W).
This reservoir was constructed on River Acauã in the hydrographic basin of River
Piranhas-Assu, which is the main hydrographic basin of Rio Grande do Norte, covering an
area of 44,000 km² (Chellappa et al., 2009; Chellappa et al., 2011). Fish samples were
captured with the help of local fishermen, who used gill nets with different mesh sizes
(varying from 6 to 12 cm) with a 12 h fishing effort per month.
Anatomy and Histology of the Digestive Tract and Feeding Habits … 41
Measurements
Fish captured were numbered, measured, weighed and samples of fish were used for
morphometric measurements to confirm the taxonomical identification of H. malabaricus.
Measures of total length (Lt1cm), total body mass (Wt1 g) and stomach weight
(Wg0.1mg) were recorded. In this study 118 individuals (51 males and 67 females) of the
study species were used, with total body length varying from 22.5 to 30.0 cm, (26.6±1.8) cm
for males, and from 22.0 to 29.0 cm (25.8±1.7) for females. The body weight varied from
101.0 to 332.0 g (194.7±42.9) for males, and from 102.0 to 290.5 g (189.9±39.5) for females.
Histological Procedures
The digestive tract was removed from the coelomic cavity, using 20 adult samples of H.
pusarum for the characterization of the morphology of the digestive tract. The position of the
mouth, type of dentition, presence or absence of gills rakers, esophagus, stomach, presence or
absence of pyloric caeca and intestine were examined for the morphological description.
For histological descriptions, the digestive tracts of 20 adult individuals of H. pusarum,
were separated and fixed in 10% formaldehyde. After 24 hours of fixation, the digestive tracts
were submitted to microtomy to obtain fragments of the digestive organs: esophagus, stomach
(cardiac, fundic, and pyloric regions) and the intestine. The fragments were submitted to
routine histological techniques (dehydration, diaphanization and paraffin embedding) and
micro sectioned at 5 μm. Samples were stained with haematoxylin-eosin (HE) and periodic
acid Schiff (PAS) following routine procedures (Smith, 1996). An Olympus microscope
CH30 coupled to an Olympus digital camera PM–10 AK was used for the slide
microphotography procedure, and the images were processed using Nikon ACT-1 software.
The Intestinal Coefficient
The intestinal coefficient (IC = length of the intestine/total length of fish) was calculated
(Bértin, 1958). Intestinal lengths (cm) and total body lengths (cm) were obtained from 118
specimens of pooled samples of males and females of H. pusarum.
Stomach Contents Analyses and Statistical Analyses
The stomach contents of 118 specimens of H. pusarum were examined under a

stereoscopic microscope Taimim TM800, up to the smallest taxonomic level possible. The
frequencies of occurrence (FO) were calculated (Hynes, 1950; Hyslop, 1980). Index of
Alimentary Importance (IAI) was applied on the values of frequency of occurrence to provide
the food items that contribute most to the diet of the species (Kawakami; Vazzoler, 1980).
The scanning electronic microscope was used for the detailed identification of the microalgae
and diatoms encountered in the stomach contents of H. pusarum (Rosecchi; Nouaze, 1987;
Chellappa; Costa, 2003; Wehr; Sheath, 2003). The food preference was analyzed using the
chi-square (χ²) test, in order to statistically identify the significant differences between the
most consumed items and the others.
RESULTS
Gross Anatomy
H. pusarum has a small ventrally situated mouth with contractible lips suitable for
suction, which helps the fish to adhere to a variety of substrates. The fish possess many small
teeth and two barbells which help in selection of food. The presence of many small
circumferential projections was observed on the lips and lip borders. All four branchial arches
are devoid of visible gill rakers (Figura 1A-C).
Figure 1. (A) Frontal view and (B) lateral view of the head of Hypostomus pusarum,
showing the teeth and barbells (arrows); (C) First left branchial arch showing only
branchial filaments.
The esophagus is in the form of a short tube, delimited anteriorly by the branchial arch
and posteriorly by the stomach. The stomach has a U shape, with the three distinct cardiac,
fundic and pyloric regions. The cardiac region is tubular and is located immediately after the
esophagus. The fundic region is long which begins immediately after the cardiac region and is
followed by the tubular pyloric region, which in turn extends up to the beginning of the
intestine (Figure 2A).
The intestine of H. pusarum is very long and is organized in the form of double spiral
loops, which are linked by conjunctive and adipose tissues. The other digestive organs could
be clearly seen only after the intestinal loops are separated out. The intestinal length
registered ranged from 190.0 to 460.0 (330.0 ± 75.0) cm. The pyloric caeca was absent
(Figure 2A-B).
Figure 2. (A) View of the structures of the digestive tract and (B) coelomic cavity of H.
pusarum: (a) esophagus, (b) stomach, (b1) cardiac stomach, (b2) fundic stomach, (b3)
pyloric stomach, (c) intestine; (d) liver and (e) ovary.
Histology of the Digestive Tract
In general, the histological analyses evidenced the presence of four distinct tunicae
composing the wall: (1) mucosa (lining the lumen of the digestive tube and consisting of an
inner epithelium), a middle lamina propria (cellular connective tissue) and outer muscularis
mucosae; (2) submucosa (with blood vessels, lymphatic tissue and nerve plexi); (3)
muscularis, an inner circular and an outer longitudinal layer; (4) serosa (composed of
connective tissue surrounded by simple squamous peritoneal epithelium).
Esophagus and Stomach
The tunica mucosa of the esophagus presents small longitudinal folds, covered by a
pseudostratified mucosecretary epithelium (Figure 3A). The mucous secretory cells were
predominant in the epithelium and were distributed in a homogenous manner along the entire
epithelium (Figure 3B). The lamina propria is formed by cellular connective tissue. The
muscular layer is composed of two layers of smooth muscle cells, an inner circular and an
outer longitudinal coat.
Figure 3. Esophagus region of H. pusarum. (A): longitudinal folds (arrow);

(B) pseudostratified mucosecretary epithelium (a); lamina propria without glands (b);
circular muscular layer (c); (C) Cardiac region of stomach: stomach folds (arrow);
internal circular muscular layer (a); external longitudinal muscular layer (b). (D) fundic
region of the stomach: gastric glands (arrow); muscular mucosa (*); aglandular
submucosa with blood vessels (a); (E) pyloric region of the stomach: blood vessel
(arrow); submucosa (a); (F) intestine: intestinal villi (a); brush border with enterocytes
(b); goblet cell (arrow).
The diameter of the esophagus becomes reduced when it approaches the stomach. The
insertion of the pneumatic duct in the dorsal region, on the left side of the esophagus, denotes
the beginning of the stomach. The transition of the esophagus to the stomach is clearly
marked by the substitution of esophageal epithelium by simple cylindrical epithelium of the
stomach and the appearance of gastric glands.
The stomach has three distinct regions, cardiac, fundic and pyloric. The lamina propria
consists of tubular gastric glands, probably constituted by enzyme and acid secretary cells,
predominant in the fundic region and in the pyloric region they become scarce. The transition
between the lamina propria and tunica submucosa is marked by the presence of muscularis
mucosa. In the stomach submucosa there are many blood vessels and is glandless. The tunica
muscular is constituted by two layers of smooth muscles, an inner circular and outer
longitudinal layer. The last layer is the serosa which is similar in all three regions of the
stomach, and is composted of connective tissue surrounded by simple peritoneal epithelium
(Figure 3C-E).
Figure 4. Microalgae encountered in the stomach contents of H. pusarum:

