ISSN 0032-9452, Journal of Ichthyology, 2016, Vol. 56, No. 2, pp. 321–324. © Pleiades Publishing, Ltd., 2016.

Original Russian Text © D.V. Prazdnikov, F.N. Shkil, 2016, published in Voprosy Ikhtiologii, 2016, Vol. 56, No. 2, pp. 240–244.

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Effects of Hyperthyroidism on the Labeobarbus (= Barbus)

intermedius (Cyprinidae) Early Larval Melanophores Development1
D. V. Prazdnikova and F. N. Shkila,b
aSevertsov Institute of Ecology and Evolution, Russian Academy of Sciences, pr. Leninskii 33, Moscow, 119071 Russia
bKoltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, 119334 Russia
e-mail: pdvfish3409@rambler.ru
Received February 24, 2015

Abstract—The experimental investigation of the effects of hyperthyroidism on the development of Labeobar-

bus intermedius pigmentation revealed that high level of thyroid hormone leads to changes in developmental
rate and timing of early larval pigment pattern. The hyperthyroidism induces the acceleration of growth of
early larval melanophores, causes changes in their physiological state, and provokes the premature onset of
the adult pigment pattern formation.

Keywords: pigment pattern, melanophores, thyroid hormone, Labeobarbus intermedius

DOI: 10.1134/S0032945216020120

INTRODUCTION (Parichy, 2006; McMenamin and Parichy, 2013).

The formation of pigment pattern in vertebrates
attracts the attention of evolutionary developmental
biologists, because it provides an outstanding oppor-
tunity for integrative studies linking the genetic and
cellular mechanisms to their organismal and evolu-
tionary consequences (Parichy, 2003). The pigment
pattern is determined by the localization, number,
composition, and physiological state of neural crest
derived pigment cells—chromatophores (Parichy et al.,
2006). Six types of chromatophores are presented in
vertebrates: melanophores, xanthophores, erythro-
phores, cyanophores, iridophores and leucophores
(Fujii, 2000). The melanophores, melanin-containing
chromatophores, have been of long-standing interest
to developmental and cell biologists, because melano-
phores are presented in all groups of vertebrates
(Parichy, 2003), and deviations of their life cycle may
lead to the morphological consequences, which are
considered as a pathology in human (Rawls and John-
son, 2001).
One of the main objects for studying the develop-
ment and evolution of pigment patterns is bony fishes
displaying a high variety of pigmentation. The major-
ity of teleosts undergo several transitions in ontogeny:
from embryo to larval stage, from larva to juvenile, and
from juvenile to adult (Balon, 2002). In many families,
these transitions are accompanied with spectacular or
subtle morphological transformations, including the
transformations of pigment patterns: changes in com-
position, localization, and ratio of pigment cells
1 The article was translated by the authors.
Regulatory mechanisms of the adult pigment pattern
development are studied well on several model fish
species. It was revealed that ontogeny of adult pigment
pattern depends upon numerous intracellular factors
and extracellular signals, particularly, neurotransmit-
ters and hormones (Kelsh et al., 2000; Aspengren
et al., 2003; Logan et al., 2006). Thus, number, distri-
bution, and physiological state of adult melanophores
in fish are under the control of catecholamines and
hormones of pituitary and pineal glands (Fujii, 2000;
Sugimoto, 2002; Logan et al., 2006). The thyroid hor-
mones (TH), one of the main regulators of fish meta-
morphic transformations, are also involved in the
development of adult pigment pattern. The level of
these hormones determines the number and develop-
mental timing of several adult-type chromatophores
(Yoo et al., 2000; Clement et al., 2001; Jegstrup and
Rosenkilde, 2003; McMenamin and Parichy, 2013;
McMenamin et al., 2014).
However, the role of endocrine signals in the
ontogeny of early larval (EL) pigment pattern remains
obscure. To clarify the involvement of endocrine sys-
tem in the formation of EL pigment pattern, we inves-
tigated the effects of hyperthyroidism on the develop-
mental timing and physiological state of EL melano-
phores in large African hexaploid barb Labeobarbus
(=Barbus) intermedius (Cyprinidae).
MATERIALS AND METHODS
The investigations were carried out in the frame-
work of studying of the role of heterochronies in the

321
322 PRAZDNIKOV, SHKIL

(а) (b)

Fig. 1. Early larval head pigmentation in the (a) control and (b) hyperthyroid groups of Labeobarbus intermedius (12 dpf). Scale:
0.5 mm.

