Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
Age and growth assessment of the near-threatened fish Rita rita (Siluriformes:
Bagridae) in the Ganges basin
Ankita Ankita1; https://orcid.org/0000-0002-3455-3557
M. Afzal Khan1*; https://orcid.org/0000-0002-8197-4311
Salman Khan1,2; https://orcid.org/0000-0001-9651-2657
1. Fish Biology and Otolith Research Laboratory, Department of Zoology, Aligarh Muslim University, India;
khanmafzal@yahoo.com (*Correspondence); ankitaverma0405@gmail.com
2. School of Life Science and Technology, Department of Zoology, IIMT University, India; sksalman21@gmail.com
Received 11-VII-2022. Corrected 03-XI-2022. Accepted 01-II-2023.
Introduction: Rita rita is a freshwater catfish under threat of extinction, mainly from loss of breeding and nurs-
ing grounds. A reliable method for age and growth estimation is needed by fishery managers.
Objective: To identify the best body structure for age and growth estimation.
Methods: We assessed estimates precision based on Average Percent Error (APE), Coefficient Variation (CV),
and Percent Agreement (PA) between readers separately analyzed each calcified structure. We used 390 fish
samples from three rivers, Ganga, Yamuna and Ramganga, from September 2018 to August 2019.
Results: The three indicators favored the use of vertebrae for age estimation; the growth band seems to be annual
and formed from May to September. The growth equations were Lt = 90.19(1-e-0.145(t+0.51)) for Ganga; Lt = 91.19
(1-e-0.14(t+0.59)) for Yamuna and Lt = 89.63 (1-e-0.15(t+0.68)) for Ramganga.
Conclusion: This species reaches moderate growth in these rivers, where vertebrae are the recommended age
estimation structure, followed, in case of need, by sectioned otoliths, whole otoliths and opercular bones. Pectoral
spines should be avoided, especially in older fish.
Key words: vertebrae; otoliths; ageing precision; VBGF; Marginal Increment Analysis.
Edad y crecimiento del pez casi amenazado Rita rita (Siluriformes: Bagridae) en la cuenca del Ganges
Introducción: Rita rita es un pez de agua dulce bajo amenaza de extinción, principalmente por la pérdida de
sitios de reproducción y crianza. Un método fiable para las estimaciones de edad y crecimiento es necesario para
los administradores de recursos pesqueros.
Objetivo: Identificar la mejor estructura corporal para estimaciones de edad y crecimiento.
Métodos: Evaluamos la precisión de las estimaciones mediante el Porcentaje de Error Promedio (APE),
Coeficiente de Variación (CV), y Porcentaje de Acuerdo (PA) entre lecturas para cada estructura calcificada.
Usamos 390 muestras de peces de tres ríos, Ganga, Yamuna y Ramganga, de setiembre 2018 hasta agosto 2019.
Resultados: Los tres indicadores favorecieron el uso de vértebras para la estimación de edad; la banda de
crecimiento parece ser anual y se forma entre mayo y setiembre. Las ecuaciones de crecimiento fueron
Lt = 90.19(1-e-0.145(t+0.51)) para Ganga; Lt = 91.19 (1-e-0.14(t+0.59)) para Yamuna y Lt = 89.63 (1-e-0.15(t+0.68))
para Ramganga.
2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
Rita rita (Hamilton, 1822) belongs to the
family Bagridae, which is distributed in the
freshwaters of India as well as many other
countries in the Asian continent. The target fish
species has high market value on account of its
consumer preference, nutritional value, orna-
mental value and palatability (Gupta & Baner-
jee, 2014; Mohanty et al., 2015). There has been
only one report (Tamubi et al., 1990) on the
age and growth characteristics of this species,
wherein no consideration of precision of age
estimates was taken into account. Molur and
Walker (1998) categorised it as a threatened
fish of the Indian subcontinent and suggested
that it could face the risk of extinction mainly
due to overfishing and loss of breeding sites.
Bangladesh, The International Union for Con-
servation of Nature (IUCN) (2015) categorizes
fish as endangered in Bangladesh but globally
as Least Concern. Therefore, the population of
Rita rita should be scientifically managed and
protected by undertaking suitable measures.