Klebsormidium sp. (a); Geminella sp. (b); Spirulina sp. (c); Ceratium sp. (d); Oscillatoria
sp. (e-h); Spirogyra sp. (i); Oedogonium sp. (j); Anabaena sp (k-l); Planktothrix sp. (m);
(n); Microcystis sp. (o); Aphanocapsa sp. (p).
Intestine
The intestinal mucosa presents finger-like processes, the villi, which consists of
absorptive epithelial covering, with mucus secreting goblet cells dispersed between the
enterocytes (absorptive cells). The cytoplasm of the goblet cells react strongly to PAS,
however, the enterocytes do not present any alteration in their color (Figure 3F).
The lamina propria has no glands, is of smooth conjunctive tissue, richly supplied with
blood vessels and in some places shows lymphatic accumulation. The intestine does not have
a submucosa, since there was no muscular mucosa. The tunica muscular is constituted by two
layers of smooth muscles, an inner circular and outer longitudinal, and between these two
layers the mioenteric plexus is well developed.
Feeding Habits
The Index of Alimentary Importance (IAI) shows that H. pusarum feeds on decomposing
organic material (88.7%), filamentous micro algae and diatoms (11.3%), characterizing it as a
detritivorous/herbivorous organism. Among the phytoplankton taxa encountered in the
stomach contents of H. pusarum, eleven were prominent: Klebsormidium sp., Geminella sp.,
Spirulina sp., Ceratium sp., Oscillatoria sp., Spirogyra sp., Oedogonium sp., Anabaena sp.,
Planktothrix sp., Microcystis sp. and Aphanocapsa sp (Figure 4).
DISCUSSION
The position of the mouth, type of dentition and the nature of the gill rakers are all related
to the feeding habits of fish (Motta, 1984; Albrecht et al., 2001; Pessoa et al., 2013). Fish with
detritivorous feeding habits present adaptive mechanisms for this type of diet (Araújo-Lima et
al., 1995). The scraping behavior of the substrate is facilitated by their morphological
characteristics, which includes the cup-shaped mouth and teeth adapted for scraping. H.
pusarum shows morphological characteristics similar to other detritivorous fish, which
indicates its food habits.
The esophagus of teleost fish are generally in the form of a short tube, wide and straight,
with great elasticity, adapted to the various diets and linking the oral cavity and pharynx to
the stomach. The esophagus of H. pusarum is short and possesses all the characteristic layers
of the digestive tract of other vertebrates: mucosa, submucosa, muscularis and serosa
(Albrecht et al., 2001; Diaz et al., 2003; Genten et al., 2009). The organization of esophageal
folds is probably related to the dispensability of the walls of this organ, adapting it for
conduction of food to the stomach (Montenegro et al., 2012). The differentiation in color and
transparency in the transitional region of esophagus to stomach was also observed in Hoplias
malabaricus (Pessoa et al., 2013).
In H. pusarum and in most other fish species, the epithelial lining of the stomach is
composed by simple columnar epithelium. The muscular layer consists mainly of smooth
muscle fibers, distinguishing an inner circular and an outer longitudinal layer (Bértin, 1958;
Liem et al., 2001). Another member of the family Loricariidae, Pterygoplichthys anisitsi,
utilizes the stomach as a respiratory organ (Oliveira et al., 2001; Cruz et al., 2009).
The intestine of H. pusarum is very long and has the form of double spiral loops,
occupying almost 2/3 of the coelomic cavity. This was also observed for three other fish
species of rio Paraná, Brazil, which is related to the time that is necessary for digestion of
decomposing organic matter (Fugi; Hahn, 1991). The pattern of the intestinal mucosal folds is
related to the function of absorption of nutrients, since it increases the area, which
compensates the low nutritive value of the ingested food. It has been observed that lower the
nutritive value of the ingested food, more elaborate are the intestinal folds.
The number of empty stomachs observed during this study could be due to the
detritivorous food habit of this fish. The use of abundantly available food, such as plant
material, which is difficult to digest and is of low nutritional value, is possibly related to the
presence of the long and looped intestine (Delariva; Agostinho, 2001). The microalgae
identified in the stomach contents of H. pusarum in this study have been also registered in the
reservoir waters of Marechal Dutra and other reservoirs in the semi arid region of Brazil,
which confirms the availability of this resource in the study area (Chellappa; Costa, 2003;
Chellappa et al., 2005; 2006; 2008).
CONCLUSION
H. pusarum has a cup shaped mouth adapted for the suction and the teeth are adapted for
scraping. The arrangement of the digestive organs is directly related to the shape of the
peritoneal cavity and the body shape. The presence of very long intestine is related to its food
habits. The anatomy and histology of the digestive tract of H. pusarum indicates its
detritivorous/herbivorous food habits, which was confirmed by the stomach content analyses
and by the anatomical characteristics of the digestive tract.
ACKNOWLEDGMENTS
Thanks are due to the National Council for Scientific and Technological Development of
the Ministry of Science and Technology of Brazil (CNPq/ MCT) for the Research grants
offered to the authors.
REFERENCES
Albrecht, M. P., Ferreira, M. F. N., Caramaschi, E. P. 2001. Anatomical features and
histology of the digestive tract of two related neotropical omnivorous fishes
(Characiformes; Anostomidae). Journal of Fish Biology, 58: 419-430.
Araújo-Lima, C. A. R. M., Agostinho, A. A., Fabré, N. N. 1995. Trophic aspects of fish
communities in Brazilian rivers and reservoirs. In: Tundisi, C. E. M.; Bicudo, C. E. M.;
Matsumura-Tundisi, T. (Eds.). Limnology in Brazil. Rio de Janeiro: ABC/SBL, p. 105-
136.
Bértin, L. 1958. Appareil digestif. In: Grassé, P.P. (Ed.). Traité de zoologie. Paris: Masson,
13: 1249-1301.
Canan, B., Pessoa, E. K. R., Volpato, G. L., Araujo, A., Chellappa, S. 2011. Feeding and
reproductive dynamics of the damselfish, Stegastes fuscus in the coastal reefs of
northeastern Brazil. Animal Biology Journal, 2: 113-126.
Canan, B., Nascimento, W. S., Silva, N. B., Chellappa, S. Morphohistology of the digestive
tract of the damsel fish, Stegastes fuscus (Osteichthyes: Pomacentridae). The Scientific
World Journal, 2012: 1-9. 2012.
Chellappa, N. T., Costa, M. A. M. 2003. Dominant and co-existing species of Cyanobacteria
from a semi-arid reservoir of Northeast Brazil. Acta Oecologica (Montrouge). Paris,
France, 24: 3-10.
Chellappa, N. T., Chellappa, S. 2005. Ecology of the Freshwater Phytoplankton and fish
commuinities from a semi-arid reservoir in Northeastern Brazil. Journal of Biology. Foz
de Figueiro, Portugal.1 (1): 14-14.
Chellappa, N. T., Chellappa, T., Lima, A. K. A., Borba, J. L.M., Souza, P. V. V., Chellappa,
S. 2006. Ecology of freshwater phytoplankton assemblages from a tropical reservoir of
Northeastern Brazil. International Journal of Lakes and Rivers. 1 (1): 61-73.
Chellappa, N. T., Borba, J. L. M., Rocha, O. 2008. Phytoplankton community structure and
physical-chemical characteristics of water in the public reservoir of Cruzeta, RN, Brazil.
Brazilian Journal of Biology. 68: 477-494.
Chellappa, S., Bueno, R. M. X., Chellappa, T., Chellappa, N. T., Val, V. M. F. A. 2009.
Reproductive seasonality of the fish fauna and limnoecology of semi-arid Brazilian
reservoirs. Limnologica, 39: 325- 329.
Chellappa, S., Nascimento, W. S., Chellappa, T., Chellappa, N. T. 2011. Impacts of anthropic
factors on native freshwater fish in Brazilian semiarid region. In: Sean P. Dempsey (Ed.).
Fish Ecology. New York, USA: Nova Science Publishers, p. 115-130.
Cruz, A. L., Pedretti, A. C. E., Fernandes, M. N. 2009. Stereological estimation of the surface
area and oxygen diffusing capacity of the respiratory stomach of the air-breathing
armored catfish Pterygoplichthys anisitsi (Teleostei: Loricariidae). Journal of
Morphology, 270: 601- 614.
Delariva, R. L., Agostinho, A. A. 2001. Relationship between morphology and diets of
neotropical loricariids. Journal of Fish Biology, 58: 832-847.
Diaz, A. O., García, A. M., Devincenti, C. V., Goldemberg, A. L. 2003. Morphological and
histochemical characterization of the mucosa of the digestive tract in Engraulis anchoita.
Anatomia, Histologia, Embryologia: Journal of Veterinary Medicine, Série C 32: 341-
346.
Genten, F., Terwinghe, E., Danguy, A. 2009. Atlas of Fish Histology. Enfield, USA: Science
Publishers.
Hugueny, B., Pouilly, M. 1999. Morphological correlates of diet in an assemblage of West
African freshwater fishes. Journal of Fish Biology, 54: 1310-1325.
Hynes, H. B. N. 1950. The food of freshwater sticklebacks (Gasterosteus aculeatus and
Pygosteus pungitius) with a review of methods used in studies of the food of fishes.
Journal of Animal Ecology, 19: 36-58.
Hyslop. E. J. 1980. Stomach contents analysis: a review of methods and their application.
Journal of Fish Biology, 17 (4): 411-429.
Kawakami, E., Vazzoler, G. 1980. Método gráfico e estimativa de índice alimentar aplicado
ao estudo de alimentação de peixes. Boletim do Instituto Oceanográfico, 29: 205-207.
Liem, K. F., Bemis, W. E., Walker-Jr., W. F., Grande, L. 2001. Metabolism and reproduction.
In: Functional Anatomy of the Vertebrates. An evolutionary perspective. Thomson (Ed.),
3rd ed., 703p.
Montenegro, L. A., Silva, N. B., Nascimento, W. S., Chellappa, S. 2012. Anatomy and
Histology of the digestive tract and feeding habits of the marbled swamp eel Synbranchus
marmoratus. Animal Biology Journal, 3 (3): 127-143.
Montoya-Burgos, J. I. 2003. Historical biogeography of the catfish genus Hypostomus
(Siluriformes: Loricariidae), with implications on the diversification of Neotropical
ichthyofauna. Molecular Ecology, 12: 1855-1867.
Motta, P. J. 1984. Mechanics and functions of jaw protrusion in Teleost fishes: a review.
Copeia, 1: 1-18.
Nascimento, W. S., Araújo, A.S., Gurgel, L. L., Yamamoto, M. E., Chellappa, N. T., Rosa, R.
S., Chellappa, S. 2011. Endemic fish communities and environmental variables of the
Piranhas-Assu hydrographic basin in the Brazilian Caatinga Ecoregion. Animal Biology
Journal, 2 (3): 97-112.
Norton, S. F., Luczkovich, J. L., Motta, P. J. 1995. The role of eco- morphological studies in
the comparative biology of fishes. Environmental Biology Fish, 44: 287-304.
Novakowski, G. C., Fugi, R., Hahn, N. S. 2004. Diet and dental development of three species
of Roeboides (Characiformes: Characidae). Neotropical Ichthyology, 2: 157-162.
Oliveira, C., Taboga, S. R., Smarra, A. L. S., Bonilla-Rodriguez, G. O. 2001. Microscopical
aspects of acessory air breathing through a modified stomach in the armoured catfish
Liposarcus anisitsi (Siluriformes, Loricariidae). Cytobios, 105: 153- 162.
Pessoa, E. K. R., Silva, N. B., Araújo, A., Chellappa, S. 2012. Anatomy and Histology of the
Digestive Tract and Feeding Habits of Hoplias malabaricus (Bloch, 1794) (Osteichthyes:
Erythrinidae). Animal Biology Journal, 3 (4): 145- 158.
Pianka, E. R. 2000. Evolutionary Ecology. Benjamin-Cummings, Addison-Wesley-Longman:
San Francisco.
Pouilly, M., Lino, F., Bretenoux, J. G., Rosales, C. 2003. Dietary-morphological relationships
in a fish assemblage of the Bolivian Amazonian floodplain. Journal of Fish Biology, 62
(5): 1137-1158.
Reis, R. E., Kullander, S. O., Ferraris-Jr., C. J. 2003. Check list of the freshwater fishes of
South and Central America. EDIPUCRS, Porto Alegre, p. 729.
Rosecchi, E., Nouaze, Y. 1987. Comparaison de cinq índices alimentaires utilisés dans
I’analyse des contenus stomacaus. Revue des Travaux de l'Institut des Peches Maritimes,
49 (3-4): 111-123.
Smith, M. P., Sansom, I. J., Repetski, J. E. 1996. Histology of the first fish. Nature, 380: 702-
704.
Weber, C. 2003. Subfamily Hypostominae. In: Reis, R. E., Kullander, S. O., Ferraris Jr. C. J.
(Eds.). Check List of the Freshwater Fishes of South and Central America. Porto Alegre,
Edipucrs, p. 729.
Wehr. J. D., Sheath, R. G. 2003. Freshwater algae of North-America: Ecology and
Classification. Academic Press. London.
Wootton, R.J. 1999. Ecology of teleost fish. The Netherlands: Kluwer Academic Publishers,
p. 386.
LABORATORY OBSERVATIONS ON BIOLOGY

OF THE TAWNY GARDEN SLUG LIMAX FLAVUS
(LINNAEUS) (LIMACIDAE: MOLLUSCA)
M. I. Mohamed and Reham F. Ali

Department of Zoology and Agricultural Nematology Faculty of Agriculture,
Cairo University; Giza, Egypt
ABSTRACT
Biological aspects of the terrestrial yellow garden slug (Tawny Garden Slug) Limax
flavus (Linnaeus) including ovipostion, hatching, generation period, life span as well as
growth parameters were studied under laboratory conditions. Results revealed that mating
is not essential for this species to lay eggs, while self-fertilization is the normal breeding
system. The oviposition period averaged 62.4 ± 30.9 days, during which the individual
laid an averaged of 41.1 ± 20.4 eggs. The incubation period averaged 21.0 ± 2,4 days,
while the generation period (from egg to egg) lasted 152,8 ± 18.8 days in average. On the
other hand, the maximum life span durated an average of 235.2 ± 27.5 days.
Growth parameters, depending on the increase and decrease of the slug body
weights, increased from hatching until the seventh month and reached its maximum
weight averaging 3.51 ± 0.86 g. The maximum change rate in weight was in the first
month reached to 78.8 ± 17.2 %. The change rate in weight decreased thereafter due to
the climatic conditions.
Keywords: Limax flavus, self-fertilization, oviposition, generation period, growth parameters
INTRODUCTION
Limax flavus becomes very abundant around houses and gardens. It becomes serious
agricultural pests (Branson, 1980). Terrestrial molluscs are major consumers and
decomposers in natural, agricultural and horticultural communities (Godan, 1983).
Crawley (1983) mentioned that herbivory is one of the dominant biological mechanisms
organizing natural habitats of land slugs. Rollo (1983) mentioned that Limax maximus is
general herbivores that eat wide variety of leaf litter, green plants and fungus. Barker and
McGhie (1984) recorded that Limax maximus is not gregarious species and is found most
readily in gardens, damp hedgerows, shelterbelts, and in waste areas such as roadsides. The
authors added that this species is strictly nocturnal, hiding during the day beneath stones,
52 M. I. Mohamed and Reham F. Ali
grounded timber, deep leaf litter, and other obscure damp places. Individuals return
repeatedly to the same daytime resting site.
Wiktor (1989) stated that slugs of Limacoidea and Zonitoidea usually prefer humid or
moderately humid habitats, (e.g., meadows, neighborhood of water reservoirs, ditches,
gardens, parks as well as cultivated fields sheltered by thick vegetation). It often hides under
stones, wood pieces, in different chinks, on compost heaps, and under rubbish. Alford (1995)
mentioned that slugs are often important pests in gardens and nurseries, attacking seedlings.
These pests cause severe damage to young shoots, foliage, and flowers.
Kerney (1999) mentioned that Limax flavus lives in crevices and under rubbish in
gardens, cellars, outhouses and farm yards; in cities, old stone-floored sculleries and damp
basements. Occasionally it is found in woods. It is strongly nocturnal and a vigorous climber.
Limax flavus is originally Mediterranean but widely spread by man. Barker (1999) recorded
that, in New Zealand L. flavus has been observed only in habitats closely associated with
human dwellings, namely gardens, crevices of walls or wood stacks, on damp carpets and
other floor coverings, and in cellars and outhouses. The author also added that L. flavus feed
on decaying vegetable matter, fungus and lichens. Bohan et al. (2000) stated that terrestrial
gastropod molluscs are generalist herbivores with a broad range of acceptable foods. They
can influence the species composition of plant communities, as well as causing considerable
crop damage. Capinera (2001) mentioned that slugs eat plant tissues, decaying organic matter
and occasionally animal tissues. Yildirim (2004) said that Limacus flavus exist near human
settlements, woods, gardens, parks, and vineyards. The land slugs live on mostly omnivorous
or herbivores diets and certain species are considered serious agricultural pests. Abbes et al.
(2010) found that different slug species in north and northwestern Tunisia lived under damp
calcareous rocks, in open habitats of various kinds including grasslands.
In Egypt, the land slugs are becoming a serious pest during the last two decades, where
they attack various types of plants. In this study, it was found that Limax flavus (Linnaeus)
was fed on lettuce leaves (Lactuca sativa L.), roots of carrot (Daucus carota L.), ripe fruits of
Tomato (Solanum lycopersicum L.), seed stalks and leaves of onion (Allium cepa L.) fruits of
strawberry (Fragaria ananassa Duchesne), leaves and fruits of broccoli (Brassica deracea
L.), chard leaves (Beta cicla L.), parsley leaves (Petroselinum crispum Mill.), broad bean
leaves and green pods (Vicia faba L.), Egyptian clover leaves (Trifolium alexandrinum L.),
stalks of maize (Zea mays L.), leaves of mulberry (Morus alba L.), leaves of grape (Vitis
vinifera L.), leaves of Ficus (Ficus nitida L.), leaves of pothos (Epipremnum auream L.), and
leaves of field bindweed (Convolvulus arvensis L.) that offer at the main sources of food.
Thus, the main objective of this investigation is to spot more light upon the biological
aspects of the dominant land slug Limax flavus under laboratory conditions.

