INTRODUCTION
Amphibians were the first group of vertebrates which were adapted to
aquatic as well as terrestrial mode of life during the course of evolution. Amphibians
have a unique place in the evolutionary history of vertebrates for being first to
establish life on land (Anderson, 2008; Frost et al., 2008). Amphibians are
intermediate between the fully aquatic fishes and the truly terrestrial amniotes. The
successful perpetuation of an amphibian species and its survival in the new
terrestrial environment depended on the development of limbs, lungs, other
anatomical modifications and more importantly on the evolution of new reproductive
strategies (Shine, 1979; Prado et al., 2005). Reproductive success for amphibians
involves spermiation, ovulation, oviposition, fertilization, embryonic development
and metamorphosis (Brown and Cai, 2007). Amphibian history suggests that the
developmental pattern evolved between the Triassic and the mid-Jurassic period
(Anderson, 2008; Boisvert, 2009). The estimate for the date of the origin of modern
amphibians (Liss amphibia: frogs, salamanders, and the limbless caecilians but not
amniota) can lie between 351 and 266 Mya (Marjanovi and Laurin, 2007) which
phylogenetically placed near the Batrachians divergence (Anderson, 2008; Frost et
al., 2008). During these years, as a consequence of interactions with the nature,
they have evolved different modes of reproduction such as oviparity, ovo-viviparity
(e.g., Pipa pipa) and viviparity (e.g., Salamandra salamandra) (Wake, 1998). It,
thus, seems that, as compared to other terrestrial vertebrates (e.g., reptiles, birds
and mammals), amphibians have radiated a wide range of diversity of reproductive
modes (Diwan, 1996). Since amphibians are poikilothermic and also that the
majority of them depend upon water for breeding, their reproductive activities are
greatly affected by the ever-changing climatic factors such as air and water
temperature, rainfall, daylength and relative humidity (Delgado et al., 1992; Sumida
et al., 2007).
In India, there are only few reports on the breeding biology of some
amphibians like Rana limnocharis (Roy and Khare, 1978), Rana alticola (Sahu and
Khare, 1983), Polypedates maculates (Mohanty and Dutta, 1986; Dutta et al.,
2000), Rhacophorus malabaricus (Sekhar, 1989), Polypedates leucomystax
(Ahmed and Lahkar, 1999; Iangrai, 2007), Rhacophorus pseudomalabaricus
(Vasudevan and Dutta, 2000), Chirixalus simus (Deuti, 2001a, b), Hyla annectans
(Ao and Bordoloi, 2001) and Rhacophorus bipunctatus (Iangrai, 2007). The
distribution and life cycle of Gegeneophis ramaswmii (Oommen et al., 2000) and
Rhacophorus pseudomalabaricus (Vasudevan and Dutta, 2000) are also well
documented in Western Ghats.
The general reproductive patterns of amphibians are (i) caecilians reproduce
biennially (Exbrayat and Delsol, 1985; Oomen et al., 2000), (ii) salamanders
reproduce annually (Cryptobranchidae, Hynobiidae and Sirenidae) or biennially
(Plethodontidae and Bolitoglossini), and (iii) anurans have seasonal reproduction
(Duellman, 1995). Reproductive cycles in amphibians are regulated by a complex
neuroendocrine mechanism involving the hypothalamus-hypophyseal-gonodal axis
(HHG) (Griffith and Wilson, 2003). The HHG axis is influenced by endogenous and
exogenous factors (Norris, 2007). Gonadotropin releasing hormone (GnRH)
secreted from the hypothalamus plays a major role in the regulation of reproductive
functions. GnRH acts on the gonadotrophic cells of the anterior pituitary to stimulate
the release of gonadotropins, namely luteinizing hormone (LH) and follicle
stimulating hormone (FSH) (Griffith and Wilson, 2003). The gonadotropins stimulate
gametogenesis and the synthesis of gonodal steroid hormones such as androgens,
estrogens and progestogens. The FSH initiates spermatogenesis in males and
follicular development in females. LH induces androgen synthesis by interstitial cell
in males and estrogen synthesis and ovulation in females (Suzuki et al., 1985).
Recent research into the relationships between testicular androgens and male
behaviors, mainly using castration/steroid treatment studies, generally supports the
conclusion that androgens are necessary but not sufficient to enhance male
behaviors. Prolactin acts synergistically with androgens and induces reproductive
development, sexual behaviors, and pheromone production (Moore et al., 2005).
This interaction between prolactin and gonodal steroids helps to explain why
androgens alone sometimes fail to stimulate amphibian behaviors. Vasotocin also
plays an important role and enhances specific types of behaviors in amphibians
(frog calling, receptivity in female frogs, amplecting and clasping, courtship
behaviors, etc.) (Moore et al., 2005).
Metamorphosis (Brown and Cai, 2007) is a post embryonic period of
profound morphological changes by which the animal alters its mode of living, gill
breathing aquatic to air-breathing terrestrial adult mode of life (Mc Diarmid and
Altig, 2000). Amphibian metamorphosis is a complex development process, and
results in reorganization of most of tissues and organs of tadpole (Galton, 1988,
Brown and Cai, 2007). Amphibian requires thyroid hormones (TH) for larval
development and metamorphosis, and secretion of TH by the thyroid gland is
greatly increased just before the onset of metamorphic climax (Dent, 1988). The
thyroid hormones help in development from the pre-metamorphic stages to the
onset of metamorphic climax. Radio Immunoassays show that levels of thyroid
hormones (TH) rise to a peak during metamorphic climax. Accompanying peaks are
reported for adrenocorticotrophic hormone (ACTH), adrenal corticoids (AC) and
prolactin (PRL) (Dent, 1988). Prolactin is widely considered to be the juvenile
hormone of anuran tadpoles and to counteract the effects of thyroid hormones (TH)
(Huang and Brown, 2000). Melatonin may also have a role in metamorphosis
(Lincoln, 1999; Wright, 2002; Udin, 2005) because it is a thyroid antagonist, whose
level falls at the metamorphic climax when the thyroid hormones peak. Melatonin
rhythms in plasma and eyes are entrained to the light/dark (LD) cycle and affected
by temperature (Wright, 2002). Consequently, melatonin could transduce
environmental information to regulate endocrine periodicity and larval circadian
organization and influence metamorphic rate.
A critical review of literature on breeding biology, breeding behaviors,
parental care, gonadal cycles, developmental biology, and metamorphosis are
given order-wise in the following sections.
Gymnophiona/Apoda:
Apodans belong to an order of amphibians with distribution in several of the
tropical and temperate zone countries (Smita et al., 2004). The Western Ghats is a
home to many Indian and regional endemic species of apodan (Oommen et al.,
2000). Fourteen out of 17 Indian species of caecilians are found in the Western
Ghats and all the species are endemic (Bhatta, 1998). Three species of
gymnophiona namely, Ichthophis garoensis, Ichthophis hussaini, and Ichthophis
sikimensis have been reported in North-Eastern region of India (Zoo Outreach
Organization, 2001; Ahmed et al., 2009). Caecilians are known to have internal
fertilization, and probably about 75% of the species are viviparous, meaning give
birth to developed young ones (Gower et al., 2008). About 25% of the species are
oviparous (egg-laying), and the eggs are guarded by the female (Wake, 1980;
1998). Caecilians, unlike other amphibians so far known, have internal insemination
and internal fertilization (Bhatta, 1998). Further, the copulatory mechanism unique
to members of the order Gymnophiona is the insertion of the male intromittent
organ (copulatory organ) in to the cloaca of the female following courtship. A major
dichotomy in caecilian reproductive modes is that of oviparity versus viviparity.
Members of aquatic family Typhlonectidae are viviparous and produce juvenile
aquatic young one (Wilkinson and Nussbaum, 1997). Viviparity has evolved from
oviparity. The transition from oviparity to viviparity requires the retention of fertilized
eggs in the female reproductive tract. Gestation requires a full year, females have
at least a biennial cycle, but males have active spermatogenesis throughout the
year (Oomen et al., 2000). Viviparity is reported for Gegeneophis seshachari
(Gymnophiona: Caeciliidae) from a gravid female containing four oviductal fetuses.
The oviducts are highly vascularized and contain patches of thickened, layered
tissue similar to fetal gut content (Gower et al., 2008). Viviparous caecilians go
through metamorphosis while inside the eggs, so they hatch with the body form of
juvenile young one. The developing young one uses the teeth to chew a nutrient
liquid made by the inner lining of the oviduct inside the mother. The fetal teeth are
shed at or near birth (Kupfer et al., 2006).
The caecilian reported so far from India are all terrestrial and oviparous
(Bhatta, 1998, Oommen et al., 2000). Oviparous caecilians lay eggs by digging a
hole close to the surface in a damped ground near water that hatch into free-living
larvae having small gills and tail fins (Bhamrah and Juneja, 1990). Many species lay
their eggs on land in burrows, crevices, under logs and debris, or at the bases of
bunch of grasses. No species of caecilians, so far known, lays the eggs in water
(Wake, 1998). Maternal care of the clutch has been mentioned in many species like
Ichthyophis glutinosus, Ichthyophis kohtaoensis, etc. Female Ichthyophis glutinosus
coils around the clutch at hatching and the larvae wriggle from the burrow to nearby
streams, where they spend approximately a year before metamorphosis (Kupfer et
al., 2006). Larval and adult caecilians are similar in morphology with the notable
exception of the presence of three pairs of external gills in the larvae. These
external gills degenerate during late embryonic or fetal life, although it is not clear
whether the gills are resorbed or broken off.