Age estimation of fish is a necessary first
step in age-based fish population assessment
and successful resource management (Maceina
& Sammons, 2006). The ability to obtain precise
age estimates is critical for estimating dynamic
rate functions like mortality and recruitment
(Khan & Khan, 2020). A precision measure is
important for evaluating the relative simplicity
of determining the age of a given hard struc-
ture, examining the reliability of an individual’s
age estimations, and measuring the ager’s level
of skill relative to others (Campana, 2001). By
incorporating a validation method, errors in
estimating the age of fish can be minimized.
There is an alternative validation technique i.e.,
comparing the age estimates between differ-
ent hard anatomical structures that potentially
provide valuable insights into the accuracy and
inaccuracy of age-estimating structures (Sylves-
ter & Berry Jr, 2006).
Researchers have compared age enumer-
ated from different hard structures in fresh-
water fishes such as Channa punctata (Khan
et al., 2013), Labeo bata (Khan et al., 2015), C.
striata (Khan et al., 2017), Sperata aor (Nazir
& Khan, 2020), Leuciscus vorax (Rashid &
Basusta, 2021), Cycleptus elongatus (Radford
et al., 2021), Salvelinus namaycush (Osborne
et al., 2022) to quantify precision and detect
biasesrelated to each structure.
Amongst several methods available to vali-
date fish age interpretations (Campana, 2001),
Marginal Increment Analysis (MIA) is one of
the extensively utilized methods (Campana,
2001). This technique is based on the notion
that growth rings are produced annually, and
the width of the outer ring will depict an
annual sinusoidal cycle if plotted against the
month of capture. (Campana, 2001; Okamura
et al., 2013).
Age estimation in several fish species has
been validated using MIA for example, Merluc-
cius hubbsi (Costa et al., 2018); Brachyplatys-
toma rousseauxii (Hauser et al., 2018). Several
studies have successfully used the von Bertalanffy
Growth Function model (VBGF) to estimate
growth in a variety of fish species, such as Schizo-
pyge niger, S. curvifrons and Schizothorax esocinus
(Sabah & Khan, 2014), Channa punctata (Khan
et al., 2019), Gerres subfasciatus (Gray, 2019),
Catla catla (Brraich & Kaur, 2022). The basic
biological information required to devise sci-
entifically sound management policies for the
Rita rita population remains warranted, par-
ticularly those involving precise age estimation
and growth trends under changing conditions
across all of the fishs primary habitats. Otoliths
have previously been used to investigate age
and growth studies in Rita rita (Tamubi et al.,
Conclusión: La especie alcanza un crecimiento moderado en estos ríos, dónde las vértebras son la estructura de
estimación etaria recomendada, seguido de, en caso de ser necesario, los otolitos segmentados, otolitos enteros y
huesos operculares. Debe evitarse el uso de espinas pectorales, especialmente en peces de mayor edad.
Palabras clave: vértebras; otolitos; precisión de la edad; VBGF; Análisis del Incremento Marginal.
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
1990) however, there is no information avail-
able on the precision of its hard structure for
age estimation.
Therefore, this study was undertaken to
assess age estimates derived from various hard
(calcified) structures (vertebrae, sectioned
otoliths, whole otoliths, opercular bones and
pectoral spines) to identify the best structure
determining accurate age estimate in Rita rita;
the annulus formation was validated using
MIA, and the VBGF equation was developed
using length-at-age data from the structure giv-
ing precise age estimate.
Sample collection: A total of 390 fish
samples of Rita rita were collected on a monthly
basis during the period September 2018 to
August 2019 from Narora site (28°19’68” N
& 78°38’14” E) of River Ganga, Mathura site
(27°49’24” N & 77°67’37” E) of River Yamuna
and Moradabad site (28°83’86” N & 78°77’33”
E) of River Ramganga (Fig. 1). Fish identifica-
tion was done according to Talwar and Jhingran
(1991). To the nearest centimeter, the total
length (TL) was measured. The total weight
(TW), which includes the gut and gonads, was
measured to the nearest gram. Otoliths, oper-
cular bones, vertebrae and pectoral spines were
extracted from the fish and prepared for age-
ing according to the protocols standardized by
Fish biology and Otolith Research Laboratory
(Khan & Khan, 2009; Khan et al., 2013; Khan
et al., 2017; Sabah & Khan, 2014). Otoliths
were sectioned according to Khan et al. (2016).