Fifty adult individuals of the tawny garden slug Limax flavus (Linnaeus) were collected
by hand during February 2008, under different types of ornamental plants such as aluminium
plant (Pilea cadierier L.), Parlour palm (Chamaedorea elegans L.), rose-scented pelargonium
(Pelargonium gravealens L'Heritier), night blooming cestrum (Cestrum noctumum L.),
Peppermint (Mentha piperita L.), garden geranium (Pelargonium hortorum Bailey), wild
wormwood (Artemisia vulgaris L.), river spiderwort (Tradescantia fluminensis Vell.), velvet
Laboratory Observations on Biology of the Tawny Garden Slug Limax flavus … 53
centaurea (Centaurea cineraria L.), ficus trees (Ficus nitida L.) and Ficus benjamina L. from
some nurseries situated in Cairo governorate. The individuals were kept in plastic boxes (27 x
27 x 25 cm) with fresh lettuce leaves (Lactuca sativa L.), which offered as a sole food source.
Boxes were covered with muslin cloth fixed with rubber band to prevent slugs from escaping
and kept under laboratory conditions then transferred to the laboratory. The soil of each box
was searched twice daily for batches or clutches (egg-masses) and remoistened as required.
The slugs were supplied with fresh lettuce leaves and the remained leaves were removed. The
newly deposited batches were left in the same box and time of oviposition was recorded,
while slug individuals were only transferred to new prepared box. The batches were observed
twice daily till hatching to determine the incubation period and hatchability.
The juveniles or off-springs (new hatch slugs) were placed singly in plastic cups (10 x 6)
cm filled with moist clay soil to depth of five centimeters with disks of lettuce leaves and
each covered with muslin cloth with rubber band to prevent escaping. Fresh lettuce leaves
were provided and moisture was added daily until juveniles reached maturity and complete
their life span.
Juveniles of L. flavus were weighed monthly from zero time (hatching time) till maturity
using digital balance to determine their growth rate, which calculated according to the
formula:
Growth rate = WF – WI x 100

WF
where WI & WF are the initial and final weights of slug individuals for given periods.
The regression analysis was carried out for weight and growth rate over age according to
the model:
Y= α + ß X
where Y is the dependent variable (slug weight or growth rate)
α is the intercept
ß is the slope of regression of Y over X
X is the age in months.
It was used also to explain the increase and the decrease of weight by month for slugs
(SAS, 1985).
RESULTS
Incubation Period and Hatchability
Forty-six slugs simultaneously deposited batches, with a range of 2 – 20 eggs for each,
were observed twice daily till hatching to determine its incubation period and hatchability.
The incubation period ranged from 18 to 25 days with an average of 21.0 ± 2.4 days under
laboratory conditions of 20.5 ± 1.7°C and 61.4 ± 7.8% R.H. (Table 1). The hatchability
ranged from 20 to 100% with an average of 74.6 ± 25.3%. It was noticed that the egg mass
hatching may completed during one day or within few days ranged from two to six days.
Life Cycle and Generation Period
After hatching, 46 juveniles were separated each in prepared plastic cups as described
before and observed daily till maturity and oviposition.
It was difficult to differentiate between immature and mature slugs by the external shape
or size. Thus, the life cycle from hatching till juveniles reach maturity including the pre-
oviposition period ranged from 110 to 165 days with an average of 130.9 ± 19.5 days (n=11)
at an average of 22.1 ± 3.0°C and 62.1 ± 7.5% R.H. While, The generation period (from egg
to egg) lasted an average of 152.8 ± 18.8 days with the range of 130 – 185 days under the
same mentioned laboratory conditions (Table 1). It was observed that the mortality ratio was
higher during juvenile period (about 46.8%) than in adult stage.
Oviposition
Individuals of L. flavus are hermaphrodites, each one possessing both male and female
sexual organs, thus self-fertilization is dominant occur in this species. Under laboratory
conditions of 20.6 ± 1.7°C and 61.3 ± 7.9% R.H., the oviposition period (from first to last
deposited egg) lasted an average of 62.4 ± 30.9 days (Table 1) (n=11) during which the slug
individual laid an average of 4.8 ± 2.0 batches each with an average size of 8.3 ± 4.6 eggs.
Table 1. Duration in days of different Limax flavus stages under laboratory conditions
oviposition period
Incubation period
Post-oviposition
Juveniles + pre-
Oviposition
Generation
Life span
Period
Average 21.0 ± 2.4 130.9±19.5 152.8±18.8 62.4±30.9 20.9±10.3 235.2±27.5
Range 18 – 25 110 – 165 130 – 185 1 – 92 6 – 32 184 – 277
Days elapsed between deposited batch and another averaged 11.4 ± 7.7 days (Table 2).
On the other hand, the total number of deposited eggs per individual averaged 41.1 ± 20.42
eggs during its oviposition period under laboratory conditions (Table 2). The eggs of L. flavus
are oval, transparent, shiny, and translucent, with a dimension averaged 6.4 ± 0.9 x 4.2 ± 0.4
mm per egg with range 5- 8.1 mm for egg length and from 3.5 – 5 mm for egg width (n = 15)
(Figure 1).
Table 2. Fecundity of Limax flavus under laboratory conditions
Total No. of deposited

Period between each
No. of batches / slug
No. of eggs / batch
No. of deposited
batches / day
eggs / slug
Items
batch
Average 1 8.3±4.6 4.8±1.99 11.43±7.66 41.1±20.42
Range 1 2–20 1–8 1-37 7-70
Post-Oviposition and Life Span Period
Post-oviposition period was estimated from last laid egg till mortality. It durated an
average of 20.9 ± 10.3 days, while the life span from hatching till death averaged 235.2 ±
27.5 days under laboratory conditions of 22.2 ± 2.9°C and 61.4 ± 8.2% R.H. (Table 1).
Figure 1. Eggs of the land slug Limax flavus.
Growth Parameters
The averages of slug weights and growth rates over age are presented in Table (3) and
figure (2).
The weight of the land slug L. flavus increased from hatching until the seventh month and
reached its maximum weight averaging 3.51 ± 0.86 g with a range of 1.69 – 5.01 g. (Table 3).
The maximum change rate in weight was recorded in the first month (78.84 ± 17.2%) and
ranged 33.33 – 95.83%. (Figure 3)
Weight = 1.03 + 0.2 * (age, month)
A significant (P ≤ 0.05) increase in weight over age (month) of 0.20 ± 0.07 g was found
in the slug (Figure 2). While the change rate in weight was highly significant (74.72 ± 8.8 %)
(Figure 3) during the first month and highly significantly decreased by -9.07 ± 1.19% every
month according to this formula:
Weight = 74.72 *** - 9.07 *** (age, month)
It has been noticed that the change rate in weight significantly increased in the first
month, and decreased thereafter due to the climatic conditions. (Table 3) The correlation
coefficients between slug weight and growth rate were in general significant and moderate to
high (Figure 4 and 5).
Figure (4) shows the regression of slug weight over age, where the slug weight at
hatching time (zero time) was not significantly estimated to be 1.03 g, but significantly
increased by 0.02 g every month.
The results indicated that the slugs had increased in weight during the activity period for
up to 7 months of age. The maximum growth rate of the slugs was 78.9 ± 17.2 % by the end
of first month and then retarded with age. Figure (5) presents a significant (P ≤ 0.0001)
regression slope of weight rate over age, where the growth retardation was by -9.07% every
month.
It is obvious that the slugs had significantly grown fast during the first month of age and
the reduction in growth rate thereafter denoted that the increase in slug weight every month
was in decreasing rate.
Table 3. Weight and weight rate of the land slug Limax flavus over age (month)
Weight Weight rate