One of the fossorial amphibians that are members of the order
Gymnophiona, Ichthyophis kohtaoensis (Southeast Asia) is an oviparous species in
which maternal care of the clutch is provided. In Ichthyophis kohtaoensis,
development from the end of neuralation to metamorphosis has been divided in to
20 developing stages (Dunker et al., 2000). In India, research work on caecilians
are mainly reported in distribution of the caecilians of the Western Ghats (Bhatta,
1998), where previtellogenic ovarian follicles of the caecilians Ichthyophis tricolor
and Ichthyophis ramaswami (Beyo et al., 2007), the assembly of ovarian follicles
and ultra structural feature of ovary (Beyo et al., 2007), distribution and abundance
of Gegeneophis ramaswami in southern Kerala have been reported (Oommen et
al., 2000). Further, the stages of spermatogenesis of two species of caecilians,
Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona)
involving light and electron microscopic studies have been conducted (Smita et al.,
2004). The spermatogenesis in these species has been divided in to three phases,
namely active spermatogenesis (July–November), early regression (December–
March) and spermatogenic quiescence (April–June) (Smita et al., 2005). There is
practically no information on the stages of development and metamorphosis of
apodans found in India.
Parental care has also been studied only in a few species of apodans. A
remarkable form of parental care and mechanism of parent-offspring nutrient
transfer has been reported in Boulengerula taitanus, which is a direct-developing,
oviparous caecilian in which the skin in brooding females is transformed to provide
a rich supply of nutrients for the developing offspring (Kuffer et al., 2006). Further,
the young individuals of this species are equipped with a specialized dentition,
which is used to peel and eat the outer layer of their mother's modified skin. This
new form of parental care provides a plausible intermediate stage in the evolution of
viviparity in caecilians (Kuffer et al., 2006; Wilkinson, 2008). So far there is no
report on any kind of parental care in caecilians in India.
Caudata/ Urodela:
Most of urodelans are four-legged and lizard-like in shape, but some are
elongate and eel-like with the degenerated limbs (e.g., Amphuima). The tail is never
lost following metamorphosis. Many salamanders have a biphasic life cycle
containing an aquatic larval form with external gills and a metamorphosed terrestrial
adult form that respires by lungs and/or through moist skin. Some species lack
metamorphosis and retain a larval appearance throughout their life (e. g., Axolotl
larva), whereas other species lack the aquatic larval stage and hatch on land as
terrestrial forms that resemble miniature adults (Buckley, 2007).
Sexual dimorphism is common with respects to breeding colors and medianfin enlargement in the males of long-toed salamanders. Breeding males having
long-toed (Ambystoma macrodactylum columbianum), which scramble for mating
opportunities, are reportedly better in recognizing and/or locating potential breeding
female mates. All the three modes of reproduction (oviparity, ovo-viviparity and
viviparity) are displayed in Urodelans (Wake, 1998; Bhamrah, 2003). Though
salamanders display lesser diversity in reproductive modes than anurans, there is
still variation in the type of fertilization, oviposition site, seasonality, oviparity/ovoviviparity/viviparity and in parental care (Nussbaum, 1987). Chemosensory cues
(Pheromones) reportedly play an important role in the daily lives of salamanders in
mediating foraging, conspecific recognition and territorial advertising (Bee and
Gerhardt, 2002; Park et al., 2004). It has been established that male newts emit
pheromones that attract females of the same species (Kikuyama et al., 1997; Watts
et al., 2004). It has been found that male Ambystoma increase their general activity
when exposed to female odorants, but that activity levels in females were not
affected by conspecific odorants of males (Park et al., 2004). It has been seen that
male newts emit pheromones by the cloacal glands that attract females of the same
species (Kikuyama et al., 1997).
Two major reproductive patterns are exhibited by Urodelans. The classical
annual breeders depended upon rise of temperature, saturation of ground by
melting snow and spring rains. The majority of the salamanders have seasonal
reproductive patterns based on cyclic climatic changes, fertilization is external and
oviposition occurs within a few hours to several days after mating (Iwasaki and
Wakahara, 1999; Osikowski and Rafinski, 2001). However, in some species mating
occurs in the autumn, and spermatozoa are stored in the spermatophores for egg
fertilization until the following spring (Wake and Dickie, 1998). Sperm competition
appears to be an important aspect of any mating system in which individual female
organisms mate with multiple males and store sperm in spermatophores (Sharon et
al., 1997). Mating usually occurs in late summer or autumn and many occur again
in the spring in the same populations (Walsh, 2007). Spawning was observed in
early spring, and hatched larvae metamorphosed by August-September, but
duration of development and metamorphosis of larva varies in different species. In
some species it takes 2 years, while in others it takes 1-year or less (Iwasaki and
Wakahara, 1999). Cycles of oogenesis and oviposition may be annual or longer,
depending on the taxon and the population location (Miller and Robbins, 2005).
Post-copulatory sexual selection may be particularly important in species
that store sperm throughout long breeding seasons, because the lengthy storage
period may permit extensive interactions among rival sperm (Adams et al., 2005).
Ocoee salamander (Desmognathus ocoee) has been reported to store sperm up to
9 months prior to fertilization (Adams et al., 2005). Multiple paternities are displayed
in a natural population of a salamander with long-term sperm storage (Liebgold et
al., 2006).
Breeding activity in urodelans is initiated by rainfall and rise of temperature
in coastal population (e.g., Ambystoma tigrinum) (Hassinger et al., 1970). Some
aquatic cold stream species (e.g., Rhyacotriton olympicus) have been reported to
breed throughout the year, but other species (e.g., Dicamptodon ensatus) exhibit
seasonal reproduction (Iwasaki and Wakahara, 1999; Osikowski and Rafinski1,
2001). Most species fertilize the eggs internally, with the male depositing a sac of
sperm in the female's cloaca (Green, 1997; Wake and Dickie, 1988). However, the
most primitive salamanders grouped together as the Cryptobranchoidea exhibit
external fertilization (Selmi et al., 1997). Some species are ovo-viviparous, with the
female retaining the eggs inside her body until they hatch (Wake, 2005).
After fertilization, a larval stage follows in which the organism is fully
aquatic or land dwelling, and possesses gills. The most noticeable morphological
changes are the resorption of the three sets of external gills and the tail fin at the
final stages of metamorphosis (Brown and Cai, 2007). Depending on species, the
larval stage may or may not possess legs (Ohmura and Wakahara, 2002). The larval
stage may last from days to years (Iwasaki and Wakahara, 1999). Some species
exhibit no larval stage at all, with the young ones hatching as miniature version of
the adult (Buckley, 2007). Many urodelans exhibit direct development, in which the
most part of ontogenesis takes place in the egg and a miniature copy of the adult
adapted to the terrestrial mode of life emerges from the egg (Brown and Zippel,
2007). The transition from the larval type to the miniature adult occurs in several
species of Urodelans (Smirnov, 2008). Neoteny has been observed in all
salamander families, in which an individual may retain gills after sexual maturity
(Shi, 2000).
Metamorphosis in the urodelans, regulatory mechanisms, amplitude of
metamorphic transformations, progressive divergence of the larval and the adult
morphology and evolution are regulated by thyroid hormones (TH) (Smirnov, 1992;
Dunn, 2004). In contrast to anurans, many salamanders do not undergo
metamorphosis in nature; however, they can be induced to undergo metamorphosis
via exposure to thyroxine (T4) (Dunn, 2004). Treatment of pre-metamorphic larvae
of urodelans with TH can lead to precocious metamorphic changes even in
facultative neotenes or pedomorphic salamanders such as axolotl that do not
undergo natural metamorphosis (Dunn, 2004). However, the obligatory neotenes
such as Necturus maculosus do not metamorphose either in nature or when treated
with exogenous thyroid hormones (Brown, 2005). The Mexican axolotl, like a
number of other urodelan species, is an obligatory neoteny, which completes its full
life cycle without undergoing metamorphosis (Rosenkilde and Ussing, 2005).
The normal stages of development in urodelans are based on Ambystoma
maculatum. The anuran tadpole changes into a tail less frog or toad, whereas the
urodelan larva hardly changes in general appearance. The existing table of stages
of the normal development of the axolotl (Ambystoma mexicanum) ends just after
hatching. At this time, the forelimbs are found as small buds (Nye et al., 2003).
Anurans/Salentia:
Anurans have a biphasic life cycle, and breed in a variety of water bodies
ranging from highly ephemeral to permanent ponds (Krishna et al., 2004). Two
basic reproductive patterns are evident in anurans. Most tropical and subtropical
anurans species are capable of reproduction throughout the year, rainfall seems to
be the primary extrinsic factor controlling the timing of reproductive activity. The
breeding cycles in anurans in tropical and temperate regions are greatly influenced
by climatic factors and latitudinal distribution (Wiens, 2006; 2007).
Tropically breeding anurans that require heavy rainfall in order to reproduce
are subject to favorable breeding conditions that are sporadic. Although there is an
increased probability of rain during the rainy season, the probability of local rainfall
is unpredictable and this may influence female anurans reproductive strategies
(Lynch and Wilczynski, 2005). In most temperate species, reproductive activity is
cyclic and dependent on a combination of temperature and rainfall. Temperate zone
anurans are explosive breeders (Miwa, 2007). Based on the annual activity,
reproductive cycle of anurans is divided into four phases namely emerging and prebreeding phase, spawning and breeding phase, post-breeding phase, and
hibernation phase (Roy, 1990; Huang et al., 1997; Pancharatna and Saidapur,
2009).