For sectioning, the other otolith of a pair
was embedded in silicon moulds consisting of
(Epothin TM Epoxy Resin and Epothin® Epoxy
Hardener in a 5:2 ratio) which were lightly
coated with release agent and allowed to solid-
ify overnight for the media to harden. Otoliths
Fig. 1. Map showing the collection sites of Rita rita from the River Ganga and its tributaries: River Yamuna and River
4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
were sectioned by using IsoMet® Low Speed
Saw (Buehler, 41 Waukegan Road, Lake Bluff,
Illinois-60044, USA) and after that each sec-
tion was polished using 1 200-grit CarbiMet®
2 Abrasive Discs on an otolith polisher, which
has been designed and developed in Fish biol-
ogy and Otolith Research Laboratory. A Nikon®
SMZ745T stereo zoom microscope was used to
analyze all the ageing structures. Two readers
separately analyzed each calcified structure. A
year’s growth was defined as an opaque and
translucent zone combined; age was defined by
the number of translucent zones.
Statistical analysis and calculations: The
average per cent error (APE), coefficient of
variation (CV), and per cent agreement (PA)
between the readers and the pair of ageing
structures were calculated to compare age esti-
mates from different hard anatomical struc-
tures. APE was calculated as per the formula
given by Beamish and Fournier (1981).
Where in xij represents the ith age estimation of
the jth fish, xj refers to the computed mean age
for the jth fish, and R denotes the frequency of
ageing for each fish.
The coefficient of variation was deter-
mined as the ratio of standard deviation over
the mean (Campana, 2001):
Where CVj is the coefficient of variation for the
jth fish.
Campana (2001) suggested APE and CV
to be reliable parameters for precise age esti-
mation in fish. While determining the most
suitable method for precise age estimation, PA
has been used as one of the precision param-
eters (Sabah & Khan, 2014). The percentage
of observations having similar age estimates
divided by the total number of observations
on age estimates is defined as percent agree-
ment. Percent agreement was calculated using
the template for ageing precision (Sutherland,
2006). A post- hoc test, Duncans Multiple Range
Test (Gomez & Gomez, 1984), was used to
determine variations in mean age readings from
different ageing structures (Khan & Khan, 2009).
Marginal Increment Analysis: Annulus
formation on vertebrae was validated by Mar-
ginal Increment Analysis using the equation:
R is the otolith radius, Rn is the last complete
growth ring radius, and Rn-1 is the previously
completed growth ring radius. Image analysis
software was used to take measurements from
the focus to the outer edge of each growth ring
(Rn). To demonstrate periodic trends in annu-
lus development, monthly mean MIR values
were plotted (Lessa et al., 2006).
Growth analysis: To evaluate variability
in growth, length-at-age data collected from
vertebrae (the most appropriate ageing struc-
ture in Rita rita) was used to develop the VBGF
equation (Ricker, 1975) by using non-linear
least-squares regression:
Where, Lt= total length (cm) of fish at age t; L
= asymptotic mean length; k = rate constant
that determines the rate at which Lt approaches
L; t = time or age of the fish; t0 = the hypotheti-
cal age at which the fish had zero length.
Comparison between observed and cal-
culated mean length-at-age was verified with
student’s t-test (Zar, 1996) for the selected fish
species. Growth parameters were estimated
using the non-linear regression method in a
Microsoft Excel-based application developed
by Cope and Punt (2007).
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
The sample size, minimum and maxi-
mum total length, used in the ageing precision
for the selected fish species are presented in
Table 1. The vertebrae in Rita rita provided a
precise age estimate. It exhibited the least APE
(1.54 %) and CV (3.17 %) values followed by
sectioned otoliths, whole otoliths, opercular
bones and sectioned pectoral spines (Table 2).