Age
Mean ± SD Mean ± SD
Hatch time 0.042 ± 0.03 --
1st month 0.25 ± 0.204 78.9 ± 17.2
2nd month 0.76 ± 0.31 65.2 ± 17.3
3rd month 1.61 ± 0.52 51.3 ± 15.9
4th month 2.62 ± 0.65 37.8 ± 14.3
5th month 3.24 ± 0.97 17.6 ± 12.7
6th month 3.28 ± 0.78 1.9 ± 17.7
7th month 3.51 ± 0.86 4.01 ± 19.7
8th month 3.33 ± 0.79 -6.01 ± 14.2
9th month 3.5 ± 0.72 5.4 ± 8.2
10th month 2.54 ± 0.55 -38.04 ± 9.8
11th month 2.23 ± 0.61 -16.1 ± 13.8
12th month 2.03 ± 0.61 -12.6 ± 18.2
Figure 2. Weight of land slug Limax flavus over age (month).
Figure 3. Weight rate of land slug Limax flavus over age (month).
***P≤0.0001
Figure 4. Regression analysis weight of land slug Limax flavus over age (month).
***P≤0.0001
Figure 5. Regression analysis weight rate of land slug Limax flavus over age (month).
DISCUSSION
Carrick (1938) stated that slugs are sexually mature before they are full grown.
According to Stephenson (1968), slugs are hermaphrodite and protandorus, that is to say, they
function as males when first sexually mature and as females later. Cross-fertilization is usual,
though self-fertilization has been seen in laboratory cultures. McCracker & Selander (1980)
reported that many species of terrestrial slugs of several families, including some of the more
ubiquitous and familiar forms are monogenic or nearly so, apparently as a consequence of
frequent or obligatory self- fertilization. They also reported that many slug species have
different breeding systems in other regions. Capinera (2001) noticed that slugs are
hermaphrodites, each individual possessing both male and female sexual organs.
Nevertheless, self-fertilization is rare, and slugs normally pair for copulation.
In this study, the laboratory studies showed that copulation in the terrestrial slug L. flavus
is not essential for oviposition, where self-fertilization was the normal breeding system in all
separated individuals.
The total number of eggs laid by slugs differs from species to species and more eggs are
laid in laboratory cultures than in the field (Carrick, 1938). Frömming (1954) mentioned from
observations made on laboratory cultures. Limax maximus L., 676 – 834 eggs, L. marginata
(Müller) 105 – 132 eggs, Agriolimax reticulatus approximately 300 eggs and Arion hortensis
105 – 203 eggs. Branson (1980) recorded that D. reticulatum deposited from 60 to 75 eggs.
The author added that the egg size of Limax maximus, Lehmannia marginata (Müller),
Deroceras laeve, and Arion intermedius was 6 x 4.5 mm, 5 x 4 mm, 2.8 mm, and 3.1 x 2.5
mm respectively.
Rollo (1983) mentioned that the terrestrial slug Limax maximus produced about half the
number of batches in the monoculture (2.02 batches / slug) compared to slugs caged with
Ariolimax columbianus (4.0 batches / slug) or Arion ater (3.57 batches / slug). While in the
laboratory, only one individual of L. maximus produced five batches of eggs in one season.
The author also added that the mean number of eggs / batch significantly differed among
species, hence in A. ater averaged 47.9 eggs / batch, L. maximus produced 59.3 eggs / batch
and A. columbianus laid 17.7 eggs / batch. While in this investigation, L. flavus recorded an
average of 8.3 ± 4.6 eggs / batch under laboratory conditions.
The total number of produced eggs per individual slug differed according to species.
Capinera (2001) mentioned that individual slugs of Deroceras reticulatum produced about
100 – 200 eggs during their life span. While in this work it was found that the total number of
deposited eggs per individual slug L. flavus averaged 41.1 ± 20.4 eggs during its life span.
The Incubation period differs according to species and temperature as well as hatching
rate. In the laboratory, the incubation period of A. reticulatus eggs kept at different
temperatures ranged from 105 days at 5ºC to 18 days at 20ºC (Carrick, 1938).
Sokolve and McCrone (1978) recorded that the incubation period of slug Limax maximus
lasted one month at 15ºC, while it was 8.7 days at 20ºC. On the other hand, Prior (1983)
recorded that this period was 24 hours at 18ºC for the same species. In 1988, Raut and
Panigerhi found that, the incubation period of the pestiferous slug Laevicaulis alte (Férussac)
eggs was 20.9 days at 20ºC and 13.3 days at 30ºC with hatchability percentage of 70.6 and
81.6% respectively. Jyoti (1991) mentioned that the incubation period of the same species at
25ºC and 35ºC was 9.3 and 7.5 days respectively with hatching rate of 72.5 and 27.5% at the
same temperature degrees respectively. According to Faberi et al. (2006) the incubation
period of D. laeve eggs was 35.3 ± 2.1 and 16.03 ± 1.6 days at 12ºC and 20ºC respectively,
with the hatching rate (Fertility %) of 89.2% at 12ºC and 66.4% at 20ºC.
Baur (1994) mentioned that hatchlings emerging from larger eggs had a longer
developmental period than those smaller ones, with hatchling size positively correlated with
egg size. While, Mohamed (1999) recorded that the incubation period of L. flavus differed
according to temperature degrees, as it was the longest 28.0 ± 3.1 days at 10ºC and decreased
to 11.9 ± 1.7 days at 30ºC during feeding on lettuce leaves. The author also added that the
hatchability was greatly affected by temperatures as it was higher at 15 to 20ºC and decreased
on 25 to 30ºC.
In this work, it was found that the incubation period of the garden slug L. flavus averaged
21.0 ± 2.4 days with hatchability of 74.64 ± 25.33% under laboratory conditions of 20.5 ±
1.7°C and 61.4 ± 7.8% R.H.
The generation period (from egg to egg) of a slug is differed according to species. Hunter
(1968) mentioned that D. reticulatum had two generations per year; A. hortensis had one
generation annually and Tandonia budapestensis required two years for single generation.
Nickles and Hoffmann (1981) stated that, the minimum generation time of D. laeve is 6
weeks.
Longevity differs from species to species. Quick (1960) mentioned that Limax maximus
lived for 3 – 4 years. A. reticulatus live 9 – 13 months, A. hortensis 7.5 – 12 months and M.
budapestensis 20 – 24 months (Frömming, 1954). Also, Capinera (2001) mentioned that,
longevity varied greatly among slugs, with D. reticulatum living 9 – 13 months, Arion spp.
living 7.5 – 12 months, and Limax spp. living 24 – 36 months.
Faberi et al. (2006) found that the longevity of D. laeve was lower at 20ºC by 21.9 ± 5.1
weeks than at 12ºC by 54.4 ± 11.2 weeks.
The period of life cycle differed according to slug species. A. reticulatus complete its life
cycle in one year but A. hortensis took nearly 2 years, while M. budapestensis usually had a
biennial life cycle (Hunter, 1966).
Alford (1984) said that the garden slug A. hortensis reached maturity in about 6 – 12
months, while in the field slug D. reticulatum, development to the adult stage took about two
months under favorable conditions, but often extended to about a year.
For the slug’s life span, Inagaki (1972) mentioned that the Limacidae are generally
longer-lived than the Arionidae. Thus, the life span of Limax maximus L. is 30 to 36 months,
Limax flavus 28 to 30 months, Lehmannia marginata 24 to 26 months, and Deroceras
reticulatum 9 to 13 months. In Arion spp., Life spans from 7 1/2 to 12 months have been
recorded for Arion hortensis Fėrussac, A. circumscriptus Johnston, A. subfuscus
(Draparnaud), and for A. intermedius (Normand) of likewise 12 months.
Godan (1983) mentioned that L. flavus L. may live for 28 – 30 months; it is, however,
sexually mature after 10 – 12 months and before it has reached maximal body weight. In our
study, the life span of L. flavus ranged form 184 – 227 days with an average of 235.2 ± 27.5
days under laboratory conditions. Rollo (1988) mentioned that Limax maximus lived 2 to 3
years and grew to 5 – 20 g compared to only 0.5 to 1.5 g for Deroceras reticulatum.
Important factors such as temperature, humidity and photoperiod influence the growth
rate of land slugs.
Nickles and Hoffmann (1981) cleared that Deroceras laeve continue to increase in size
throughout their lives. Maximum size of laboratory reared D. laeve was 0.51g, compared to
0.43g as a maximum in the nature.
Foltz et al. (1982) mentioned that the mean weight of young slugs Arion intermedius at
hatching was 2.46 ± 0.04 mg. the growth of young slugs was slow during the autumn and
winter months but increased rapidly from September to October onwards. Estimates of
growth rates by regression analysis tended to confirm the initial very slow growth.
South (1982) mentioned that growth rate, reproductive, development, fecundity, and
longevity of Deroceras reticulatum differed according to temperature-sensitive processes.
Deroceras reticulatum grew and produces over a wider range of temperatures than Arion
intermedius.
Barker and McGhie (1984) reported that the maximum size of Limax maximus appeared
to be 95 mm resting (150 mm extended), in length.
Hommay et al. (2001) mentioned that the weight increase of Limax valentianus occurred
in three distinct phases: an initial phase of rapid growth (infantile phase), followed by a phase
with a smaller increase in weight (juvenile phase) and finally a phase with minimal growth,
during which reproduction occurred (adult phase). This phase ended with a weight decrease,
due to senescence. Growth rate was fastest in isolated individuals reared under long days and
warm nights, while reproduction was greatest in isolated individuals reared under long days
and cool nights. The authors added that the land slug Arion circumscriptus showed only two
growth phases: an initial phase associated with a rapid development of the hermaphrodite
gland, and a second slow phase during which eggs were laid. The growth rate of Deroceras
reticulatum increased at 15ºC and reached its maximal value at 18ºC.
Faberi et al. (2006) reported that the growth rate of Deroceras laeve was lower at 12ºC
than 20ºC. The inflection point (the mean body weight at which the growth rate started to
decrease) was reached when the slugs Deroceras laeve were 2.5 months old and the mean
body weight was 204, 7 mg at 20ºC. At the same age at 12ºC the body weight was 132.9 mg.
In addition, slugs reached a greater mean body weight at 12ºC (936.2 ± 18.9 mg) than at 20ºC
(409.4 ± 16.02 mg) i.e., approximately half the weight of the slugs kept at 12ºC. The growth
curve for Deroceras laeve has two phases: a juvenile phase of pre-oviposition, and a mature
phase of oviposition during which the slugs lay their eggs.
REFERENCES
Abbes, I.; Liberto, F.; Castillejo, J. and Nouira, S. (2010). A review of slugs and semi slugs of
Tunisia (Testacellidae, Milacidae and Limacidae). Journal of Cochology,. 40(2):219-231.
Alford, D.V. (1984). A colour atlas of fruit pests their recognition, biology and control. A
wolfe science Book, 320 p.
Alford, D.V. (1995). A colour atlas of pests of ornamental trees, shrubs and flowers. Halsted
Press, Third Avenue, New York, 448 p.
Barker, G. M. (1999). Naturalised Terrestrial Stylommatophora (Mollusca: Gastropoda).
Fauna of New Zealand, Number 38. Manaaki Whenua Press, Canterbury, New Zealand,
254 p.
Barker, G. M. and McGhie, R. A. (1984). The biology of introduced slugs (Pulmonata) in
New Zealand 1. Introduction and notes on Limax maximus. New Zealand Entomologist,
8:106-111.
Baur, B. (1994). Inter-population differences in propensity for egg cannibalism in hatchlings
of the land snail Arianta arbustorum. Animal Behaviour, 48:851-860.
Bohan, D. A.; Glen, D. M.; Wiltshire, C. W. and Hughes, L. (2000). Parametric intensity and
the spatial arrangement of the terrestrial mollusk herbivores Deroceras reticulatum and
Arion intermedius. Journal of Animal Ecology, 69:1031-1046.
Branson, B.A. (1980). The recent gastropoda of Oklahoma, Part VIII. The slug families
Limicidae, Arionidae, Veronicellidae, and Philomycidae. Proc. Okla. Acad. Sci., 60:29-
35.
Capinera, J. L. (2001). Handbook of vegetables pests. Academic Press, New York, London,
729 p.
Carrick, R.(1938). The life history and development of Agriolimax agrestis L., the grey field
slug. Trans. R. Soc. Edinb., 59:563-597.
Crawley, M. J. (1983). Herbivory. The dynamics of animal-plant interactions. Blackwell
Scientific Publications, Oxford.
Faberi, A. J.; López, A. N.; Manetti, P. L.; Clemente, N. L. and Álvares Castillo, M. A.
(2006). Growth and reproduction of the slug Deroceras laeve (Müller) (Pulmonata:
Stylommatophora) under controlled conditions. Spanish Journal of Agricultural
Research, 4(4):345-350.
Foltz, D. W.; Ochman, H.; Jones, J.S.; Evangelisti, M. and Selander, R.K. (1982). Genetic
population structure and breeding systems in arionid slugs (Mollusca, Pulmonata). Biol.
J. Linnean Soc. 17: 225-242.
Frömming, E. (1954). Biologie der Mitteleuropäischen Landgastropoden. Duncker &
Humblot, Berlin.
Godan, D. (1983). Pest slugs and snails, Bbiology and control. Springer-Verlag; Berlin,
Germany. 445 p.
Hommay, G.; Kienlen, J. C.; Gertz, C. and Hill, A. (2001). Growth and reproduction of the
slug Limax valentianus Férussac in experimental conditions. J. Moll. Stud., 67:191-207.
Hunter, P. J., (1966). The distribution and abundance of slugs on an arable plot in
Northumberland. J. Anim. Ecol., 35:543-557.
Hunter, P. J. (1968). Studies on slugs of arable ground II. Life cycles. Malacologia, 6:379-
389.
Inagaki, H. (1972). Sénescence dans la croissance chez le male de Littorina saxatilis var.
rufus (Olivi), Mollusque Gastéropode, mise en evidence par la regression sexuelle et la
diminution de l'intensité respiratoire. C. R. Hebd. Séance Acad. Sci. Sér. D. Sci. Nat.,
274:1828-1831.
Jyoti, M. (1991). Effect of moisture and temperature on the biology of land slug Laevicaulis
alte (Férussac) Geobuios, 18:41- 42.
Kerney, M. (1999). Atlas of the land and freshwater molluscs of Britain and Ireland. - pp. 1-
264. Colchester. (Harley).
McCracken, G. F. and Selander, R. K. (1980). Self-fertilization and momogenic strains in
natural populations of terrestrial slugs. Proc. Natl. Acad. Sci. USA., 77(1):684-688.
Mohamed, M. F. (1999). Ecological and biological studies on land snails and slugs in Egypt.
Ph.D. Thesis, Fac. Agric. Cairo Univ. 223 p.
Nicklas, N. L. and Hoffmann, R. J. (1981). A pomictic parthenogensis in a hermaphroditic
terrestrial slug, Deroceras laeve (Müller). Biol. Bull., 160:123-135.
Prior, D. J. (1983). The relationship between age and body size of individuals in isolated
clusters of the terrestrial slug, Limax maximus. J. Exp. Zool., 225: 321-324.
Quick, H.E. (1960). British slugs (Pulmonata; Testacellidae, Arionidae, Limacidae). Bulletin
of the British Museum (Natural History), Zoology, 6 (3):103-226.
Raut, S. K. and Panigarhi, A. (1988). Infulence of temperature on hatching of eggs of the
pestiferous slug Laevicaulis alte (Férussac). Boll. Malacol., 24:61- 65.
Rollo, C.D. (1983). Consequences of competition on the reproduction and mortality of three
species of terrestrial slugs. Res. Popul. Ecol., 25:20- 43.
Rollo, C. D. (1988). The feeding of terrestrial slugs in relation to food characteristics,
starvation, maturation and life history. Malacologia, 28(1-2):29 -39.
SAS., (1985). SAS/ STAT user's Guide: statistics. SAS Institute Inc., Cary, NC, USA.
Sokolove, P. G. and McCrone, E.J. (1978). Reproductive maturation in the slug Limax
maximus and the effects of artificial photoperiod. J. Comp. Physiol. A. sensory, Neural
Behav. Physiol., 125:317-326.
South, A. (1982). A comparison of the life cycles of Deroceras reticulatum (Müller) and
Arion intermedius Normand (Pulmonata: Stylommatophora) at different temperatures
under laboratory conditions. J. Molluscan Studies, 48:233-144.
Stephenson, J. W. (1968). A review of the biology and ecology of slugs of agricultural
importance. Proc. Malac. Soc. Lond., 38:169-178.
Wiktor, A. (1989). Slugs of Limacoidea and Zonitoidea (Gastropoda: Stylommatophora).
Fauna Polski, 12:157-161.
Yildirim. M. Z. (2004). Slugs (Gastropoda: Pulmonata) of the lakes region (Göller Bölgesi) in
Turkey. Turk. J. Zool., 28:155-160.
A BRIEF REVIEW OF NONINVASIVE METHODS

OF DNA SAMPLING FOR WILDLIFE CONSERVATION
Haseeb A. Khan1and Ibrahim A. Arif 2

1
Department of Biochemistry, College of Science, King Saud University,
Riyadh, Saudi Arabia
2
Prince Sultan Research Chair for Environment and Wildlife, Department of Botany
and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
ABSTRACT
Availability of good quality genomic DNA is a primary requirement for conducting
molecular analyses for various applications in wildlife conservation. Blood and tissues
are commonly used for extraction of genomic DNA. However, the dependency on
noninvasive sources of DNA such as feces, hair, feathers and buccal cells is particularly
important in studies where conventional sampling is hard to practice, as in the case of
wild animals. This short review summarizes the advantages and disadvantages of some
noninvasive methods of DNA sampling with special reference to their application in
wildlife conservation.
Keywords: DNA sampling, noninvasive methods, molecular markers, population genetics,

molecular conservation, wildlife, endangered animals
INTRODUCTION
Molecular phylogeny using mitochondrial DNA sequences provides valuable insights for
wildlife conservation (Arif et al. 2009, Arif and Khan 2009, Arif et al. 2011a, Khan et al.
2008a, 2008b, 2008c, Khan et al. 2011). Microsatellite or short sequence repeats (SSR) are
powerful molecular markers for various purposes in wildlife conservation (Arif et al. 2010b,
2010c, Arif et al. 2011a). Whereas DNA barcoding using cytochrome oxidase subunit 1
(COI) is commonly used for molecular identification of bird species (Khan et al. 2010, Arif et
al. 2011b, 2012b, Khan and Arif 2013). There are several factors that influence the DNA
quality and quantity including environmental exposure, diet and methods of sample
collection, DNA isolation and its storage. The significant interaction found between the