Since anurans are poikilothermic and the majority of them depend upon
water for breeding, their reproductive activities are greatly affected by the changing
external climatic factors such as temperature (Saidapur and Hoque, 1995), rainfall
(Grafe et al., 2004; Lynch and Wilczynski, 2005), daylength (Saidapur, 1989;
Edwards and Pivorun, 1991), environmental iodine levels (Dunn, 2004) and pool
desiccation (Lind et al., 2008). Lunar cycle has also been reported to influence
breeding cycle of some species of anurans (Granta et al., 2009). The administration
of exogenous hormones and hibernation increases the breeding behavior and
gamete release by boreal toads, Bufo boreas borea (Roth et al., 2010). The three
major environmental factors temperature, rainfall and photoperiod have been
implicated in the regulation of the amphibian breeding cycles (Lofts, 1984; Dodd
and Dodd, 1976; Roy, 1994).
The environmental temperature plays a key role in regulating population
density, physical activity, metamorphosis and developmental process of anurans
(Dodd and Dodd, 1976; Reading, 2003; Brown and Cai, 2007). In ectothermic
vertebrates, environmental temperature is believed to play a key role in the control
of metabolic activity, sexual behavior and reproductive activity (Fraile et al., 1989).
Latitudinal distribution and temperature are dependent for embryonic survival,
growth and developmental rates in the common frog, Rana temporaria (Laugen et
al., 2003). Thermal acclimatization at higher temperature increases reproductive
activity such as calling and amplexus where as at lower temperature decreases
reproductive activities in Limnodynastes peronii (Rogers et al., 2007).
Growth, sexual maturation and body size dimorphism in the Indian bull frog,
Hoplobatrachus tigerinus depends upon ambient temperature (Gramapurohit et al.,
2004). Formation of growth marks in the bones of the tropical frog, Rana
cyanophlyctis takes place under natural temperature and daylength (Kumbar et al.,
2002). Effect of temperature on development time and energy expenditure was
studied in terrestrially breeding moss frog, Bryobatrachus nimbus (Mitchell and
Seymour, 2000). Environmental temperature is positively correlated with the rate of
metamorphosis and growth of anurans (Dodd and Dodd, 1976; Hayes et al., 1993).
There is paucity of information on the effects of low temperature on amphibians
metamorphosis. The thermal acclimatization ability in tropical and subtropical
amphibians is dependent on seasons (Chang and Lucy, 2005; Newman, 1998).
The effects of variation in climatic temperature were studied on breeding
activity and metamorphosis in the common toad, Bufo bufo (Reading, 2003).
Breeding activity is highly correlated with variation in climatic temperature in
common toad, Bufo bufo (Reading, 2003). Although the specific response to
temperature can vary widely between species, the most frequent observation in
amphibians with a potentially continuous cycle is that exposure to mild
temperatures (15-200C), stimulates spermatogenesis even during the period of
testicular quiescence (Paniagua et al., 1990). Effects of photoperiod and
temperature on testicular function seems to be the most important external factors
involved in the regulation of breeding cycle in many amphibian species (Lehman,
1997).
The histological evidence indicates that although proliferation of cell nests is
present throughout the year, the most important spermatogenetic activity is initiated
in summer (Delgado et al, 1989). Low temperature and short photoperiod
(daylength)
in
winter
induced
the
arrest
of
spermatogenesis/spermiogenesis
and
activation
the
of
maturation
primary
phase
of
spermatogonia
proliferation in the frog, Rana perezi (Delgado et al., 1992). Thereby, temperature
and photoperiod regulate seasonal testicular activity. Further, sexual differentiation
of gonads has been shown to be temperature-sensitive in many species of
amphibians (Dournon et al., 1990).
Ovarian cycle is also under the control of temperature in bull frog, Rana
tigerina (Pancharatna and Saidapur, 1990). Ovary mass is larger in the temperate
than in the subtropical population (Huang and Yu, 2005). Therefore, seasonal
changes in the first growth phase oocytes (FGP) and second growth phase oocytes
(SGP) in anurans may be influenced either by a change in the gonadotrophic
hormones of the pituitary.
Rainfall seems to be another important environmental factor in regulation of
the breeding activity and reproductive cycle in both temperate and tropical anurans
(Grafe et al., 2004; Lynch and Wilczynski, 2005; Brown and Shine, 2007). The first
rain triggers the anurans to come out from their hibernation/hiding and feeding
place. Since their early life history passes in aquatic system, hence water is the
most essential for breeding cycle and metamorphosis of the anuran species.
Reproductive modes also depend upon availability of water (Touchon and
Warkentin, 2008). Water is also essential for male calling, male calls from wetter
nests are more significant for embryonic development (Mitchell, 2000). Further,
males occupying drier nests may have risked of dehydration by calling, and so were
less able to signal to females. Hydration states, therefore, have the potential to
influence the reproductive success of terrestrial male frogs (Mitchell, 2001). There
was a significant interaction between rainfall and sex, dry weather having a stronger
negative effect on males than females as in afro-tropical pig-nosed frog, Hemisus
marmoratus (Grafe et al., 2004).
As in other vertebrates (Reptiles, Birds and Mammals), daylength
(photoperiod) plays an important role in regulation of the annual breeding cycle in
tropical and subtropical anurans. Available evidence suggests that photoperiod,
temperature and rainfall, as the proximate factor determine their seasonal variation
in physiology and physical activity. Environmental temperature and photoperiod
regulate seasonal testicular activity in the frog, Rana perezi (Delgado et al., 1992).
Strong temperature
vs. photoperiod significantly interacts in growth and
development of Rana temporaria tadpole southern populations (Laurila, 2001).
An experiment with blinding and exposure to red light stimulated ovarian
growth and demonstrated that melatonin counteracts blinding or red-light-induced
stimulation of ovarian activity (Joshi and Udaykumar, 2000). A deep brain
photoreceptor molecule „pinopsin‟ discovered in the toad hypothalamus is
reportedly responsible for photoreception (Yoshikawa et al., 1988). In Rana pipiens,
the incidence of both ovulation and normal embryonic development were increased
following exposure of the animals to low temperatures and short daylength
(Lehman,
1997).
However,
light
had
no
positive
role
in
regulation
of
spermatogenesis in the frog, Rana cyanophlyctis (Shivakumar, 1999). Most
amphibians exhibit an annual testicular cycle characterized by a quiescent period
(late autumn-winter) and a spermatogenic period (spring and summer) (Saidapur,
1989). At the end of the period of spermatogenesis, undifferentiated interstitial cells
transform into steroid-secreting leydig cells which regress at the beginning of the
new spermatogenetic cycle (Paniagua et al., 1990). Experiments performed during
the period of germ-cell proliferation indicated that low temperatures (below 11 0C) as
well as short photoperiods (less than 8 hrs.) hinder germ-cell proliferation where as
moderately high temperatures (about 30 0 C) and long photoperiod (above 12 hrs.)
accelerate this proliferation (Paniagua et al., 1990). The spermatozoa are normally
retained in the testis in winter (low photoperiod) where as spermatozoa are
released during breeding period (high photoperiod) in bullfrog, Rana catesbeiana
(Sprando and Russel, 1988). Continuous normal light for 30 days increased
gonado-somatic index (GSI), whereas continuous injections of melatonin decreased
the GSI in the skipper frog, Rana cyanophlyctis (Udaykumar and Joshi, 1996,
1997).
The sense of olfaction (odor/pheromone/chemosensory) is another cue for
migration to breeding sites by anurans (Ishii et al., 1995; Park et al., 2004). Odor or
scent is the main factor in recognisition of sex and species (Diwan, 1995; Ishii et al.,
1995). Odor seems to be the major factor in orientation and movements to the
breeding sites for some species like Amazonian frog (Ishii et al., 1995). The sexual
selection has been reported to drive behavioral isolation and speciation among
populations of an Amazonian frog, Physalaemus petersi. Sex-pheromone secreted
by males and females also attract for mating in frogs (Kikuyama, 2002). Moreover,
each pheromone secreted by the male acts on conspecific females (Kikuyama et
al., 2005; Kikuyama, 2008).
The major factor in anuran courtship is the production of advertisement calls
(vocalization) by males (Kelly, 2004). Male advertisement vocalization in frogs is
known to be one of the energetically most expensive activities of ectothermic
vertebrates (Emerson and Hess, 2001). Vocalizations attract female anurans to
breeding sites, and there is growing experimental evidence to support auditory
orientation in anurans (Ryan et al., 2001, 2007). The vocalization of frogs has
provided means for investigating acoustic communication (Emerson and Boyd,
1999; Kelly, 2004). Vocal communication plays an important role in behavior
ranging from territorial defense to reproduction. The anuran calls are classified
according to the particular behaviors that they serve. Vocal advertisement is
generally the domain of males (Kelly, 2004; Shen, 2008). Sexual communication in
anuran amphibians has focused heavily on the advertisement call made by
reproductive males (Bowcock et al., 2007). Adult male anurans produce a speciesspecific mating call which is used to attract conspecific females during their mating
season, and this call serves as a mechanism to maintain reproductive isolation from
other sympatric species (Roy, 1994; Ryan, 2007). Males produce specific calls as
an attractive courtship signal. In addition other kinds of calls are also emitted such
as territorial call, encounter call and mating call (Lode, 2001; Filho et al., 2008). The
recognition of courtship calls in a chorus may play a useful role in long-term
regulation of anuran breeding activity, especially in distantly placed partner. Male
produce three distinct vocalizations: (1) an advertisement call that attracts both
males and females, (2) an encounter call which is used in territorial interactions and
(3) a courtship call that is only produced when males perceive a female in their
immediate vicinity (Robertson, 2006). Male bullfrogs emit multicroak, quasi
harmonic advertisement calls that function in mate attraction and neighbor
recognition (Simmons, 2004).