The vertebrae had the highest percentage of
reader agreement (Table 2). A comparison of
vertebrae age estimates was analyzed with those
from other structures (i.e., sectioned otoliths,
opercular bone, whole otoliths and sectioned
pectoral spines) suggested the least variation
between vertebrae vs sectioned otoliths age
estimates followed by vertebrae vs opercular
bone, vertebrae vs whole otoliths and vertebrae
vs sectioned pectoral spine (Table 2).
Mean age readings from vertebrae were
significantly (P < 0.05) different from pectoral
spines but similar (P > 0.05) to the age estimates
from sectioned otoliths, whole otoliths and
opercular bones. Thus, in the absence of the
most appropriate age estimate (vertebrae), we
may use age data from sectioned otoliths, whole
otoliths and opercular bones but the use of pec-
toral spines may be avoided to the maximum
possible extent (Table 3).
The von Bertalanffy growth model was fit
using the ages estimated from vertebrae. The von
Bertalanffy growth parameters of Rita rita from
the three rivers are presented in Table 4. Readabil-
ity scores presented in Table 5 showed that ver-
tebrae had the highest readability score (64.1)
followed by sectioned otoliths (56.41), whole
otoliths (41), opercular bones (14.36) and sec-
tioned pectoral spines (2.05). Fig. 2 shows the
number of specimens in each age group in all
the studied rivers. The age group 3 and 4 were
most dominant in the sampling from all three
rivers while a smaller number of samples were
found in age group 7. Ageing structures in
Rita rita vertebrae, sectioned otoliths, whole
otoliths, opercular bones, and sectioned pecto-
ral spines are shown in Fig. 3. Age bias graphs
between vertebral age estimations and each of the
other ageing structures are shown in Fig. 4.
Marginal Increment Analysis has been
used to validate age estimation in the target
fish species. The monthly mean of Marginal
Increment Ratio (MIR) declined from a high-
est of 1.02 in March to the lowest of 0.32 in
July (Fig. 5). The results of MIR revealed that
the annulus formation occurred from May to
September (Fig. 5). The proportion of opaque
Table 1
Estimated parameters of Rita rita collected from the
selected rivers.
Rivers Sample size TL Range (cm) Mean (cm)
Ganga 140 9.3 - 57.2 36.4
Yam u n a 120 10.2 - 56.1 33.6
Ramganga 130 9.8 - 54.4 34.2
TL: Total length.
Table 2
Measures of precision in Rita rita.
Ageing structures APE CV PA
Ver tebr ae 1.54 3.17 88.3
Sectioned otoliths 1.61 3.51 86.8
Whole otoliths 2.06 4.10 84.6
Opercular bones 3.38 6.12 80.2
Pectoral spines 5.40 8.44 76.3
Between structures
Vertebrae- sectioned otoliths 1.66 3.38 87.2
Vertebrae - whole otoliths 2.00 3.83 85.4
Vertebrae - opercular bones 3.53 5.65 81.9
Vertebrae–pectoral spines 5.93 7.52 79.0
PA: Percentage of Agreement, APE: Average Percentage
Error, and CV: Coefficient of Variation (CV).
Table 3
Comparison of mean values of age estimates from different
hard structures in Rita rita.
Ageing Structures Mean values of age estimates
Ver tebr ae 4.4223b
Sectioned otoliths 4.2896b
Whole otoliths 3.9918ab
Opercular bones 3.1854ab
Pectoral spines 2.876a
*Values having similar superscripts are insignificantly
different (P > 0.05) from each other.
6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
bands on vertebrae increased regularly from
August to March and then decreased from
April to July. These results suggest that the
narrow translucent band formed during the
monsoon season (May to September) and the
wide opaque band formed during the rest of
the months.
The von Bertalanffy growth function equa-
tions derived using length at age data of verte-
brae of Rita rita were Lt = 90.19(1-e-0.145(t+0.51))
for River Ganga; Lt = 91.19 (1-e-0.14(t+0.59)) for
River Yamuna; Lt = 89.63 (1-e-0.15(t+0.68)) for
River Ramganga. There were no significant
variations between Rita ritas calculated and
observed lengths of the River Ganga (R2 =
0.9781; d.f. = 6, P > 0.05), River Yamuna (R2 =
0.9686; d.f. = 6, P > 0.05) and River Ramganga
(R2 = 0.9731; d.f. = 6, P > 0.05) The von Berta-
lanffy growth curves of Rita rita from the three
rivers: River Ganga, River Yamuna and River
Ramganga showed significant differences (P <
0.05) with each other (Fig. 6).