130 larvae reared under different hormonal regimes fixed

Diameter of melanophores, micrometers

daily in 10% formalin. The diameter of chromato-

120 phores was measured by ocular micrometer embedded
110 in MBS-9 stereoscopic microscope. The sizes of early
100 melanophores’ pseudopodia were not taken into
account in measurement. The effects of formalin were
90 neglected because all procedures—fixation, storage
80 and processing of samples—were carried out identi-
70 cally. We processed the pictures with a Canon EOS 7D
photographic camera. In total, 470 individuals of
60 L. intermedius reared under both hormonal regimes
50 were analyzed. Statistical analysis was performed
using the Statistica v. 6.0 software package (Statsoft,
40
United States).
30
20 RESULTS
5 7 9 11 13 15 17 19 21 23 25 27 29 31
Age, day post-fertilization Newly hatched prolarvae (3–4 days post-fertiliza-
tion (dpf)) from the control group had no chromatho-
Fig. 2. Dynamics of melanophore diameters in the control
phores. The first pigment cells, EL melanophores,
(―) and hyperthyroid (- - -) groups of Labeobarbus inter- appeared at 5 dpf on the head, yolk sac, and dorsal
medius. (h) standard error, (I) mean square deviation. part of trunk. They had pseudopodia and were capable
of amoeboid movements. At the prolarval stage, EL
melanophores displayed the dispersed state, and sig-
evolution of the species flock of Lake Tana large Afri- nificantly increased in size and number. The transition
can barb genus Labeobarbus (Cyprinidae; Teleostei) to larval stage (beginning of exogenous feeding,
(Smirnov et al., 2012). The fertilized eggs of L. inter- 10 dpf) was accompanied by the changes in the mor-
medius were divided into two groups reared under dif- phology of EL melanophores. They acquired a
ferent hormonal regimes: control group reared in pure rounded shape with outgrowths, and changed their
water; hyperthyroid group reared in water with physiological state to aggregation (Fig. 1a). Further,
triiodothyronine (T3, an active form of TH) (Sigma, EL melanophores remained in an aggregation state.
United States) at a 1 ng/mL concentration. The concen- Their size (Fig. 2) and number slightly increased on
tration of T3 was selected earlier experimentally. All the head and trunk ventral and dorsal pigmentation lines
other conditions (background of the bottom and side up to the onset of adult pigment pattern formation,
walls, light regime, temperature, aeration, feeding, stock- appearance of adult-type melanophores (32 dpf).
ing density, etc.) were standard in both groups (detailed In barbs reared under hyperthyroid conditions at
experimental design in Shkil et al., 2010). prolarval stage and at transition to larval stage, timing
and sequence of EL melanophore developmental
Prolarvae (developmental stage from hatching up events were similar to those in the control group.
to transition to exogenous feeding, characterized by However, after transition (11–12 dpf), EL melano-
the presence of the yolk sac and larval fin fold) and phores in the majority of larvae (>96%) reared under