Correspondence to: Haseeb A. Khan PhD, MRACI (Aus), FRSC (UK), Associate Professor, Department of
Biochemistry, College of Science, Bldg. 5, Room 1B20, King Saud University, P.O. Box 2455, Riyadh 11451,
Saudi Arabia, E-mail: khan_haseeb@yahoo.com
64 Haseeb A. Khan and Ibrahim A. Arif
collection, preservation methods and the extraction methods reinforces the importance of
conducting a factorial design experiment, rather than examining each factor separately, as a
pilot study before initiating a full-scale noninvasive research project (Renan et al. 2012). Use
of noninvasive sources of DNA such as feces, hair and feathers for genetic analyses is
particularly important in studies where conventional sampling is difficult due to animal size,
behavior, conservation status and ethical considerations. Capturing of wild animals for
sampling often causes a considerable amount of stress. Nocturnal animals, marine mammals
or animals living in dense forest are particularly difficult to sample because they are hard to
trace. Moreover, elusive nature of some wild animals such as leopards makes their monitoring
difficult (Mondol et al. 2009). Due to fragmented populations of certain endangered animals
such as Bos javanicus and B. gaurus, it has become almost impossible to capture them in the
wild and non-invasive investigations are the only feasible approach to obtain data on these
populations (Rivière-Dobigny et al. 2009). Recent advances in DNA methods along with non-
invasive sampling techniques can be used to monitor populations and individuals across large
landscapes including human dominated ones. Recently, noninvasive methods of collection of
biological samples have been successfully employed for population estimation of wildlife
species (Piggott et al. 2006, Bhagavatula and Singh 2006, Prugh et al. 2005, Kurose et al.
2005). Although the technique can be potentially useful for wild animals, there are some
disadvantages such as small amount of DNA present in the feces or the DNA can be highly
degraded due to passing through the intestinal track. This review provides an updated
information on our viewpoint reported earlier (Arif et al. 2010a). Some of the important
issues related to noninvasive methods of DNA sampling for molecular markers analysis are
summarized in Table 1.
FECAL DNA
The collection of fecal pellets (scats) from the forest ground presents an opportunistic
sampling strategy so that the DNA can be collected without capturing or even sighting a wild
animal. Wedrowicz et al. (2013) have presented a protocol for producing a reliable individual
koala genotype from a single fecal pellet suggesting that this method could readily be adapted
for genetic studies of mammals other than koalas, particularly those whose diet contains high
proportions of volatile materials that are likely to degrade DNA. Arandjelovic et al. (2011)
collected wild chimpanzee fecal samples for genetic capture-recapture analyses over a four-
year period in Loango National Park, Gabon. Since chimpanzee males are patrilocal and
territorial, they genotyped samples from males using variable Y-chromosome microsatellite
markers and concluded that seven chimpanzee groups are present in the area. Mondol et al.
(2009) described a DNA-based method for leopard individual identification using fecal DNA
samples for obtaining the genetic material. They collected the samples in a human-dominated
landscape to estimate the minimum number of leopards in this human-leopard conflict area in
Western India and demonstrated that the selected panel of 8 microsatellite loci can
conclusively identified leopards from various kinds of biological samples. Rivière-Dobigny et
al. (2009) used bovine fecal samples for species identification using cytochrome b fragment
between positions 867 and 1140, which distinguished the four species including B. gaurus, B.
indicus, B. javanicus and B. taurus. They also evaluated the feasibility of non-invasive
microsatellite genotyping on fecal samples collected in Vietnamese nature reserves.
A Brief Review of Noninvasive Methods of DNA … 65
Table 1. Some noninvasive sources of DNA sampling and their advantages

and disadvantages for molecular analyses
Source Taxa Marker Remark Reference

Feces Indian 12S rRNA For field analysis, authentication of Pandey et al.
leopard collected scat is necessary before analysis. 2007
Feces Leopard Cyt b, SRY It is sometimes difficult to analyze long Kurose et al.
fragments using fecal samples, because 2005
fecal DNA is often fragmented due to time
process and environmental conditions.
Feces Leopard SSR Amplification success of field collected Mondol et al.
scats was up to 72%, and genotype error 2009
ranged from 0-7.4%.
Feces Bengal tiger SSR The fecal samples collected from captive Bhagavatula &
tigers were amplified with 100% success Singh 2006
versus 60% with fecal samples from field.
Feces Andean cat 16S rRNA Fecal DNA is often of low quality. Cossios &
Short segments of genes should be selected. Angers 2006
Hairs and other undigested rests from preys
present on fecal samples may interfere and
produce hybrid patterns.
Feces Bovids Cyt b, SSR Fecal DNA, especially in tropics, is usually RivièreDobigny
characterized by low concentrations, poor et al. 2009
quality and contamination from alien DNA.
Feces Oryx CR No difference in the quality of DNA Masembe et al.
between droppings and tissue samples. 2006
Feces Oryx CR, SSR For fecal samples, heterozygous genotypes Iyengar et al.
were repeated twice and homozygous were 2007
repeated 4-6 times. Failure to amplify large
product with fecal DNA.
Feces Giant otter CR, Cyt b Fecal DNA is not suitable for microsatellites Garcia et al.
and intron sequencing. 2007
Feces Bighorn SSR With only outer pellet material, PCR was Wehausen et al.
sheep excellent. When inner pellet material was 2004
included PCR success declined. Fecal
pellets of herbivore species that live in wet
environments may undergo more DNA
degradation if not collected fresh.
Feces Brown bear Sex When female carnivores consume male Murphy et al.
determine mammalian prey, all are incorrectly scored 2003
as male due to amplification of X and Y
chromosome fragments.
Feces Monkey CR, SSR First attempt to provide genetic information Oliveira et al.
for captive yellow-breasted capuchin 2011
monkey, Cebus xanthosternos in Brazil.
Feces Chimpanzee SSR The poor amplification success rate resulted Arandjelovic et
in a limited number of recaptures. al. 2011
Feces Sage grouse Sex PCR success rates were higher for females Baumgardt et
determine than males. al. 2013
Feather Kakerori SSR Success in developing microsatellite Chan et al.
markers from feather samples. 2008
Table 1. (Continued)
Source Taxa Marker Remark Reference