Females always select advertisement calls of a heavier male (Robertson,
2006). Males are always combating for sexual partners, but some turgid female
toads give males the slip: a new mechanism of female mate choice in the anurans
(Bruning et al., 2010). Males occupying drier nests may have risked dehydration by
calling and so were less able to signal to the females toad lets (Mitchell, 2001).
Females responded faster to high call rates, and female vocal activity was greater
in response to low-frequency male calls in Iberian midwife toad, Alytes cisternasii
(Bee and Gerhardt, 2002). Females usually exhibit strong and unequivocal
recognition of conspecific mating signals and reject those of other sympatric heterospecifics (Bee and Gerhardt, 2002; Ryan et al., 2007). Receptive females and
males of Bufo terrestris responded positively at a distance up to 40 m to a recording
of a conspecific chorus (Duellman and Trueb, 1994). Female poison frogs prefer to
mate with good caller because calling performance is an honest indicator of
paternal genetic quality of the male (Frosman and Hagman, 2006). Females are
typically silent, but in a few anuran species they can produce a feeble reciprocal call
or rapping sounds or rapid trills during courtship (Watson and Kelly, 1992; Elliott
and Kelly, 2007; Shen et al., 2008). Androgen levels in females at this time are
significantly higher than even those levels in males (Emerson and Boyd, 1999;
Burmeister and Wilczynski, 2001). Arginine-8 vasotocin inhibits the call by causing
an accumulation of water and internal pressure (Diakow, 1978).
In India, advertisement call (vocalization) has been studied in Polypedates
maculatus (Kanamadi et al., 1993), Ramanella montana (Kadadevaru et al., 1998),
Kaloula pulchara (Kanamadi et al., 2002), and Polypedates leucomystax (Roy,
2002). Morphological and acoustic comparisons of Microhyla ornata, Microhyla
fissipes, and Microhyla okinavensis (Anura: Microhylidae) are well described for
species identification in Western Ghats (Kuramoto and Joshy, 2006). The
advertisement calls of three Indian frogs, Ramanella triangularis (Microhylidae),
Indirana gundia (Ranixalidae) and Fejervarya rufescens (Dicroglossidae) have been
analyzed and species are characterized in Western Ghats (Kuramoto and Dubois,
2009).
Mating calls of three frog species abundant in North East India Rana
tigerina, Rana cyanophlyctis, and Rana limnocharis were recorded and analyzed in
the fields of Assam and Meghalaya during their breeding season (Roy, 1994; Roy
and Elepfand, 2007; Roy, 2008). A comparison of the mating calls of Rana
cyanophlyctis with those of the sibling Rana ehrenbergi showed differences in their
temporal and spectral characters, supporting the suggestion that these two species
are distinct species, rather than subspecies of the same species (Roy and
Elepfand, 2007). Rana limnocharis in Northeast India is composed of several sub
species. Vocalizations of Rana limnonectes/ Fejervarya limnonectes were studied
in Eastern Himalayas and accordingly species were characterized on the basis of
ossilogram (Borthakur et al., 2007). In Meghalaya, mating calls of Polypedates
leucomystax and Rhacophorus bipunctatus were studied (Iangrai, 2007). The
ossilogram of Polypedates leucomystax showed that each call was composed of
three notes, while that of Rhacophorus bipunctatus each call was composed of six
notes. The difference in the number of notes per call indicates that the call is
species specific (Roy et al., 1998; Iangrai, 2007).
In Orissa, Microhyla ornata and Ramanella variegata usually call from grass
stems or leaves and small branches (Dash and Mahanta, 1993). Polypedates
maculatus commonly calls from branches of trees from low vegetation and from
ground near the water during breeding season (Das and Dutta, 2006). In some
species (e.g., B. melanostictus, B. stomaticus, P. maculatus, Τ. Breviceps, R.
tigerina) the croaking sounds were observed only during the monsoon period (rainy
season). But other two species, (e.g., R. limnocharis and R. Cyanophlyctis) the
croaking was observed throughout the year (Dash and Mahanta, 1993).
The role of gonadotropins in the vitellogenic process and in ovarian
steroidogenesis has been investigated through in vitro experiments in Rana
esculenta (Polzonetti-Magni et al., 1998). The anuran testis is considered as a
model to study germ cell progression during spermatogenesis (Pierantoni et al.,
2002). In the bullfrog, Rana catesbeiana, testicular weight is constant throughout
the year, but the volume and densities of germinal and interstitial compartments
undergo inverse changes from winter (non-breeding) to summer (breeding) (Sasso
et al., 2004). A study in Scandinavian Peninsula found that relative testicular weight
varies and testis weight declines towards the subarctic in the frog, Rana temporaria
(Hettyey et al., 2005). In the reproductive cycle of hylid frog, Dendropsophus
minutus, testicular morphometry is characterized by a continuous gametogenesis
(Santos and Oliveira, 2007). In Rana temporaria, testicular steroid metabolism of
winter and spring frogs showed marked seasonal differences (Antila and Saure,
1979). The spermatogenic activity of Rana ridibunda living in the East Marmara
region was found to be potentially continuous type. Further, the components of
thumb pads exhibited structural changes with respect to the activities of Leydig cells
(Kaptan and Murathanoglu, 2008).
In Bufo melanostictus, plasma androgen, plasma testosterone and changes
in the weights of testes, liver, and fat bodies activity were higher during breeding
period (Huang et al., 1997). Histological evidence indicated that the spermatogenic
cycle of Bufo melanostictus is of a fluctuating continuous type. Although cell nests
of all spermatogenic cell types were present throughout the year, however, the
highest intensity of spermatogenic activity occurred in the month of March (Huang
et al., 1997). Studies have been undertaken on changes in the cytomorphology of
gonadotrophs during the breeding cycle of the male bull frog, Rana tigerina
(Pancharatna and Saidapur, 1990). The gonado-somatic index (GSI) of bullfrogs,
Rana catesbeiana showed no significant variations during different months of the
year (Sasso-Cerri et al., 2004), whereas in Rana cyanophlyctis exposure to
continuous light for 30 days stimulated the GSI and melatonin treatment for 30 days
decreased the GSI (Udaykumar and Joshi, 1997).
Temperate zone female anurans typically have annual ovarian cycles that
are seasonally correlated. In contrast, tropical anurans have diverse patterns of
ovarian cycles (Jorgensen et al., 1979; Rastogi et al., 1983). Another remarkable
characteristic of anurans is the change of ovarian cyclicity in correlation with the
variation in environmental conditions (Kanamadi and Saidapur, 1982; Pancharatna
and Saidapur, 1992). Among anuran amphibians, cyclic ovarian changes have
been reported in Xenopus laevis (Dumont, 1972), Bufo bufo (Jorgensen et al.,
1979), Rana esculenta (Rastogi et al., 1983), Rana cyanophlyctis (Pancharatna and
Saidapur, 1985), Bufo melanostictus (Kanamadi et al., 1989), Rana perezi (Delgado
et al., 1990), and Polypedates maculatus (Kanamadi and Jirankali, 1991).
The classification of developing oocytes of anurans had been carried out by
many workers (Saidapur and Hoque, 1995). Role of temperature in regulation of
ovarian cycle in bull frog, Rana tigrina was studied by exposing them to different
temperature (Pancharatna and Saidapur, 1990). Depending upon the phase of the
oogonial proliferation and the reproductive cycle and/or the pattern of oogenetic
activity, the ovary contains oogonia, first growth phase oocytes (FGP), second
growth phase oocytes (SGP) and matured ovum (Saidapur and Hoque, 1995;
Khanna and Yadav, 2005). The rate of somatic development of the ovary in
anurans is correlated with the rate of gonad differentiation and varies from species
to species (Khanna and Yadav, 2005). FGP and SGP or vitellogenic oocytes were
produced in both the captive and wild caught frogs (Rana cyanophlyctis) throughout
the year (Pancharatna and Saidapur, 1992). Ovarian follicular kinetics and
gravimetric changes in the ovary were studied in the skipper frog, Rana
cyanophlyctis (Pancharatna and Saidapur, 1992; Udaykumar and Joshi, 1996). A
quantitative study of follicular kinetics in relation to body mass, oviduct, and fat body
cycles were studied in Rana cyanophlyctis (Pancharatna and Saidapur, 2009). The
progression of ovarian cycles has been studied in, Rana tigrina (Girish and
Saidapur, 2000).
The temperate anurans exhibit breeding activity throughout the year while
subtropical anurans breed only from March to September (Huang and Yu, 2005).
The annual reproductive pattern of anurans has been studied from the temperate
and subtropical regions. Studies on the tropical anuran species are comparatively
less. Mating success of individual male frogs within explosive breeding species can
depend on their ability to compete for a mate and to hold onto that mate during
amplexus. Such importance of amplexus has resulted in the evolution of sexual
dimorphism in the morphology and anuran forelimb muscles used during amplexus
(Navas and James, 2007).