In this study, the vertebrae of Rita rita
showed regularly formed annual rings, which
were clear to count, compared to other hard
anatomical structures (whole otoliths, sectioned
otoliths, opercular bones, pectoral spines). Our
Table 4
von Bertalanffy growth parameters in Rita rita collected from the Gangetic River System.
Species Selected rivers Sampling sites Ageing structures used L(cm) k t 0Max. Age (years)
Rita rita Ganga Narora Vertebrae 90.19 0.145 0.51 7
Rita rita Yamu n a Mathura Vertebrae 91.19 0.14 0.59 7
Rita rita Ramganga Moradabad Vertebrae 89.63 0.15 0.61 7
Fig. 2. Number of individuals in each age group from the three rivers: Ganga, Yamuna and Ramganga.
Table 5
Distribution of readability scores for different hard structures in Rita rita.
Readability score 12345
Ver tebr ae 7.69 64.1 15.38 8.97 3.85
Sectioned otoliths 5.64 56.41 16.4 12.31 9.23
Whole otoliths 3.08 41 11.03 18.97 25.9
Opercular bones 1.79 14.36 4.62 30.77 48.46
Sectioned pectoral spines 2.05 6.15 40 51.79
Revista de Biología Tropical, ISSN: 2215-2075, Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
results corroborate with the findings of Li and
Xie (2008) that in Siluriformes fishes, the best
ageing structure is vertebrae that were easy to
process and exhibited regularly formed clear
rings. Precise age estimation using vertebrae
has been reported in a variety of freshwater
fishes, such as Heteropneustes fossilis (Khan
et al., 2013), Schizothorax esocinus (Sabah &
Khan, 2014), Mastacembelus armatus, and
Ompok pabda (Khan et al., 2015), Capoeta
banarescui (Bostanci et al., 2015), S. aor (Nazir
& Khan, 2020).
Next to vertebrae, otoliths provided better
age estimates than other ageing structures. Sec-
tioned otoliths after polishing exhibited clearer
annuli as compared to whole otoliths and there-
by requiring less effort and time by the readers
to estimate age. Several studies reported sec-
tioned otoliths to provide precise age estimates
in Paralichthys dentatus (Sipe & Chittenden,
Fig. 3. Ageing structures in Rita rita A. vertebrae, B. sectioned otoliths, C. whole otoliths, D. opercular bones and E. sectioned
pectoral spines. Scale bar: 1 mm.
8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 71: e51734, enero-diciembre 2023 (Publicado Feb. 22, 2023)
2001), C. striata (Khan et al., 2017). Because
of its distinctive attributes of being metaboli-
cally inert, otoliths are usually utilized as an
ageing structure, not showing reabsorption and
exhibiting acellular growth throughout fish life.
(Phelps et al., 2007).
Whole otoliths in Rita rita did not show
distinct annuli because whole otoliths are thick
at the center which reduces the clarity of growth
lines. As a result, the opaque rings on otoliths
could not be distinguished clearly. Since fish
growth shows declining trends with age, the
annuli come closer to each other near the edges
of the ageing structures and hence it becomes
difficult to distinguish annuli in small and thick
whole otoliths used in this study. Whole otoliths
underestimated the fish age compared to ver-
tebrae, opercular bones, and sectioned otoliths.
There are several findings in support of our study
that the use of whole otoliths rather than sec-
tioned otoliths can lead to an underestimation of
Fig. 4. Age bias graphs for Rita rita between two independent readers: A. vertebrae, B. sectioned otoliths, C. whole otoliths,
D. opercular bones and E. pectoral spines. Each error bar represents the 95 % confidence interval, and the solid line
indicates the theoretical 1:1 agreement line of age estimates between readers. Points above the line indicate ages that were
overestimated, whereas points below the line indicate ages that were underestimated.