JOURNAL OF ICHTHYOLOGY Vol. 56 No. 2 2016

 EFFECTS OF HYPERTHYROIDISM
hyperthyroid conditions changed their physiological
state from aggregation to dispersion and displayed
rapid growth (Fig. 1b and 2). The remaining larvae
had EL melanophores similar in size and physiologi-
cal state with EL melanophores in larvae from the
control group. At 16 dpf, EL melanophores in all lar-
vae reared under hyperthyroid conditions were at an
aggregation state and significantly decreased the
growth rate. The adult pigment pattern formation
began at 25 dpf.
DISCUSSION
The obtained data indicated the effects of hyper-
thyroidism on the development of EL pigment pattern
in L. intermedius. We failed to find any significant dif-
ferences between groups reared under different hor-
monal regimes in timing and sequence of EL melano-
phore developmental events at prolarval stage and at
transition to larval stage, but observed differences in
further development. In the control group after transi-
tion to larval stage, EL melanophores gradually
increased in size and number, and they were in an
aggregated physiological state up to the onset of for-
mation of adult pigment pattern, the replacement of
EL melanophores by the adult-type melanophores. In
contrast, in the majority of fish reared under hyper-
thyroid conditions after transition to larval stage, EL
melanophores immediately changed their physiologi-
cal state to dispersion, and they demonstrated acceler-
ated growth in several days (Fig. 2). Then, EL melano-
phores in hyperthyroid fish changed their physiologi-
cal state to aggregation again and drastically decreased
the growth rate. Further, in this group, EL melano-
phores remained in an aggregated state up to onset of
adult pigment pattern formation, which began signifi-
cantly earlier than in the control group.
Thus, hyperthyroidism causes the changes in
developmental rate and timing of EL pigment pattern
and provokes the premature onset of adult pigment
pattern formation in L. intermedius. One may propose
that these changes are associated with the TH-induced
changes in developmental rate and timing of larval
skin development. Previously, it was shown that teleost
skin undergoes serious developmental transformations
in larval periods (Le Guellec et al., 2004), which can
be regarded as metamorphic. Some of these processes
are under direct or indirect control of the hypotha-
lamic-pituitary-thyroid (HPT) axis (McMenamin
and Parichy, 2013). For example, the onset of squama-
tion, one of the main indicators of late metamorphic
transformations in bony fish skin (Sire and Akimenko,
2004), begins in cyprinids reared under hyperthyroid
conditions, including L. intermedius, significantly ear-
lier than in cyprinids reared under normal conditions
(Smirnov et al., 2006; Levin, 2011; Levin and Levina,
2014). This fact indicates significant acceleration of
skin developmental rate and timing caused by high
level of TH. Given that pigment cells are components
JOURNAL OF ICHTHYOLOGY Vol. 56 No. 2 2016
323
of integumentary system, one may propose that accel-
erated development of skin, as a system, in L. interme-
dius reared under hyperthyroid conditions, induces
the accelerated growth of EL melanophores and pre-
mature appearance of adult-type melanophores. Pre-
viously, dependence of pigment pattern developmen-
tal rate and timing on the TH-level was reported for
bony fish with pronounced metamorphosis: Para-
lichthyidae and Anguillidae (Yoo et al., 2000; Jegstrup
and Rosenkilde, 2003). Our experimental data
revealed that HPT axis may control the development
of pigment pattern in fish with subtle metamorphic
transformations as well.
The revealed acceleration of EL melanophores
developmental growth in L. intermedius reared under
hyperthyroid conditions was accompanied by the
transition of their physiological state from aggregation
to dispersion. The physiological state of adult-type
melanophore depends on the concentrations of cyclic
adenosine monophosphate (cAMP) and ions of Ca2+
in the cytosol (Fujii, 2000). These concentrations are
under the control of different signal factors, including
hormones. Thus, melanin-concentrating hormone
induces the decrease of cytosolic cAMP concentration
and promotes the aggregation. In contrast, melatonin,
adrenocorticotropic, and melanocyte-stimulating
hormones cause the increase of cAMP level and pro-
voke dispersion. Also, melatonin and noradrenaline
stimulate the increase of cytosolic Ca2+ concentration
and activate dispersion (Fujii, 2000; Sugimoto, 2002;
Aspengren et al., 2003; Logan et al., 2006). The
dependence of the level of above-mentioned hor-
mones on the activity of HPT axis is unclear. How-
ever, the complex and diverse relations between pineal
gland and different endocrine axes (Norris and Carr,
2013) allow proposing the reciprocal dependence of
the levels of these hormones. Thus, negative feedback
linking pineal and thyroid glands leads to increase of
the melatonin synthesis in pineal gland in response to
the increase of TH-level in blood. At the same time,
the high level of melatonin inhibits the synthesis of
thyroid-stimulating hormone in pituitary and, conse-
quently, decreases the activity of HPT axis (Nir and
Hirschmann, 1978; Vaughan and Vaughan, 1992;
Lewinski and Karbownik, 2002). Thus, we may pro-
pose the cross-talk between HPT and other endocrine
axes affects the physiological state of melanophores.
Taking into account that EL melanophores are genet-
ically distinct from adult-type melanophores (Parichy,
2006), we cannot exclude other direct or indirect
mechanisms of TH influence on the EL melano-
phores physiological state. However, these hypotheses
require additional experimental investigations.
ACKNOWLEDGMENTS
This work was partially funded by Russian Founda-
tion for Basic Research, project nos. 14-04-00590 and
13-04-00031. We thank D.V. Kapitanova (Severtsov
324 PRAZDNIKOV, SHKIL
Institute of Ecology and Evolution, Russian Academy
of Sciences) for help and assistance throughout the
work. Moreover, we thank Yu.Yu. Dgebuadze and
S.V. Smirnov (Severtsov Institute of Ecology and Evo-
lution, Russian Academy of Sciences) for providing
helpful comments on the manuscript.
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