In India, based on observations on the annual breeding cycle of
Rhacophorus maculatus (Mishra and Das, 1984), Rana limnocharis (Roy, 1990),
Bufo melanostictus (Huang et al., 1997), Polypedates maculatus (Das et al., 2001),
Hyla annectans (Ao and Bordoloi, 2001), Chirixalus simus (Deuti, 2001), Paa
annandalii (Bordoloi et al., 2001), Polypedates leucomystax (Iangrai 2007), and
Rhacophorus bipunctatus (Iangrai, 2007) have been established. It has been found
that the annual testicular cycle of these species consists of four phases, though the
breeding timings differ from species to species. Based on observations on annual
activity cycle and gonadal histology, the annual breeding cycle of male anurans has
been divided into four phases, namely emerging and pre-breeding period, spawning
and breeding period, post-breeding period and hibernation period (Huang et al.,
1997; Roy, 2003; Iangrai, 2007; Pancharatna and Saidapur, 2009).
Generally the female frogs select oviposition sites based on factors such as
water depth, water temperature, water pH, presence or absence of predators
(Khanna and Jadav, 2005). According to Duellman and Trueb (1994), there are 29
ways of egg deposition. But according to Haddad and Prado (2008), there are more
than 29 reproductive modes in anurans. Based on daily monitoring of data on
anuran oviposition, it has been reported that there can be 69 types of natural
oviposition sites during a complete reproductive season (Rudolf and Rodel, 2005).
The most common and phylogenetically widespread site of oviposition is in standing
water (Mode 1), or flowing water (Mode 2), eggs arboreal and tadpoles aquatic
(Mode 4, 6, 18 19 & 20), and eggs terrestrial and tadpoles aquatic (Mode 12, 13, 14
& 24) (Duellman and Trueb, 1994). Many anurans evolved reproductive modes to
meet the special conditions. Such modes include breeding terrestrially and
arboreally, making foam nests, parental transport of eggs and/or tadpoles, direct
development. Other modes are ovo-viviparity and viviparity (Wake and Dickie,
1998, Buckley et al., 2007).
In Western Ghats, diversity of egg laying had been reported. In
Rhacophorus pseudomalabaricus, foam nest construction is done by females, and
eggs are fertilized by sperm secreted by male malabaricus (Vasudevan and Dutta,
2000). In case of a rare microhylid frog, Ramanella montana while in axillary
amplexus, the male clasped the female and pressed her abdomen against the tree
trunk, which apparently facilitated egg deposition. Eggs were deposited and
attached to the surface of the tree trunk just above the water and on the floating,
dried leaves (Krishna et al., 2004). In case of Nyctibatrachus humayani, the female
determines oviposition sites, and lays eggs exactly at the spot from where the male
had been calling. There was no amplexus or any physical contact between the
sexes (Kunte, 2004). In case of shrub frog (Philautus glandulosus), eggs
development is direct and takes place in egg membrane, and there are no free
swimming tadpole stages. Further, the eggs undergo direct development and
hatching of frog lets occurs after 28 days (Biju, 2003).
In North eastern region there is less information on egg laying habits of
anurans. All the Ranid frogs like Rana cyanophlyctis (Mahanta-Hejmadi and Dutta,
1979), Rana limnocharis (Roy, 1990; Borthakur et al., 2007), Bufo melanostictus
(Huang et al., 1997), and Paa annandalii (Bordoloi et al., 2001) laid eggs upon the
water surface attached to a substratum, especially to aquatic plants. After courtship,
the amplecting pair lays the eggs in batches covered with jelly capsules.
In contrast to apodans and urodelans, practically all anurans exhibit external
fertilization (Sever, 2002; Beck, 2002; Marjanovi and Laurin, 2007; Buckley et al.,
2007). Internal fertilization is known only in Ascaphus truei (Sever, 2002) and two
species of Eleutherodactylus (Townsend et al., 1981). In India, so far no anuran
species with internal fertilization has been reported.
In anurans, maternal care is restricted in species with internal fertilization,
and male parental care is limited in species with external fertilization (Beck, 2002).
Male parental care is prevalent in neotropical frog, Eleutherodactylus coqui, where
males brood clutches of direct-developing eggs in non-aquatic nest sites and
defend eggs against cannibalistic nest intruders (Townsend, 1986). The male
Australian pouched frog (Assa darlingtoni) has pouches along its side in which the
tadpoles reside until metamorphosis. Male parental care has also been reported in
the genus Alytes (Rafel, 1993). The care of the strings of eggs is carried by male
partner in Alytes cisternasii (Iberian midwife toad), A. dickhilleni (Bentic midwifw
toad), A. obstetricans (Common midwife toad), and A. mulelensis (Mallorcan
midwife toad) (Rafel, 1993). A unique example of parental care is found in the
female gastric brooding frogs, Rheobatrachus silus from Australia (Tyler et al.,
1983). The female carries embryos and young-ones in the stomach and gives births
to the juveniles orally without any injury to young ones (Tyler et al., 1983). There is
no report regarding the male parental care in Indian species.
Metamorphosis (Gr. meta- "change" + morphe "form") is a biological process
generally attributed to amphibians (Bishop et al., 2006). Metamorphosis in anurans
involves spectacular changes such as resorption of tail, development of fore and
hind limbs, changes in organ system such as gill breather to lung breather, etc.
(Dodd and Dodd, 1976). The transformation of tadpole into frog is one of the most
spectacular processes in nature and, consequently, one of the most thoroughly
investigated event (Shi, 2000; Mc Diarmid and Altig, 2000). Anuran metamorphosis
is divided into three specific periods: pre-metamorphosis, pro-metamorphosis and
metamorphosis climax (Mc Diarmid and Altig, 2000; Brown and Cai, 2007).
In Anurans, the metamorphosis and developmental stages reach a higher
degree of modifications and specialization in comparison to apodans and urodelans
(Mc Diarmid and Altig, 2000). Metamorphosis is a model system to study anuran
organogenesis (Brown and Cai, 2007). Metamorphosis has been studied as a
series of transcriptional programs controlled by thyroid hormones (TH). During
metamorphosis, distinct remodeling has been reported in tail resorption (Huang
and Brown, 2000; Yaoita and Nakajina,1997), muscles (Nicolas et al., 1998;
Gaillord et al., 1999; Cai et al., 2007), intestine (Shi and Brown, 1993), pancreas
(Shi and Brown, 1990; Maake et al., 1998), kidney (pronephros to metanephros),
respiratory organs (gills to lungs) (Dodd and Dodd,1976), liver (Atkinson et al.,
1998), immune system (Rollins-Smith, 1998), brain and spinal cord (Kollros, 1981),
eyes (Hoskins, 1986; Mann and Holt, 2001), nose (Higgs and Burd, 2001), pituitary
gland (Kikuyama et al., 1993; Huang et al., 2001), hematopoetic system (Weber,
1996) and most of the skeleton (Trueb and Hanken, 1992).
In nature, anuran metamorphosis is accelerated by a number of ecological
factors (extrinsic factors) such as increasing temperature (Saidapur and Hoque,
1995), rainfall (Lynch and Wilczynski, 2005), photoperiod (Saidapur, 1989), pool
desiccation (Lind et al., 2008), diet quality (Nicieza et al., 2006), environmental
iodine levels (Dodd and Dodd, 1976) and pond hydrology (Ryan and Winne, 2001).
These factors play an important role in determining the rate and fate of
metamorphosis (Hayes, 1997). The iodine is essential for the synthesis of thyroid
hormones. Hence, sufficient amounts of iodine must be present in the diet and/or
water. Another factor that is likely to function through a neuroendocrine pathway is
light, which regulates melatonin synthesis, and hence thyroid physiology and
metamorphosis (Wright et al., 1990).
Metamorphosis is mainly controlled by thyroid hormones (TH) secreted by
the thyroid gland (Hanken and Hall, 1988; Huang and Brown, 2000). The anuran
metamorphosis is controlled by the hypothalamus-pituitary-thyroid axis involving
actions of several hormones (Hanken and Hall, 1988; Page et al., 2008; Huang and
Brown, 2000). Environmental factors stimulate release of thyroid releasing hormone
(TRH) by the hypothalamus, which stimulates secretion of thyroid stimulating
hormone (TSH) from the pituitary. TSH stimulates secretion of thyroid hormones
(TH) namely 3, 5, 3‟-triiodothyronine (T3) and 3, 5, 3‟5‟-tetraiodothyronine (T4) from
the thyroid gland. An increased concentration of T4 has been reported to accelerate
metamorphosis of anuran tadpoles (Page et al., 2008).
Thyroid hormones play a critical role in the morphological transformations
during metamorphosis in larval bullfrogs, Rana catesbeiana (Galton, 1988;
Fernandez-Mongil et al., 2009). Its effects are mainly mediated through
transcriptional regulation by T3 receptor (TR) (Das et al., 2008). Anuran
metamorphosis serves as an excellent model to study T3 function during postembryonic development in vertebrate due to its total dependence on TH (Wang et
al., 2006; Page et al., 2009). The thyroid hormone receptor functions as a master
control factor that can both activate and repress genes in controlling the
transformation of the larval tadpole to the adult frog. Transcription studies have
shown that TR activates or represses TH-inducible genes by recruiting co-activators
or co-repressors in the presence or absence of TH, respectively. However, the
developmental roles of the co-activators or co-repressors of TR remain largely
unexplored (Lorenz et al., 2009).
Besides thyroid hormones, prolactin also plays a critical role in regulation of
anuran larval development and metamorphosis (Dodd and Dodd, 1976; Takada and
Kasai, 2003). Prolactin mainly helps in metamorphosis in early part of the life
history. It has not been detected after 34 day of developing tadpole in gray tree
frog, Hyla versicolor (Beachy et al., 1999). The growth of post-metamorphic
anurans is stimulated by somatotropin but not by prolactin (Frye et al., 2004).