Feather Harpy eagle SSR, Sex The sex determination markers easily and Banhos et al.
determine consistently differentiated males from 2008
females even with highly degraded DNA
extracted from naturally shed feathers.
Feather Sea eagle CHD Fairly reproducible. Naim et al.
2011
Feather Pigeon CR Risk of repeated sampling from the same Seki 2006
individuals. High rate of genotyping error
due to low DNA quantity and quality.
Feather Birds SSR PCR success is higher from extracts of Segelbacher
plucked feathers than molted feathers. 2002
Feather Parrot SSR Production of microsatellite alleles of Presti et al.
poorer quality and had to be processed 2013
immediately after DNA extraction.
Feather Macaws COI, SSR Useful for forensic identification and future Abe et al. 2001
conservation of large macaws that have
been lost due to deforestation.
Hair Monkey CR, SSR First attempt to provide genetic information Oliveira et al.
for captive yellow-breasted capuchin 2011
monkey, Cebus xanthosternos in Brazil.
Buccal Amphibians Cyt b, SSR Greatly increase the accessibility of genetic Pidancier et al.
swab studies in small vertebrates. Only frozen 2003
storage allowed microsatellite genotyping
Buccal Fish SSR, RFLP 2-fold to 3-fold time and costs saving. Error Lavia et al.
swab rate for SSR ranged from 1.9 to 3.3%. 2006
Buccal Fish SSR Samples stored at room temperature in the Campanella et
swab dark for over 200 days could still yield al. 2006
DNA suitable for PCR
Abbreviations are: CHD, chromo helicase DNA-binding gene; COI, cytochrome oxidase 1; CR,
control region; Cyt b, cytochrome b; RFLP, restriction fragment length polymorphism; SSR,
short sequence repeat; SRY, sex-determining region Y.
Garcia et al. (2007) have suggested that the noninvasive samples such as feces and mucus
for molecular genetic analyses are informative DNA sources to analyze giant otter (Pteronura
brasiliensis populations (Garcia et al. 2007). The mucus quality and amount obtained from
feces depends on the sample age; when feces are fresh it is possible to collect only the mucus
using a syringe without needle. When feces are old or dry, the surface is scraped off targeting
epithelial cells sloughed from the gut lining during gut passage of the fecal bolus (Garcia et
al. 2007). Wehausen et al. (2004) have got best results while using 60 mg of scraped outer
pellet material from Bighorn sheep feces whereas the use of 30 mg caused a 54% decline in
signal strength, though PCR success was little affected suggesting that the outer pellet layer
contained a significant amount of high quality template DNA. The fecal samples collected
from captive tigers have been amplified with 100% success as compared to 60%
amplification success with fecal samples from the field; moreover the rate of errors generated
from captive animal fecal samples is considerably lower than the errors generated from fecal
samples collected from the jungle (Bhagavatula and Singh 2006).
There is a potential concern over using fecal DNA from herbivorous animals because of
inhibitory effects of plant secondary compounds on PCR reactions; consequently the
amplification success depends on maximizing the concentration of DNA from target animal
while minimizing secondary compounds originating from dietary plant materials (Fernando
2003). Error in sex identification using fecal DNA of carnivorous animals would result if they
have consumed prey of opposite sex (Murphy et al. 2003). Baumgardt et al. (2013) measured
the PCR success rates for amplifying the W and Z chromosomes from greater sage-grouse
(Centrocercus urophasianus) fecal samples and found that PCR success rates were higher for
females than males. Moreover, it is sometimes difficult to analyze long fragments of DNA
with fecal samples because fecal DNA is often degraded due to the time process and
environmental conditions (Kurose et al. 2005).
FEATHER DNA
Molted feather sampling is a useful tool for genetic analyses of endangered species, but it
is often very laborious due to the low quality and quantity of the DNA obtained. Recently,
molted feathers have been used as noninvasive samples for studies on the genetic structure of
birds (Horvath et al. 2005, Seki 2006). Unfortunately, molted feather samples might result a
genotyping error due to low DNA quality and quantity as well as poor extraction yields
(Taberlet et al. 1999; Presti et al. 2013). When compared with DNA extracted from blood
samples, DNA extracted from feathers produced microsatellite alleles of poorer quality and
had to be processed immediately after extraction (Presti et al. 2013). Segelbacher (2002) have
observed that only 50% of molted feather samples are reliably genotyped. Genomic DNA has
been extracted from a 1 cm segment at the root end of feathers (Hoglund et al. 2007). A
feather with a rachis <0.2 mm in diameter may contain several hundred pulp cells,
corresponding to roughly 105-106 copies of mtDNA, 104-105 copies of nuclear ribosomal
DNA and 102-103 copies of single locus nuclear genes hence the amplification can be carried
out on both mitochondrial and nuclear DNA (Taberlet and Bouvet 1991). Although blood and
tissues are the most commonly used sources of DNA for isolating microsatellites, recently
Chan et al. (2008) have successfully used the feather DNA for developing microsatellite
markers for the endangered forest bird, kakerori (Pomarea dimidiata).
Naim et al. (2011) used a PCR test with feathers as the DNA source for amplifying two
homologous fragments of Chromo Helicase DNA-binding (CHD) gene including CHD-W
gene, unique to females, and the CHD-Z gene, occurring in both sexes. This test was applied
to five individuals of H. leucogaster from the Malacca Zoo and to male and female domestic
chickens, Gallus domesticus, for comparison. All individuals were sexed successfully with
high reproducibility concluding that this test is a reliable sexing method for H. leucogaster
(Naim et al. 2011). Speller et al. (2011) applied vigorous contamination controls and sensitive
PCR amplification protocols to obtain accurate mitochondrial DNA-based species
identification with as few as two feather barbs. This minimally destructive DNA approach
was successfully tested on different bird species, including North American wild turkey
(Meleagris gallopavo), Canada goose (Branta canadensis), blue heron (Ardea herodias) and
pygmy owl (Glaucidium californicum). The DNA was successfully obtained from fresh
feathers, historic museum specimens and archaeological samples, demonstrating the
sensitivity and versatility of this technique (Speller et al. 2011). Species-level identification
by matching the cytochrome oxidase subunit 1 (COI) sequences with Barcode of Life Data
(BOLD) systems showed that seized feathers shared the highest similarity with scarlet
macaws (A. macao) whereas microsatellite profiles showed that patterns of allelic distribution
in the seized feathers were apparently distinct from those of red-and-green macaw (A.
chloropterus), but were overlapped with those of A. macao, suggesting that all of seized
feathers were derived from several individuals of A. macao (Abe et al. 2012). These
investigators also determined the parentage of hybrid macaws by the combination of COI
barcodes and microsatellite profiles and suggested that this technique will contribute to
forensic identification and future conservation of large macaws that have been lost due to
deforestation.
BUCCAL DNA
Buccal swab sampling has been suggested as a useful alternative method for DNA
sampling for both mitochondrial (cytochrome b) and nuclear (microsatellites) DNA markers
in amphibians (Pidancier et al. 2003). However, only frozen storage allowed microsatellite
genotyping. They concluded that this method could greatly increase the accessibility of
genetic studies in small vertebrates and could be preferred in the field of conservation
genetics (Pidancier et al. 2003). Swabbing body mucus provides a less invasive method of
DNA collection from small fish while fin clips may not be a method of choice as it might
affect the behavior or survival in wild (Le Vin et al. 2011). A comparison of multilocus
microsatellite genotypes from the same individuals when collected at low and high density
and compared this with fin clips did not show any differences between these categories, with
a genotyping error rate of 0.42%, validating the use of body mucus swabbing for DNA
collection in fish (Le Vin et al. 2011). Lavia et al. (2006) reported a rapid, nondestructive,
reproducible and cheap DNA extraction method from body mucus and buccal cells of
northern pike and brown trout. They used the captured DNA directly for microsatellite and
restriction fragment length polymorphism (RFLP) analyses. Although the genotyping error
rate for microsatellite ranged from 1.9% to 3.3%, this methodology combines speed of
sampling and processing, with a twofold to a threefold time and costs saving (Lavia et al.
2006). Campanella et al. (2006) developed a field sampling method for obtaining high quality
DNA from sunfish (Lepomis) and other freshwater fish by employing a variation on the
buccal swab method. They also tested the potential storage conditions of swabbed samples
and whether those conditions affect DNA extraction and PCR amplification. It was found that
samples stored at room temperature in the dark for over 200 days could still yield DNA
suitable for PCR amplification and polymorphism detection (Campanella et al. 2006). Reid et
al. (2012) validated the buccal cell DNA collection for small-bodied fish. Overall, the buccal
cell sampling can be an effective tool for collecting DNA from small and sensitive fish
species without physical damage and the risk of other lethality.
CONCLUSION
Non-invasive samples such as feces and feather that are readily available in their foraging
ground can be useful for understanding the structure and composition of populations.
Obtaining DNA by noninvasive sampling can be used to rapidly sample a large proportion of
a population. Although the DNA obtained from noninvasive sources such as feces and
feathers may not be of such high quality as extracted from fresh blood or tissue samples, it
offers a more convenient and practically feasible alternative for fingerprinting of wild
animals. By applying appropriate contamination controls, sufficient quantities of
mitochondrial DNA can be reliably recovered and analyzed from feather barbs. Nevertheless,
special care and caution should be undertaken at sample collection, DNA extraction and
analysis levels while working with fecal or feather specimens to avoid spurious results and to
achieve high level of accuracy and reproducibly.
REFERENCES
Arif IA, Khan HA. Molecular markers for biodiversity analysis of wildlife animals: A brief
review. Anim. Biodiv. Conserv. 2009; 32: 9-17.
Arif IA, Khan HA, Bahkali AH, Al Homaidan AA, Al Farhan AH, Shobrak M, Al Sadoon M.
Comparison of Neighbor-joining and maximum-parsimony methods for molecular
phylogeny of Oryx species using 12S rRNA and 16S rRNA gene sequences. Anim. Biol.
J. 2009; 1: 117-125.
Arif IA, Khan HA, Al Homaidan AA, Al Farhan AH, Bahkali AH, Shobrak M, Al Sadoon M.
Usefulness of noninvasive methods of DNA sampling but with a caution. Anim. Biol. J.
2010a; 1: 151-155.
Arif IA, Khan HA, Shobrak K, Al Homaidan AA, Al Sadoon M, Al Farhan AH. Measuring
the genetic diversity of Arabian Oryx using microsatellite markers: implication for
captive breeding. Genes. Genet. Syst. 2010b; 85: 141-145.
Arif IA, Khan HA, Shobrak M, Al Homaidan AA, Al Sadoon M, Al Farhan AH, Bahkali AH.
Interpretation of electrophoretograms of seven microsatellite loci to determine the genetic
diversity of the Arabian Oryx. Genet. Mol. Res. 2010c; 9: 259-265.
Arif IA, Khan HA, Bahkali AH, Al Homaidan AA, Al Farhan AH, Al Sadoon M, Shobrak M.
DNA marker technology for wildlife conservation. Saudi J. Biol. Sci. 2011a; 18: 219-
225.
Arif IA, Khan HA, Shobrak M, Williams J. Cytochrome c oxidase subunit I barcoding of the
green bee-eater (Merops orientalis). Genet. Mol. Res. 2011b; 10: 3992-3998.
Arif IA, Bakir MA, Khan HA. Inferring the phylogeny of bovidae using mitochondrial DNA
sequences: resolving power of individual genes relative to complete genomes. Evol.
Bioinform. Online. 2012a;.8:.139-150.
Arif IA, Khan HA, Williams JB, Shobrak M, Arif WI. DNA barcodes of Asian houbara
bustard (Chlamydotis undulata macqueenii). Int. J. Mol. Sci. 2012b; 13: 2425-2438.
Abe H, Hayano A, Inoue-Murayama M. Forensic species identification of large macaws using
DNA barcodes and microsatellite profiles. Mol. Biol. Rep. 2012; 39: 693-699.
Arandjelovic M, Head J, Rabanal LI, Schubert G, Mettke E, Boesch C, Robbins MM,
Vigilant L. Non-invasive genetic monitoring of wild central chimpanzees. PLoS One.
2011; 6: e14761.
Banhos A, Hrbek T, Gravena W, Sanaiotti T, Farias IP. Genomic resources for
theconservation and management of the harpy eagle (Harpia harpyja, Falconiformes,
Accipitridae). Genet. Mol. Biol. 2008; 31: 146-154.
Baumgardt JA, Goldberg CS, Reese KP, Connelly JW, Musil DD, Garton EO, Waits LP. A
method for estimating population sex ratio for sage-grouse using noninvasive genetic
samples. Mol. Ecol. Resour. 2013; 13: 393-402.
Bhagavatula J, Singh L. Genotyping faecal samples of Bengal tiger Panthera tigris tigris for
population estimation: A pilot study. BMC Genetics. 2006; 7: 48.
Campanella JJ, Smalley JV. A minimally invasive method of piscine tissue collection and an
analysis of long-term field-storage conditions for samples. BMC Genetics. 2006; 7: 32.
Chan CH, Zhao Y, Cheung MY, Chambers GK. Isolation and characterization of
microsatellites in the kakerori (Pomarea dimidiata) using feathers as source of DNA.
Conserv. Genet. 2008; 9: 1067-1070.
Cossios D, Angers B. Identification of Andean felid feces using PCR-RFLP. Mastozool.
Neotrop. 2006; 13: 239-244.
Fernando PT, Vidya NC, Pajapakse C, Dangolla A, Melnick DJ. Reliable noninvasive
genotyping: fantasy or reality. J. Hered. 2003; 94: 115-123.
Garcia DM, Marmontel M, Rosas FW, Santos FR. Conservation genetics of the giant otter
(Pteronura brasiliensis (Zimmerman, 1780)) (Carnivora, Mustelidae). Braz. J. Biol.
2007; 67: 819-827.
Hoglund J, Larsson JK, Jansman HA, Segelbacher G. Genetic variability in European black
grouse (Tetrao tetrix). Conserv. Genet. 2007; 8: 239-243.
Horváth MB, Martínez-Cruz B, Negro JJ, Kalmár L, Godoy JA. An overlooked DNA source
for non-invasive genetic analysis in birds. J. Avian. Biol. 2005; 36: 84-88.
Iyengar A, Gilbert T, Woodfine T, Knowles JM, et al. Remnants of ancient genetic diversity
preserved within captive groups of scimitar-horned oryx (Oryx dammah). Mol. Ecol.
2007; 16: 2436-2449.
Khan HA, Arif IA. COI barcodes and phylogeny of doves (Columbidae family).
Mitochondrial DNA. 2013; 24: 689-696.
Khan HA, Arif IA, Al Farhan AH, Al Homaidan AA. Phylogenetic analysis of oryx species
using partial sequences of mitochondrial rRNA genes. Genet. Mol. Res. 2008a; 7: 1150-
1155.
Khan HA, Arif IA, Al Homaidan AA, Al Farhan AH. Application of 16S rRNA, cytochrome
b and control region sequences for understanding the phylogenetic relationships in Oryx
species. Genet. Mol. Res. 2008b; 7: 1392-1397.
Khan HA, Arif IA, Bahkali AH, Al Farhan AH, Al Homaidan AA. Bayesian, maximum
parsimony and UPGMA models for inferring the phylogenies of antelopes using
mitochondrial markers. Evol. Bioinform. Online. 2008c; 4: 263-270.
Khan HA, Arif IA, Shobrak M. DNA barcodes of Arabian partridge and Philby's rock
partridge: implications for phylogeny and species identification. Evol. Bioinform. Online.
2010; 6: 151-158.
Khan HA, Arif IA, Shobrak M, Al Homaidan AA, Al Farhan AH, Al Sadoon M. Application
of mitochondrial genes sequences for measuring the genetic diversity of Arabian oryx.
Gene. Genet. Syst. 2011; 86: 67-72.
Kurose N, Masuda R, Tatara M. Fecal DNA analysis for identifying species and sex of
sympatric carnivores: A noninvasive method for conservation on the Tsushima Islands,
Japan. J. Hered. 2005: 96; 688-697.
Le Vin AL, Adam A, Tedder A, Arnold KE, Mable BK. Validation of swabs as a non-
destructive and relatively non-invasive DNA sampling method in fish. Mol. Ecol. Resour.
2011; 11: 107-109.
Livia L, Antonella P, Hovirag L, Mauro N, Panara F. A nondestructive, rapid, reliable and
inexpensive method to sample, store and extract high-quality DNA from fish body mucus
and buccal cells. Mol. Ecol. Notes. 2006; 6: 257-260.
Masembe C, Muwanika VB, Nyakaana S, Arclander P, Siegismund HR. Three genetically
divergent lineages of the Oryx in eastern Africa: evidence for an ancient introgressive
hybridization. Conserv. Genet. 2006; 7: 551-562.
Mondol S, Navya R, Athreya V, Sunagar K, Selvaraj VM, Ramakrishnan U. A panel of
microsatellites to individually identify leopards and its application to leopard monitoring
in human dominated landscapes. BMC Genet. 2009; 10: 79.
Murphy MA, Waits LP, Kendall KC. The influence of diet on faecal DNA amplification and
sex identification in brown bears (Ursus arctos). Mol. Ecol. 2003; 12: 2261-2265.
Naim DM, Nor SA, Baharuddin MH. Non-invasive sex identification of the white-bellied sea
eagle (Haliaeetus leucogaster) through genetic analysis of feathers. Genet. Mol. Res.
2011; 10: 2505-25110.
Oliveira CG, Gaiotto FA, Costa MA, Martinez RA. Molecular genetic analysis of the yellow-
breasted capuchin monkey: recommendations for ex situ conservation. Genet. Mol. Res.
2011; 10: 1471-1478.
Pandey PK, Dhotre DP, Dharne MS, et al. Evaluation of mitochondrial 12S rRNA gene in the
identification of Panthera pardus fusca (Meyer, 1794) from field-collected scat samples in
the Western Ghats, Maharashtra, India. Curr. Sci. 2007; 92: 1129-1133.
Pidancier N, Miquel C, Miaud C. Buccal swabs as a non-destructive tissue sampling method
for DNA analysis in amphibians. Herpatol. J. 2003; 13: 175-178.
Piggott MP, Banks SC, Stone N, Banffy C, Taylor AC. Estimating population size of
endangered bush-tailed rock-wallaby (Petrogale penicillata) colonies using faecal DNA.
Mol. Ecol. 2006; 15: 81-91.
Presti FT, Meyer J, Antas PT, Guedes NM, Miyaki CY. Non-invasive genetic sampling for
molecular sexing and microsatellite genotyping of hyacinth macaw (Anodorhynchus
hyacinthinus). Genet. Mol. Biol. 2013; 36: 129-133.
Prugh LR, Ritlands CE, Arthur SM, Krebs CJ. Monitoring coyote population dynamics by
genotyping faeces. Mol. Ecol. 2005; 14: 1585-1596.
Reid SM, Kidd A, Wilson CC. Validation of buccal swabs for noninvasive DNA sampling of
small-bodied imperiled fishes. J. Appl. Ichthyol. 2012; 28: 290-292.
Renan S, Speyer E, Shahar N, Gueta T, Templeton AR, Bar-David S. A factorial design
experiment as a pilot study for noninvasive genetic sampling. Mol. Ecol. Resour. 2012;
12: 1040-1047.
Rivière-Dobigny T, Doan LP, Quang NL, Maillard JC, Michaux J. Species identification,
molecular sexing and genotyping using non-invasive approaches in two wild bovids
species: Bos gaurus and Bos javanicus. Zoo Biol. 2009; 28: 127-136.
Segelbacher G. Noninvasive genetic analysis in birds: testing reliability of feather samples.
Mol. Ecol. Notes 2002; 2: 367-369.
Seki SI. Application of molted feathers as noninvasive samples to studies on the genetic
structure of pigeons (Aves: Columbidae). J. For. Res. 2006; 11: 125-129.
Speller CF, Nicholas GP, Yang DY. Feather barbs as a good source of mtDNA for bird
species identification in forensic wildlife investigations. Investig. Genet. 2011; 2: 16.
Taberlet P, Bouvet J. A single plucked feather as a source of DNA for bird genetic studies
Auk. 1991; 108: 959-960.
Taberlet P, Waits LP, Luikart G. Noninvasive genetic sampling: look before you leap. Trends
Ecol. Evol. 1999; 14: 323-327.
Wedrowicz F, Karsa M, Mosse J, Hogan FE. Reliable genotyping of the koala (Phascolarctos
cinereus) using DNA isolated from a single faecal pellet. Mol. Ecol. Resour. 2013; 13:
634-641.
Wehausen JD, Ramey RR, Epps, CW. Experiments in DNA Extraction and PCR
Amplification from Bighorn Sheep Feces: the Importance of DNA Extraction Method. J.
Hered. 2004; 95: 503-509.
ANATOMY AND HISTOLOGY OF THE DIGESTIVE

TRACT OF A RARE ANNUAL FISH HYPSOLEBIAS
ANTENORI (RIVULIDAE) FROM BRAZIL
Wallace Silva do Nascimento1, Naisandra Bezerra da Silva2,