Corticoids (e.g., corticosterone) and the sex steroids (especially 17ßestradiol)
potentially
regulate
thyroid
hormone activity
both
by
affecting
hypothalamic and pituitary control of thyroid hormone secretion and also by
interacting with thyroid hormones peripherally (Hayes, 1997). Corticosteroids disrupt
amphibian metamorphosis by complex modes of action including increased
prolactin expression (Lorenz et al., 2009).
In India, out of 303 species of amphibians, the developmental stages of only
few species have been studied and documented (Das and Dutta, 2007, Ahmed et
al., 2009). The total duration of metamorphosis of anurans varies from species to
species as reported in Bufo melanostictus (35-50 days, Khan, 1965), Polypedates
maculates (55 days, Mohanty and Dutta, 1986), Rhacophorus malabaricus (68
days, Sekar, 1989), Rana pipens (90 days, Taylor and Shumway, 1990), Hyla
annectans (64 days, Ao and Bordoiloi, 2001), Philautus glandulosus (28 days, Biju,
2003), Polypedates leucomystax (60-61 days, Iangrai, 2007), and Rhacophorous
bipunctatus (59-60 days, Iangrai, 2007).
Appropriate staging of the larval period is fundamental to life history of
anurans. Gosner (1960) gave a simplified table for staging anuran embryos and
larvae with notes of identification. Mc Diarmid and Altig (2000) suggested the
complete tables of development for accurate comparison of development stages in
different anurans with 46 Gosner stages. There is paucity of information on the
development and metamorphosis in Indian anuran species.
According to the IUCN Red Data Book (Version 2009.1), there are 787
species of amphibians worldwide listed in the endangered category (Frost et al.,
2008). These species need serious attention for their in situ conservation. As a first
step in this direction, the breeding biology of the listed endangered species needs
to be investigated in their natural habitat as well as under captivity (Daniels, 1990;
Hoffman, 2009).
The global amphibian crisis has resulted in renewed interest in captive
breeding as a conservation tool for amphibians (Gupta, 1998; Griffiths and
Pavajeau, 2008). Captive breeding programme and reintroduction of anurans are
employed for species which are locally extinct and might help in sustaining
populations (Daniels, 1990). Tropical frogs and toads are disappearing worldwide
due to habitat damage or destruction. The tropical forests of India are also under
human pressure, and many species of anurans are believed to be locally extinct or
at the verge of extinction. The bronzed frog (Rana temporalis) and Malabar torrent
toad (Ansonia ornate) in south India are extinct, but were present 50 years ago
(Daniels, 1992).
Most captive breeding and reintroduction programme for
amphibians have focused on threatened species in industrialized countries with
relatively low amphibian diversity (Griffith and Pavajeau, 2008). The conservation
status of all the amphibians in China is analyzed, and the country has shown
priority for conservation in comparison to many other countries of the world (Xie et
al., 2007).
Significant advances have been made during the last decade for amphibian
assisted reproduction including the use of exogenous hormones for induction of
spermiation and ovulation, in vitro fertilization, short-term cold storage of gametes
and long-term cryopreservation of spermatozoa (Kouba and Vance, 2009). The
endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive
breeding and reintroduction programme. Because Wyoming toads in captivity rarely
ovulate spontaneously, and therefore, hormonal induction is used to ovulate
females or to stimulate spermiation in males (Browne et al., 2006). The Mullorcan
mid wife toad (Alytes muletensis) are conserved by captive breeding programmed
by Jersy Wildlife Preservation Trust, Jersy (Morgan et al., 2008).
In India, captive breeding of common Indian frogs and toads has been
undertaken
in
Bufo
melanostictus,
Euphylyctis
cyanophlyctis,
Euphylyctis
hexadactylus, Hoplobatrachus crassus, Limnonectes keralensis, Limnonectes
limnocharis, Rana temporalis and Tomopterna breviceps (Gupta, 1998). The
captive breeding programme is being introduced to conserve the near-threatened
species Ramanella montana endemic to the Western Ghats (Krishna et al., 2004)
and an endangered tree frog, Rhacophorus lateralis located in coffee plantation in
Kerala (Dinesh et al., 2010).
A critical review of the literature clearly indicates that most of the studies on
breeding biology, reproductive behavior, development and metamorphosis of
amphibians have been conducted on temperate zone species. Limited information
is available on the breeding cycle, reproductive behavior, development and
metamorphosis of amphibian species found in India. Further, the available
information on breeding biology of amphibians in India is fragmentary in nature. So
far no attempt has been made to study the breeding biology of any endangered
and/or threatened amphibian species in any part of the country. Rana leptoglossa is
one of the rare and endangered anuran amphibian species in India (Biodiversity
Conservation Prioritization Project, India, CAMP Workshops REPORT, 2001).
There is paucity of information on population density, breeding biology, gonadal
cycle, developmental stages and metamorphosis of the frog, Rana leptoglossa
under its natural habitat as well as under captive condition in India. Therefore,
keeping in view the endangered and data deficient status of the frog, it was thought
worthwhile to investigate reproductive biology, gonadal cycle, developmental stages
and metamorphosis of the frog, Rana leptoglossa at the Kakoijana Reserve Forest
(KRF), Bongaigaon, Assam. The present dissertation will provide basic information
on reproductive biology, development and metamorphosis of the endangered frog,
Rana leptoglossa (Cope, 1868).
DECLARATION
NORTH- EASTERN HILL UNIVERSITY
SHILLONG -793 022
I, Mr. Biplab Kumar Saha, hereby declare that the subject
matter of this thesis is the record of the work done by me, that the
contents of this thesis did not form the basis of the award of any
previous degree to me or to the best of my knowledge to any body
else, and that the thesis has not been submitted by me for any
research degree in any other University /Institute.
This is being submitted to the North-Eastern Hill University for
award of the degree of Doctor of Philosophy in Zoology.
Prof. R. N. K. Hooroo
(Head)
Prof. B. B. P. Gupta
(Supervisor)
Mr. B. K. SAHA
(Candidate)
Fig.1.1: Male Rana leptoglossa (Cope, 1868)
Fig.1.2: Female Rana leptoglossa (Cope, 1868)
Fig.1.3: Dorsal view of Rana leptoglossa (Cope, 1868)
AIE VALLEY DIVISION

KAKAIJANA RESERVE
FOREST
B
K
O
H
U
T
A
N
MANAS RIV E R
K
R
A
J

MANAS RES E RV E D FORE S T
H
B
A
R
P
E
T
A

A
MANAS RIV E R
R
NH 31
To Guwahati

NH31
BONGAIGAON
INDEX
D
H
U
B
R
I
International Boundary
State Boundary
Rivers

BRAHMAP UTRA RIV ER

Roads
Railway
Map1.1: Map of Kakoijana Reserve Forest in Aie Valley Division, Assam, India.
Map1.2: Map of Kakoijana Reserve Forest (KRF) showing selected breeding sites.
Table 1. 1: Latitudes, longitudes and altitudes of selected breeding sites of
Rana leptoglossa at Kakoijana Reserve Forest (K. R. F)
Site/ Plot No.
Latitudes
Longitudes
Altitude
(ASL)
Site 1
260 28' 6.7″ N
900 38' 33.5″ E
59 m
Site 2
260 27' 50″ N
900 38 ' 35.9″ E
46 m
Site 3
260 27' 46.1″ N
900 38' 35.6″ E
49 m
Site 4
260 27 ' 2″ N
900 38' 35.3″ E
57 m
Site 5
260 27 ' 20″ N
900 38' 36″ E
67 m
Site 6
260 28' 8″ N
900 38' 36.4″ E
48 m
Site 7
260 28 ' 16″ N
900 37' 6.9″ E
45 m
Site 8
260 27' 50″ N
900 37 ' 9″ E
71 m
Site 9
260 27 ' 57.9″ N
900 36' 50.9″ E
70 m
Site 10
260 28' 5.4″ N
900 37' 10.9″ E
76 m
Table 1.2: Criteria for morphometric measurements (Chanda, 1994)
Criterion
Snout-vent length
Head length
Head width
Eye Diameter
Inter-orbital space
Snout length
Tympanic Diameter
Length of Forelimb
Length of Hand
1st Finger length
2nd Finger length
3rd Finger length
4th Finger length
Hind limb length
Length of Tibia
Foot length
1st toe length
2nd toe length
3rd toe length
4th toe length
5th toe length
Abbrevi
ations
SVL
HL
HW
ED
IOS
SL
TD
LF
LH
F1
F2
F3
F4
HLL
TBL
FL
T1
T2
T3
T4
T5
Details of the morphometric parameters
From tip of snout to vent
From the angle of the jaw to tip of snout
At angle of jaw
Distance from posterior corner to anterior corner of eye
Maximum gap between two eyes
From tip of snout to anterior corner of eye
Greatest tympanum diameter along horizontal plane
From the proximal end of forelimbs to tip of longest finger
From the base of the palm to tip of longest finger
From the base of palm to tip of 1st finger
From the base of palm to tip of 2 nd finger
From the base of palm to tip of 3rd finger
From the base of palm to tip of 4 th finger
From mid-ventral line of leg with body to tip of longest toe
Distance between surface of knee to surface of heel
From the base of foot to tip of longest toe
From the base of phalange to tip of 1st toe
From the base of phalange to tip of 2 nd toe
From the base of phalange to tip of 3 rd toe
From the base of phalange to tip of 4 th toe
From the base of phalange to tip of 5th toe
Table 1.3: Morphometric measurements of Rana leptoglossa
Sl.
No.
1
Criterion
Snout-vent
Males (N=10)
Females (N=10)
Range
(mm)
Mean ±
S. E
Range
(mm)
Mean ±
S. E
41-59.1
49.83
32.5-69
57±3.4*
Results of one-way
ANOVA
F-ratio Level of
significance
3.158
0.092
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
length (SVL)
Head
length
(HL)
Head
width
(HW)
Eye
diameter
(ED)
Inter-orbital
space (IOS)
Snout
length
(SL)
Tympanic
diameter (TD)
Length of
forelimb (LF)
Length of Hand
(LH)
1st Finger length
(F1)
2nd Finger
length (F2)
3rd Finger length
(F3)
4th Finger length
(F4)
Hind limb length
(HLL)
Length of Tibia
(TBL)
Foot length (FL)
1st Toe
(T1)
2nd Toe
(T2)
rd
3 Toe
(T3)
4th Toe
(T4)
5th Toe
(T5)
±1.9*
17.52±0.69c
14.02
0.001
14.5±0.25
15.619.5
14-18
16.0±0.70a
7.505
0.013
5.5-6.3
5.95±0.09
6.1-6.5
6.3±0.07
c
17.64
0.001
3.1-3.9
3.49±0.08
3.5-6
5.2±0.53
c
43.22
0.001
6.5-7.5
7.04±0.10
7.6-9
8.83±0.27c
260.967
0.001
3.1-4.1
3.6±0.10
3.8-5
4.6±0.21c
32.609
0.001
25-26.1
25.55±0.10
28.18±0.65
39.348
0.001
11-14.75
13.17±0.44
14.4±0.31b
10.608
0.004
8-8.9
8.5±0.1
13.24±0.25c
807.542
0.001
6.9-7.8
7.35±0.09
11.04±0.09c
1410.183
0.001
10.5-11.5
10.99±0.11
15.7±0.07c
2018.776
0.001
8.3-9.2
8.75±0.09
14.28±0.23c
1261.935
0.001
78.1-87.2
82.58±0.97
26.430.1
13.515.3
12.513.8
10.811.3
15.515.9
13.814.9
88-92
90.0±0.70c
52.095
0.001
25.4-26.3
25.85±0.09
28-31.5
29.9±0.64
c
102.196
0.001
20.8-21.8
21.32±0.10
27-31
29.0±0.70c
275.963
0.001
length
4.6-5.5
5.05±0.09
10-12
10.8±0.37c
663.462
0.001
length
9.6-10.6
10.1±0.11
13-15
14.2±0.37c
268.245
0.001
length
14.8-15.9
15.37±0.11
18-22
20.0±0.70c
101.006
0.001
length
20.5-21.8
21.08±0.14
27-31
29.0±0.70c
296.183
0.001
length
16-16.9
16.45±0.09
20-23
21.7±0.53c
221.781
0.001
14.5-17.2
15.75±0.35
13.5-15.7
c
*All values are expressed as mean ± Standard error (S.E.); N = 21.
a, b, c
Differ significantly from the respective parameter of the male: p < 0.05, 0.01 and 0.001,
respectively.
Table 1.4: Environmental parameters of Kakoijana Reserve Forest during 2005
Year/
Month
Minimum
Temperature
Maximum
Temperature
Average
Temperature
Rain
fall
Daylength
(hour)
Relative
Humidity
(oC)
(oC)
(oC)
(mm)
12.54 ± 0.25*
21.08 ± 0.47*
16.81 ±
0.30*
0
10.70 ±
0.02*
81.35 ±
1.47*
FEB
17.06± 0.44
25.83 ± 0.25
21.45± 0.25
0.6
11.26±
0.03
73.92 ±
1.09
MAR
21.38 ± 0.33
28.76 ± 0.17
25.07 ± 0.12
22.8
11.96 ±
0.03
63.27±
1.01
APR
23.19 ± 0.13
27.53 ± 0.07
25.36 ± 0.06
30.5
12.65 ±
0.05
77.78 ±
0.50
MAY
22.28 ± 0.24
28.85 ± 0.38
25.46 ± 0.28
75.6
13.40±
0.02
JUN
24.57 ± 0.25
30.77 ± 0.36
27.73 ± 0.23
16.1
13.73
±0.006
76.90 ±
1.22
JUL
25.74 ± 0.26
30.64 ± 0.50
28.19 ± 0.35
52.3
13.6 ±
0.02
87.0 ±
0.86
AUG
26.82 ± 0.15
31.26 ± 0.33
29.04 ± 0.21
263.2
13.05 ±
0.03
83.1 ±
1.00
SEP
26.12 ± 0.31
32.01 ± 0.47
29.06 ± 0.35
99.0
12.31 ±
0.04
79.22 ±
2.73
OCT
22.36 ± 0.21
25.30 ± 0.26
23.83 ± 0.13
66.4
11.55 ±
0.03
84.85 ±
1.15
NOV
18.99 ± 0.28
25.00 ± 0.20
21.99 ± 0.20
1.4
10.88 ±
0.03
77.4 ±
1.89
DEC
14.44 ± 0.25
23.46 ± 0.14
18.92 ± 0.16
0
10.53
±0.006
73.7.9 ±
0.60
2005
JAN
(%)
78.67±
0.83
* All values are expressed as mean ± standard error (S. E.), N=12.
Table 1.5: Environmental parameters of Kakoijana Reserve Forest during 2006
Year/
Month
Minimum
Temperature
(0C)
Maximum
Temperature
(0C)
Average
Temperature
(0C)
Rain
fall
(mm)
Day
Length
(hour)
Relative
Humidity
(%)
12.54 ±
0.25*
21.08 ± 0.47*
16.81 ±
0.30*
51.2
10.70
±
0.02*
81.35 ±
1.47*
FEB
16.85 ± 0.41
25.72 ± 0.24
21.28 ± 0.22
9.1
11.26
± 0.03
74.19 ±
1.29
MAR
19.88 ± 0.27
30.63 ± 0.37
25.25 ± 0.21
16.8
11.96
± 0.04
56.56 ±
1.69
APR
21.45 ± 0.35
29.04 ± 0.64
25.24 ± 0.44
37.8
12.73
± 0.03
73.15 ±
1.98
MAY
24.14 ± 0.30
32.03 ± 0.42
28.09 ± 0.32
119.1
13.40
± 0.02
70.25 ±
1.73
JUN
25.52  0.18
30.5  0.24
28.01  0.16
136.1
13.72

0.006
80.88 
1.00
JUL
26.97 ± 0.13
32.31 ± 0.18
29.64 ± 0.12
53.6
13.58
± 0.02
75.58 ±
2.6
AUG
27.24 ± 0.17
33.20 ± 0.30
30.22 ± 0.21
18.3
13.02
± 0.03
75.95 ±
1.07
SEP
25.41± 0.18
30.25 ± 0.47
27.83 ± 0.30
7.8
12.29
± 0.03
80.06 ±
1.09
OCT
23.28 ± 0.33
29.57  0.18
26.43 ± 0.22
0
11.52
± 0.03
77.53 ±
0.73
NOV
18.83 ± 0.39
25.96 ± 0.34
22.39 ± 0.35
3.1
10.87
± 0.28
79.26 ±
0.97
DEC
14.70 ± 0.17
22.93 ± 0.17
18.81 ± 0.14
0
10.53
±
0.007
2006
JAN
* All values are expressed as mean ± standard error (S. E.), N=12.
81.90 ±
1.04
Table 1.6: Environmental parameters of Kakoijana Reserve Forest during 2007
Year/
Month
Minimum
Temperature
(0C)
Maximum
Temperature
(0C)
Average
Temperature
(0C)
Rain
fall
(mm)
Day
Length
(hour)
Relative
Humidity
(%)
2007
JAN
11.6 ± 0.30*
21.32 ±
0.29*
16.46 ±
0.25*
0
10.38 ±
0.31*
81.74 ±
0.73*
FEB
15.04 ± 0.23
22.63 ± 0.53
18.84 ± 0.31
2.6
11.25 ±
0.03
77.5 ±
1.9
MAR
18.69 ± 0.57
28.12 ± 0.38
23.40 ± 0.38
8
11.98 ±
0.04
62.53 ±
2.36
APR
22.13 ± 0.38
30.15 ± 0.52
26.14 ± 0.41
25.2
12.76 ±
0.03
76.56 ±
1.09
MAY
24.97 ± 0.23
33.30 ± 0.43
29.14 ± 0.30
38.6
13.40 ±
0.02
69.56 ±
1.24
JUN
26.27  0.31
30.56  0.50
28.41  0.36
70.3
13.69 
0.008
76.55 
0.59
JUL
26.90 ± 0.19
30.64 ± 0.58
28.77 ± 0.38
55.5
13.55 ±
0.024
81.51 ±
0.80
AUG
26.48 ± 0.20
31.66 ± 0.40
29.07 ± 0.28
15.8
13.43 ±
0.06
82.98 ±
0.85
SEP
26.50± 0.07
28.15 ± 0.20
27.32 ± 0.12
6
12.30 ±
0.04
72.42 ±
1.4
OCT
25.15 ± 0.30
30.38  0.38
27.76 ± 0.33
15.4
11.56 ±
0.04
80.59 ±
0.69
NOV
19.46 ± 0.38
26.46 ± 0.31
22.96 ± 0.32
0
10.88 ±
0.02
77.4 ±
0.80
DEC
13.84 ± 0.28
22.36 ± 0.30
18.10 ± 0.27
0
10.54 ±
0.04
* All values are expressed as mean ± standard error (S. E.), N=12.