Maria Emília Yamamoto1 and Sathyabama Chellappa1
1
Programa de Pós-Graduação em Psicobiologia, Centro de Biociências, Universidade
Federal do Rio Grande do Norte, Lagoa Nova, Natal, Rio Grande do Norte, Brasil
2
Departamento de Morfologia, Centro de Biociências, Universidade Federal do Rio
Grande do Norte, Lagoa Nova, Natal, Rio Grande do Norte, Brazil
ABSTRACT
This study investigated the anatomy and histology of the digestive tract of a rare
annual fish, Hypsolebias antenori captured from seasonal freshwater pools located in
Northeastern Brazil. Scientific information about this rare species is very limited. The
digestive tract of H. antenori shows a dorsally situated mouth, small conical teeth, first
branchial arch with anatomically visible gill rakers, a short tubular esophagus constituted
by pseudo stratified epithelium and lamina propria, stomach with cardiac, fundic and
pyloric regions, and an intestine which opens out through the anus. The three stomach
regions are constituted by simple cylindrical epithelium with mucous cells. The stomach
and intestine are well vascularized. The anterior part of the intestine has well developed
folds, presence of goblet cells and glands for absorption, while the posterior part of the
intestine lacks them. This pioneering study gives details about anatomical and
histological characters of the digestive tract of H. antenori, which indicate omnivorous
feeding habits.
Keywords: Hypsolebias antenori; annual fish; morphology; histology; digestive tract
INTRODUCTION
The family Rivulidae is one of the largest families of freshwater fish of the Neotropical
region. It is a diverse group of annual fish, most popularly known as "killifish", which occurs
in seasonal freshwater pools of tropical and subtropical areas of South America (Costa, 2011).
Brazil has the greatest diversity of the family Rivulidae and is represented by 30 genera with
approximately 120 species (Fricke and Eschmeyer, 2012). Despite the richness of killifishes,

chellappa.sathyabama63@gmail.com
74 W. Silva do Nascimento, N. Bezerra da Silva, M. Emília Yamamoto et al.
this family is considered rare and endangered in Northeastern Brazil (Nascimento et al.,
2012). The annual fish complete their life cycle in temporary freshwater pools which dry out
during the warm season, and as a result the entire population dies from desiccation (Wourms,
1972; Errea and Danulat, 2001; Nascimento et al., 2012). These temporary pools are
environments that have large fluctuations in temperature, level of dissolved oxygen
concentration and volume of water. To survive under these extreme conditions, the fish eggs
deposited in the sediment of the pools go through diapause stages, during which time the
embryonic development becomes temporarily arrested. With the onset of the next rainy
season these eggs hatch out and a new generation is formed (Nascimento et al. 2012).
Hypsolebias antenori (Costa, 2006) is an annual fish of family Rivulidae, which was first
registered in 1945 in Ceará, Northeastern Brazil, and is currently considered as a rare and an
endangered fish in Brazil (Nascimento et al., 2012).
Interpretation of feeding habits based only on analysis of gastrointestinal contents might
lead to incorrect conclusions, since the frequency of these items are usually related to their
availability in the environment. The digestion rate of different food items varies, and as such,
the presence of some food items which are difficult to be digested, may overestimate their
importance in the diet of a given species (Ross et al., 2006).
Morphological and histological studies are very useful for the characterization of the
digestive tract, which contribute important information for understanding the feeding habits
and physiology of fish. These investigations also assist in environmental impact assessments
(Morrison and Wright, 1999). Morphological characters, such as, different shapes of the teeth,
size of the stomach, length of the intestine in relation to the total length of the fish, could
indicate the type of feeding: herbivory, carnivory and omnivory (Becker, 2010).
The present work investigated the anatomy and histology of the digestive tract of H.
antenori which has a short span of life. The following question was addressed: Does the
digestive tract of H. antenori conform to the standard pattern of the teleost fish? This question
was addressed due to its short span of life.
Study Site and Fish Sampling
The capture of the rare annual fish H. antenori had to be authorized by a competent
authority in Brazil. As such, the sample collection of H. antenori was authorized by ICMBIO-
MMA (Chico Mendes Institute for Biodiversity Conservation of the Brazilian Ministry of
Environment), and SISBIO (Authorization system and biodiversity information), with the
official authorization number of 27451. Fish were captured from the temporary pools, during
the period of June to October, 2011 and from April to June, 2013. Fish were not captured
during 2012, since there were no temporary pools due to the severe and extended drought
period in Northeastern Brazil.
During 2011 and 2013, the temporary pools were located in the municipality of Russas,
Ceará, Northeastern Brazil (04°57’39.8” S and 37°54’26.2” W). Fish were captured utilizing
small hand trawl nets (50 x 150 cm) and sieves (60 x 60 cm) of 2 mm mesh size. Fish
specimens captured were transported to the laboratory conserved on ice.
Anatomy and Histology of the Digestive Tract of a Rare Annual Fish Hypsolebias … 75
Measurements
Each fish was numbered, measured (total length Lt±1cm), and weighed (total weight
Wt±1g). The taxonomical identification of the fish species was confirmed (Costa, 2006).
Total body length (Lt 1cm) and total body mass (Wt 1 g) of all individuals were measured.
In this study ten individuals of the study species were used, with total body length varying
from 3.7 to 5.0 cm (4.68± SD 0.44). The body weight varied from 0.5 to 2.1g (1.2g ± 0.4).
The Intestinal Coefficient (IC = length of the intestine/total fish length) was calculated
(Bertin, 1958).
Histological Procedures
Each fish was dissected to expose the digestive tract, which was removed from the
coelomic cavity. Tissue samples were fixed in 10% Bouin’s fluid. After 24 hours of fixation,
the digestive tract was submitted to microtomy to obtain fragments of the digestive organs:
esophagus, stomach (cardiac, fundic and pyloric regions), and the intestine (anterior and
posterior regions). The fragments were submitted to routine histological techniques
(dehydration, diaphanization and paraffin-embedding) and microsectioned at 5µm. Samples
were stained with Haematoxylin-Eosin and periodic acid Schiff following routine procedures.
For anatomical analysis a stereoscope (PZO Labimex) was used. For the slide
microphotography procedure an Olympus microscope CH30 coupled to an Olympus digital
camera PM – 10 AK was used and the images were processed using Nikon ACT-1 software.
RESULTS
Gross Anatomy
H. antenori has a high, convex and laterally compressed body. The mouth is superior in
position having thin lips, with a lower jaw slightly larger than the upper. The mouth is
slightly protractile, with small and conical teeth. There are four pairs of gill arches with gill
filaments on the outer side. Only the first gill arch has long gill rakers, while the other arches
have very short gill rakers (Figure 1A).
The digestive tract presents the esophagus, stomach (cardiac, fundic and pyloric regions),
and the intestine (anterior and posterior regions) (Figure 1B). The intestine is short, with an
estimated mean intestinal coefficient of 0.61±0.09, and could be subdivided into proximal and
distal intestine, in relation to the stomach. Caeca are absent in the entire intestinal region.
Histology of the Digestive Tract
The esophagus has thick walls, is short and cylindrical, with a small diameter. The
internal surface posses small longitudinal folds, parallel to each other. There is no sphincter
between the esophagus and stomach. Histologically, the mucosa lining of the esophagus folds
consist of pseudostratified epithelium and lamina propria, without glands, overlying the
muscle layer. There is a great quantity of epithelial and mucous secretary cells. Externally
there is a muscular layer containing striated muscular fibers (Figure 2).
Figure 1. A. Lateral view of the head of Hypselobias antenori: (a) mouth closed; (b)
mouth open showing the teeth; (c) the first branchial arch showing the (a) gill rakers, (b)
branchial arch, (c) gill filaments; (d) the second branchial arch showing the short gill
rakers; B. Structures of the digestive tract of H. antenori: (a) esophagus; (b1) cardiac
stomach, (b2) fundic stomach, (b3) pyloric stomach, (c) anterior intestine (d) posterior
intestine.
Figure 2. Esophagus of H. antenori showing: A. mucosa with folds consisting of (a1)

lamina propria (PAS) and (*) lumen of the esophagus; B. (b1) lamina propria and (b2)
muscular layer containing striated muscular fibers (PAS); C. (c1) pseudo stratified
epithelium (PAS); D. (d1) pseudo stratified epithelium containing numerous goblet cells
(arrow) and (*) mucous.
The stomach shows three distinct regions: the cardiac, fundic and pyloric. The cardiac
region is directly linked to the esophagus, and is responsible for the initial stage of food
digestion. The fundic region is large and is linked to the pyloric region. The cardiac region
histologically presents numerous mucosal folds, and the epithelium consists of simple
cylindrical cells with many mucous cells situated between enterocytes. Gastric glands were
absent in this region which is highly vascularized. The stroma of the mucosa contains
lymphocytes. The submucosa is absent, and the lamina propria continues to the muscular
layer which is composed of inner circular and outer longitudinal coats of smooth muscle cells
(Figure 3).
The fundic region presents thin walls with small mucosal folds, formed by an epithelium
similar to the cardiac region. The epithelium invaginates to give origin to few structurally
simple gastric glands, which possibly produces both hydrochloric acid and pepsin. The
submucosa is characteristically absent. The muscular layer of the fundic region is divided into
an inner circular, thicker than in the cardiac region, and outer longitudinal coat (Figure 4).
Figure 3. Cardiac stomach of H. antenori: A. short folds of the stomach (arrow) and (*)
lumen of the organ; B. (b1) lamina propria, (b2) blood vessels, (b3) lymphatic vessels; C.
(c1) longitudinal muscular layer; lymphatic vessels (long arrow); blood vessels (short
arrow); D. (d1) simple cylindrical mucosecretory epithelium goblet cells (long arrow) and
the brush border (short arrow) (PAS).
Figure 4. Fundic stomach of H. antenori: A. (a1) simple cylindrical mucosecretory

epithelium with globet cells; (a2) muscular circular layer, (a3) muscular longitudinal
layer and gastric glands (arrow) (PAS); B. (b1) simple cylindrical epithelium, (b2) gastric
glands and lamina propria with lymphatic vessels (arrow) (HE).
Figure 5. Pyloric stomach of H. antenori: A. pyloric folds in the stomach mucosa; blood
vessels (arrow); (a1) muscular circular layer, (a2) muscular longitudinal layer; B (b1)
simple cylindrical epithelium with goblet cells, gastric glands (long arrow) and lymphatic
vessels (short arrow) (PAS).
Figure 6. Anterior intestine de of H. antenori: A. intestinal villus and lumem of intestine