83.8 ±
0.56
Table 1.7: Average temperature at KRF, Bongaigaon, Assam during three consecutive
years (2005, 2006 and 2007)
Temperature (0C)
Temperature (0C)
Temperature (0C)
2005
2006
2007
Average
Temperature
(0C)
Jan
16.81 ± 0.30
16.81 ± 0.30
16.46 ± 0.25
16.69 ± 0.28*
Feb
21.28 ± 0.22
21.28 ± 0.22
18.84 ± 0.31
20.44 ± 0.25
Mar
25.25 ± 0.21
25.25 ± 0.21
23.40 ± 0.38
24.63 ± 0.26
Apr
25.24 ± 0.44
25.24 ± 0.44
26.14 ± 0.41
25.52 ± 0.43
May
28.09 ± 0.32
28.09 ± 0.32
29.14 ± 0.30
28.19 ± 0.31
Jun
28.01  0.16
28.01  0.16
28.41  0.36
28.14 ± 0.68
Jul
29.64 ± 0.12
29.64 ± 0.12
28.77 ± 0.38
29.35 ± 0.20
Aug
30.22 ± 0.21
30.22 ± 0.21
29.07 ± 0.28
29.83 ± 0.23
Sept
27.83 ± 0.30
27.83 ± 0.30
27.32 ± 0.12
27.66 ± 0.24
Oct
26.43 ± 0.22
26..43 ± 0.22
27.76 ± 0.33
26.87 ± 0.25
Nov
22.39 ± 0.35
22..39 ± 0.35
22.96 ± 0.32
22.58 ± 0.34
Dec
18.81± 0.14
18.81± 0.14
18.10± 0.27
18.57 ± 0.18
Months
* All values are expressed as mean ± standard error (S. E.), N=12.
Table 1.8: Average daylength at KRF, Bongaigaon, Assam during three consecutive
years (2005, 2006 and 2007)
Months
Daylength (hrs)
2005
Daylength (hrs)
2006
Daylength (hrs)
2007
Average
Daylength (hrs)
Jan
10.70 ± 0.02
10.70 ± 0.02
10.38 ± 0.31
10.59 ± 0.11*
Feb
11.26 ± 0.03
11.26 ± 0.03
11.25 ± 0.03
11.25 ± 0.03
Mar
11.96 ± 0.03
11.96 ± 0.04
11.98 ± 0.04
11.96 ± 0.03
Apr
12.65 ± 0.05
12.73 ± 0.03
12.76 ± 0.03
12.71 ± 0.03
May
13.40 ± 0.02
13.40 ± 0.02
13.40 ± 0.02
13.40 ± 0.02
Jun
13.73 ± 0.006
13.72  0.006
13.69  0.008
13.71 ± 0.006
Jul
13.6 ± 0.02
13.58 ± 0.02
13.55 ± 0.024
13.57 ± 0.02
Aug
13.05 ± 0.03
13.02 ± 0.03
13.43 ± 0.06
Sept
12.31 ± 0.04
12.29 ± 0.03
12.30 ± 0.04
12.30 ± 0.03
Oct
11.55 ± 0.03
11.52 ± 0.03
11.56 ± 0.04
11.54 ± 0.03
Nov
10.88 ± 0.03
10.87 ± 0.28
10.88 ± 0.02
10.87 ± 0.11
Dec
10.53 ± 0.006
10.53 ± 0.007
10.54 ± 0.04
10.53 ± 0.017
13.16 ± 0.04
* All values are expressed as mean ± standard error (S. E.), N=12.
Table 1.9: Average rainfall at KRF, Bongaigaon, Assam during three consecutive
years (2005, 2006 and 2007)
Months
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sept
Oct
Nov
Dec
Rainfall
2005
Rainfall
2006
Rainfall
2007
Average
Rainfall (mm)
0
51.2
0
17.06
0.6
9.1
2.6
4.10
22.8
16.8
8
15.86
30.5
37.8
25.2
31.16
75.6
119.1
38.6
77.60
16.1
136.1
70.3
74.14
52.3
53.6
55.5
53.80
263.2
18.3
15.8
99.10
99.0
7.8
6
37.60
66.4
0
15.4
27.26
1.4
3.1
0
1.50
0
0
0
0.0
Table 1.10: Average relative humidity at KRF, Bongaigaon, Assam during three
consecutive years (2005, 2006 and 2007)
Months
Relative
Humidity (%)
2005
Relative
Humidity (%)
2006
Relative
Humidity (%)
2007
Average
R. H. (%)
Jan
81.35 ± 1.47
81.35 ± 1.47
81.74 ± 0.73
81.48 ± 0.1.2*
Feb
73.92 ± 1.09
74.19 ± 1.29
77.5 ± 1.9
75.20 ± 1.42
Mar
63.27 ± 1.01
56.56 ± 1.69
62.53 ± 2.36
60.78 ± 1.68
Apr
77.78 ± 0.50
73.15 ± 1.98
76.56 ± 1.09
75.83 ± 1.19
70.25 ± 1.73
69.56 ± 1.24
72.82 ± 1.26
May
78.67 ± 0.83
Jun
76.90 ± 1.22
80.88  1.00
76.55  0.59
78.11 ± 0.93
Jul
87.0 ± 0.86
75.58 ± 2.6
81.51 ± 0.80
81.36 ± 1.42
Aug
83.1 ± 1.00
75.95 ± 1.07
82.98 ± 0.85
80.67 ± 0.97
Sept
79.22 ± 2.73
80.06 ± 1.09
72.42 ± 1.4
77.23 ± 1.74
Oct
84.85 ± 1.15
77.53 ± 0.73
80.59 ± 0.69
80.99 ± 0.85
Nov
77.4 ± 1.89
79.26 ± 0.97
77.4 ± 0.80
78.02 ± 1.22
Dec
73.79 ± 0.60
81.90 ± 1.04
83.8 ± 0.56
79.83 ± 0.73
*All values are expressed as mean ± standard error (S. E.), N=12.
Table 1.11: Correlation of average population density with different climatic factors
such as temperature, daylength, rainfall and relative humidity
Months
Average
Coefficient of correlation (r)
population
density
± S.E
(2005 - 2007)
March
0.49 ± 0.029*
April
0.53 ± 0.033
May
0.59 ± 0.029b
June
0.62 ± 0.029c
July
0.55 ± 0.029a
August
0.40 ± 0.020
Temperature
vs.
Population
density
Daylength
vs.
Population
density
Rainfall
vs.
Population
density
Relative
Humidity
vs.
Population
density
0.639d
0.863d
0.676d
-0.336
*All values are expressed as mean ± standard error (S. E.).
d
Significant positive correlation: p < 0.05 level (N=6).
a, b, c
Differ significantly from the values of August (Minimum): P < 0.05, 0.01 and 0.001,
respectively.
Fig.1.4: Average population density of Rana leptoglossa during 2005, 2006 and
2007 at KRF, Bongaigaon.
b
a
c
c
c
c
c
c
c
c
c
c
c
c
c
a
Fig.1.5: Monthly variations between average temperature and population density.
a, b, c
All values are expressed as mean ± standard error (S. E.).
Differ significantly from the values of August: P < 0.05, 0.01 and 0.001, respectively.
b
a
c
c
c
c
c
c
c
c
c
c
c
c
c
a
Fig.1.6: Monthly variations between average daylength and population density.
All values are expressed as mean ± standard error (S. E.).
Differ significantly from the values of August: P< 0.05, 0.01 and 0.001,
respectively.
a, b, c
b
a
c
c
c
c
c
c
c
c
c
c
c
c
c
a
Fig.1.7: Monthly variations between average rainfall and population density.
All values are expressed as mean ± standard error (S. E.).
Differ significantly from the values of August: P < 0.05, 0.01 and 0.001,
respectively.
a, b, c
b
a
c
c
c
c
c
c
c
c
c
c
c
c
c
a
Fig.1.8: Monthly variations between average relative humidity and population density.
a, b, c
All values are expressed as mean ± standard error (S. E.).
Differ significantly from the values of August: P < 0.05, 0.01 and 0.001, respectively.
35
Temperature (0C)
30
25
20
2005
15
2006
10
2007
5
0
0 J F M A M J J A SO N D J F M A M J J A SO N D
Months
J F M A M J J A SO N D
Fig. 1.9: Monthly variations of air temperature at KRF, Bongaigaon during 2005,
2006 and 2007.
Daylength
(hrs)
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
2005
2006
2007
0 J F M A M J J A SO N D
J F M A M J J A SO N D
J F M A M J J A SO N D
Months
Fig. 1.10: Monthly variations of daylength at KRF, Bongaigaon during 2005, 2006
and 2007.
300
2005
250
2006
2007
Rainfall
(mm) (mm)
((mmin
mmmmmm
m
200
150
100
50
0
0 J F M A M J J A SO N D
J F M A M J J A SO N D
J F M A M J J A SO N D
Months
Fig. 1.11: Monthly variations of rainfall at KRF, Bongaigaon during 2005, 2006 and
2007.
90
Relative humidity (%)
80
70
60
50
2005
40
2006
30
2007
20
10
0
0 J F M A M J J A SO N D
J F M A M J J A SO N D
Months
J FM A M J J A SO N D
Fig. 1.12: Monthly variations of relative humidity at KRF, Bongaigaon during 2005,
2006 and 2007.