(*); B. (b1) Intestinal glands (long arrow) and blood vessels (short arrow); C. (c1)
absorptive simple cylindrical epithelium with brush border and goblet cells, intestinal
gland (arrow); D. (d1) muscular circular layer and (d2) longitudinal muscular layer, blood
vessels (short arrow) (PAS).
Figure 7. Posterior Intestine of H. antenori: A. short intestinal folds (arrow) and lumen
(*); B. (b1) absorptive simple cylindrical epithelium, (b2) lamina propria with blood
vessels (arrow), (b3) longitudinal muscular layer and (b4) muscular circular layer (PAS).
In the pyloric region, the mucosa was constituted by simple cylindrical epithelium, which
is mucous secretary and absorptive, followed by the lamina propria. These two layers formed
long folds and are joint to the muscular layer (Figure 5). Pyloric caecae were absent. The
morphology and histology of the stomach of H. antenori indicate that this organ is
specifically involved with the absorption of micronutrients.
The stomach was followed by the intestine, and this region showed the presence of
mucosal folds, some were arched and the others resembled the intestinal villi. These were
finger-like processes of the intestinal mucosa extending in the lumen. They are composed of
simple cylindrical epithelium with goblet cells and microvilli. The mucosa is devoid of
glands, however, there were some diffused pseudo glands. The submucosa was absent, and
the lamina propria was well vascularized and continued to the muscular layer, composed of
inner circular and outer longitudinal coats of smooth muscle cells (Figure 6). The wall of the
distal intestinal region had a similar structure as described for the initial part of the intestine,
however, the lumen was ample and the muscular layers were thin (Figure 7).
DISCUSSION
Fish feed on the various resources found at different depths of the water column and their
feeding habits, along with the digestive tract descriptions, provide useful ecological and
biological information. In tropical aquatic ecosystems, most of the fish species show
considerable plasticity in their diets despite their anatomical specializations for feeding
(Mazzoni and Rezende, 2003; Shibatta and Bennemann, 2003). The position, shape, size and
protrusion of the mouth, types of dentition and gill rakers are related to the feeding habits of
fish (Albrecht et al., 2001).
The small mouth of H. antenori indicates the consumption of small sized food particles
and the conical teeth, present on the inferior maxilla, are generally used to retain the food.
The gill rakers are well developed only on the first branchial arch in order to retain the food
particles. Earlier works published with other species of annual fish from the south of Brazil,
characterize them as omnivores, feeding on allochthonous and autochthonous food items
found in the water. The principal food items of annual fish reported are zooplankton,
phytoplankton, insect larvae, adults and parts of insects (Shibatta and Rocha, 2001; Shibatta
and Bennemann, 2003; Abilhoa et al., 2010; Gonçalves, 2011).
The herbivorous and detrivorous fish species tend to have long, thin and narrow intestines
than the carnivorous species, while the omnivorous species have intestines of intermediary
length (Fugi, et al., 2001). A functional explanation for the long intestine of herbivores is that
the components of their diet are digested slowly, and as such require a longer time in the gut.
Generally, the value of the intestinal coefficient of carnivores varies from 0.2 to 2.5, of
omnivores from 0.6 to 8.0 and that of herbivores from 0.8 to 15. The value of the intestinal
coefficient of H. antenori registered in this work is comparable to that of other omnivores,
which feed on animal and vegetable matter, showing a mixed diet and possessing digestive
structures which are not specialized (Rotta, 2003; Ward-Campbell et al., 2005).
The pyloric caecae are finger like structures which possess a type of intestinal epithelium
with multi-folds in order to increase the surface area for the retention and absorption of food
in the intestine (Genten et al., 2009). These structures are well developed in carnivores while
they are either reduced or absent in herbivores. The absence of pyloric caecae in H. antenori
could be related to their low protein diet, since these structures play a role in the digestion and
absorption of proteins and lipids.
In general, the histological analyses of the digestive tract of teleost fish typically consists
of four main layers: (1) mucosa (lining the lumen of the digestive tube and consisting of an
inner epithelium), a middle lamina propria (cellular connective tissue) and an outer
muscularis mucosae; (2) submucosa (a less cellular connective tissue layer with blood
vessels, lymphatic tissue and nerve plexi); (3) a muscular coat, an inner circular and an outer
longitudinal layer; (4) serosa (composed of connective tissue surrounded by simple squamous
peritoneal epithelium) (Genten et al. 2009). In the present study it was observed that the
digestive tract of H. antenori does not strictly comply with the standard pattern of other
teleost fish, specifically due to the absence of muscularis mucosa. The muscular layers of the
mucosa help in the movement of the folds which mix the food, facilitating the mixing of
gastric acids in the stomach and absorption in the intestine. The absence of this layer could be
compensated by the development of the circular muscle layer and smooth muscle, as observed
clearly in the micrographs. The absence of muscularis mucosa has also been observed in
some other omnivorous fish species (Hale, 1965; Kessler et al., 1979; Shibatta and
Bennemann, 2003).
The epithelium of the stomach and intestinal mucosa is characterized as simple
cylindrical with absorptive and goblet cells. This has also been observed in other omnivorous
fish species, such as, Leporinus taeniofasciatus (Albrecht et al., 2001) and Oreochomis
niloticus (Morrison and Wright, 1999). The mucous-secreting or globular cells are continuous
and abundant, and the mucous secreted by them facilitates the passage of food along the tract,
and may be related to mucosal protection. The mucosa of the stomach showed little tubular
gastric glands in the lamina propria, which are possibly responsible for the synthesis of
hydrochloric acid and pepsin. The presence of these glands is related to the food habits
(Mohsin, 1962) and enzyme activity (Medeiros et al., 1970). The low incidence of these
glands suggests that the synthesis and release of substances for digestion are produced at a
higher intensity by the hepatopancreas (Genten et al., 2009).
It could be stated that the stomach of H. antenori is different from other teleost fish, since
it does not exclusively function for gastric digestion. Based on its mucosal structure it is
possible to predict that it is also responsible for promoting intense nutritional absorption. The
presence of lymphatic vessels is a strong indicator to show that the stomach of H. antenori is
also involved in absorption of nutrients.
During the larval stages of fish, the digestive tube is simple and is undifferentiated, and
the digestive organs develop later on. These changes characterize the different functional
adaptations to varied diets (Govoni et al., 1986). During the larval development, changes
occur in the process of digestion, absorption and assimilation of chemical components
(Dabrowski, 1984). Fish of the family Rivulidae present a short life span with rapid larval
stage. H. antenori possess a simple digestive tract and probably this is related to the necessity
of rapid growth and gonad development to complete its life cycle within a short span of time.
This strategy possibly guarantees its survival and reproduction in the tropical temporary
pools.
CONCLUSION
This pioneering study gives details about anatomical and histological characters of the
digestive tract of H. antenori, an annual fish from temporary pools in Northeastern Brazil.
The digestive tract of this species shows anatomical and histological adequacy for
omnivorous feeding habit, indicating its importance in the food chain in the ecosystem
dynamics of temporary pools. This species is an omnivore, which feeds on a variety of animal
and vegetable matter, and this feeding strategy is well suited to life in temporary pools. The
stomach and intestine are adapted for digestion and absorption of nutrients, and this is
important for its fast growth and reproduction within a short life span.
ACKNOWLEDGMENT
This study was supported by the National Council for Scientific and Technological
Development of the Ministry of Science and Technology of Brazil (CNPq/MCT) in the form
of research grants, and by the Post-Graduate Federal Agency of the Ministry of Education and
Culture of Brazil (CAPES/MEC).
REFERENCES
Abilhoa, V., Vitule, J., Bornatowski, H., Lara, F., Kohler, G., Festti, L., Carmo, W. and
Ribeiro, I. 2010. Effects of body size on the diet of Rivulus haraldsiolii (Aplocheiloidei:
Rivulidae) in a coastal Atlantic Rainforest island stream, southern Brazil. Biotemas, 23:
59-64.
Albrecht, M. P., Ferreira, M. F. and Caramaschi, N. E. P. 2001. Anatomical features and
histology of the digestive tract of two related neotropical omnivorous fishes
(Characiformes; Anostomidae). Journal of Fish Biology, 58:419-430.
Becker, A. G., Gonçalves, J. F., Garcia, L. O., Behr, E. R., Graça, D. L., Filho, M. K.,
Martins T. and Baldisserotto, B. 2010. Morphometric parameters comparisons of the
digestive tract of four teleosts with different feeding habits. Ciência Rural, 40: 862-866.
Bertin, L. 1958. Digestive tract,” in Traité de Zoologie, P.P.Grasse, Ed., vol. 13, 1249–1301,
Masson, Paris, France.
Costa, W. J. E. M. 2006. Taxonomy and phylogenetic relationships among species of the
seasonal, internally inseminating, South American killifish genus Campellolebias
(Teleostei: Cyprinodontiformes: Rivulidae), with the description of a new species.
Zootaxa, 1227: 31-55.
Costa, W. J. E. M. 2011. Comparative morphology, phylogenetic relationships and historical
biogeography of plesiolebiasine seasonal killifishes (Teleostei: Cyprinodontiformes:
Rivulidae). Zoological Journal of the Linnean Society, 162:1-18.
Errea, A. and Danulat, E. 2001. Growth of the annual fish, Cynolebias viarius
(Cyprinodontiformes), in the natural habitat compared to laboratory conditions.
Environmental Biology of Fishes, 61: 261-268.
Fricke, R. and Eschmeyer, W. N. 2012. A guide to Fish Collections in the Catalog of Fishes
database, Online Version, Updated 7 August.
Fugi, R., Agostinho, A. A. and Hahn, N. S. 2001. Trophic morphology of five benthic-
feeding fish species of a tropical floodplain. Brazilian Journal of Biology, 61: 27-33.
Genten, F., Terwinghe, E. and Danguy, A. 2009. Atlas of Fish Histology. Science Publishers,
Endfild, NH, USA.
Gonçalves, C. S., Souza, U. P. and Volcan, M. V. 2011. The opportunistic feeding and
reproduction strategies of the annual fish Cynopoecilus melanotaenia
(Cyprinodontiformes: Rivulidae) inhabiting ephemeral habitats on southern Brazil.
Neotropical Ichthyology, 9: 191-200.
Govoni, J. J., BoehleR, G.W. and Watanabe, Y. 1986. The physiology of digestion in fish
larvae. Environmetal Biology of Fishes, 16: 59-77.
Hale, P. A. 1965. The morphology and histology of the digestive systems of two freshwater
teleosts, Poecilia reticulata and Gasterosteus aculeatus. Journal of Zoology 146:132–
149.
K. Dabrowski, 1984. The feeding of fish larvae: present "state of the art" and perspectives.
Reproduction Nutrition and Development, 24: 807-833
Kessler, R. O., Dias, M. I. and Oliveira, E. F. 1979. Estudo histológico do estômago de
Prochilodus sp. Acta Biologica Leopoldensia. 1: 55–69.
Mazzoni, R. and Rezende, C. F. 2003. Seasonal diet shift in a tetragonopterinae
(Osteichthyes, Characidae) from the Ubatiba River, RJ, Brazil”. Brazilian Journal of
Biology, 63: 69 – 74.
Medeiros, L., Ferri, S., Longhi, L. and Worsman, T. 1970. Histochemical study of protein in
epithelial tissue of the digestive tract of Pimelodus maculatus Lacepéde, 1803. Acta
Histochemica, 37: 113-117.
Mohsin, S. M. 1962. Comparative morphology and histology of the alimentary canals in
certain groups of indian teleostes. Acta Zoologica, 43: 79-133.
Morrison, C. M. and Wright Jr, J. R. 1999. A study of the histology of the digestive tract of
the Nile tilapia. Journal of Fish Biology, 54: 597-606.
Nascimento, W. S., Yamamoto, M. E and Chellappa, S. 2012. Proporção sexual e Relação

peso-comprimento do peixe anual Hypsolebias antenori (Cyprinodontiformes: Rivulidae)
de poças temporárias do Semiárido brasileiro. Biota Amazônia, 2:37-44.
Ross, L.G., Martinez-Palacios, C. A., Aguilar Valdez, M. C., Beveridge, M. C. M.and Chavez
Sanchez, M. C. 2006. Determination of feeding mode in fishes: the importance of using
structural and functional feeding studies in conjunction with gut analysis in a selective
zooplanktivore Chirostoma estor estor Jordan 1880. Journal of Fish Biology, 68: 1782-
1794.
Rotta, M. A. 2003. Aspectos Gerais da Fisiologia e Estrutura do Sistema Digestivo dos Peixes
Relacionados à Piscicultura,” Corumbá: Embrapa Pantanal.
Shibatta, O. A. and Rocha, A. J. A. 2001. Alimentação em machos e fêmeas do pirá-brasília,
Simpsonichthys boitonei Carvalho (Cyprinodontiformes, Rivulidae). Revista Brasileira
de Zoologia, 18: 381-385.
Shibatta, O. A. and Bennemann, S. T. 2003. Plasticidade alimentar em Rivulus pictus Costa
(Osteichthyes, Cyprinodontiformes, Rivulidae) de uma pequena lagoa em Brasília,
Distrito Federal, Brasil. Revista Brasileira de Zoologia, 20: 615-618.
Ward-Campbell, B.M.S., Beamish, F. W. H. and Kongchaiya, C. 2005. Morphological
characteristics in relation to diet in five coexisting Thai fish species. Journal of Fish
Biology. 67: 1266-1279.
Wourms, J. P. 1972.Developmental biology of annual fishes. I. Stages in the normal
development of Austrofundulus myersi. The Journal of Experimental Biology, 182: 143–
168.
View publication stats

1949-498X 4 1limax

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

1949-498X 4 1limax

Uploaded by

Copyright:

Available Formats

See discussions, stats, and author profiles for this publication at: https://www.researchgate.

Laboratory Observations on Biology of the Tawny Garden Slug Limax ﬂavus

Article · January 2013

Biological aspects of the common terrestrial gastropods in Egypt View project

The user has requested enhancement of the downloaded file.

Profiles of Carbohydrates During Osmoionic Regulation of the Stenohaline

Seasonal Changes in Condition Factor, Gonadossomatic and Hepatosomatic

Histomorphology of Digestive Tract in Two Closely Related Mountain Newts

Anatomy and Histology of the Digestive Tract and Feeding Habits

Laboratory Observations on Biology of the Tawny Garden Slug Limax flavus

A Brief Review of Noninvasive Methods of DNA Sampling for Wildlife

Anatomy and Histology of the Digestive Tract of a Rare Annual Fish

Nova Science Publishers, Inc.

Institutional Subscription Rate per Volume

Additional color graphics might be available in the e-version of this journal.

EDITORIAL BOARD MEMBERS

PROFILES OF CARBOHYDRATES DURING OSMOIONIC

Fauzia Anwar Sherwani and Iqbal Parwez

Keywords: Carbohydrate profiles, teleost fish, fresh water, osmoregulation

MATERIALS AND METHODS

Collection and Care of Fish

Artificial Salt Water

Artificial SW was prepared in dechlorinated TW according to Goswami et al. (1983) and

Plasma osmolality was measured in 10 l sample with vapour pressure osmometer

Plasma glucose was assayed by the glucose-O-toluidine method (Hyvarinen and

Groups of catfish were transferred from TW to aquaria containing 30% (320

Catfish acclimated for 15 days in 35% SW were transferred to TW and 35% SW

Figure 1. Changes in plasma osmolality of the catfish, Heteropneustes fossilis following

Figure 2. Changes in plasma glucose concentration of the catfish, Heteropneustes fossilis

Figure 3. Changes in liver glycogen concentration of the catfish, Heteropneustes fossilis

Figure 4. Changes in muscle glycogen concentration of the catfish, Heteropneustes

Figure 5. Changes in plasma osmolality of the catfish, Heteropneustes fossilis following

Figure 6. Changes in plasma glucose concentration of the catfish, Heteropneustes fossilis

Figure 7. Changes in liver glycogen concentration of the catfish, Heteropneustes fossilis

Figure 8. Changes in muscle glycogen concentration of the catfish, Heteropneustes

SEASONAL CHANGES IN CONDITION FACTOR,

Nirlei Hirachy Costa Barros1, Arrilton Araujo de Souza1

Keywords: Synbranchus marmoratus; Synbranchiformes; length-weight relationships;

MATERIALS AND METHODS

Figure 1. Relative frequency of occurrence of different sex types of S. marmoratus during

The hepatosomatic index (HSI) of females and secondary males of S. marmoratus

Figure 5. Analysis of the Principal Component Analysis (PCA) of the environmental

Chellappa, N.T., Costa, M.A.M. 2003. Dominant and co-existing species of

HISTOMORPHOLOGY OF DIGESTIVE TRACT

P. Parto 1, S. Vaissi 2 and M. Sharifi 2

Keywords: Neurergus kaiseri, Neurergus microspilotus, digestive system, anatomy,

MATERIALS AND METHODS

Figure 1. General view of digestive tract in N. microspilotus (A and C) and N. kaiseri

Figure 2. A.The pharynx is lined with pseudostratified ciliated epithelium (EP)

Figure 3. Section of the esophagus (A, B in N. microspilotus and B, C in N. kaiseri).

Stomach in N. kaiseri and N. microspilotus is a straight, expanded conical tube, laying

Figure 4. Stomach in N. kaiseri (A and D) and N. microspilotus (B and C). A.Cardia is

Figure 5. Duodenum in N. kaiseri (A) and N. microspilotus (B), these micrographs

species of North American salamanders showed that in Siren lacertian, Notophthalmus v.

Sharifi, M. & Assadian, S. (2004). Distribution and conservation of Neurergus microspilotus.

ANATOMY AND HISTOLOGY OF THE DIGESTIVE

Emilly Kataline R. Pessoa1, Naisandra Bezerra da Silva2,

Keywords: Hypostomus pusarum, anatomy, histology, feeding habits

MATERIALS AND METHODS

Study Site and Fish Sampling

The Intestinal Coefficient

Stomach Contents Analyses and Statistical Analyses

The stomach contents of 118 specimens of H. pusarum were examined under a

Weight = 74.72 * - 9.07 * (age, month)