THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275:374-382 (1996)

Hormonal Induction of Sex Reversal and Progeny

Testing in the Zebra Cichlid CichZasorna
nigrufasciatum
T. GEORGE AND T.J. PANDIAN
Department of Genetics, School of Biological Sciences, Madurai Kamaraj
Uaiuersity, Madurai 625 021, India

ABSTRACT Hormonal sex reversal was induced in the ornamental cichlid C. nigrofasciatum
by dietary administration of estradiol-P or 17a-methyltestosterone. The treatments involving 200
mg estradiol-P and !ZOO mg 17a-methyltestosterone/kg diet for 20 days in the 4-day posthatchling
C. nigrofasciatum resulted in 100% feminization and 82% masculinization, respectively. Superop-
timal doses led to higher mortality and stunted growth, especially among the surviving males,
which resisted feminization. Progeny testing of the feminized and masculinized individuals indi-
cated that sex was determined by XX and XY (male heterogamety) sex chromosomes; however, the
role played by autosomes in these individuals was also apparent. Live homogamous males (YY)
could not be produaed. Progeny testing further showed that hormonal treatment impaired growth
and reproductive performance of the sex-reversed females and males. F1 progenies sired by the
sex-reversed females as well as males also suffered higher mortality, extended interspawning pe-
riod and lower fecundity. @ 1996 Wiley-Liss, Inc.

Studies on hormonal sex control have focused
two major objectives: (1) establishment of an ef-
fective treatment protocol for sex reversal, and (2)
elucidation of the mech,anisms of sex determina-
tion and differentiation. Among the economically
important fish studied so far, the cichlids are per-
haps the most amenable group to hormonal in-
duction of sex reversal; complete masculinization
or feminization has been achieved in many cichlid
species (see Hunter and Donaldson, '83; Pandian
and Sheela, '95). However, recent publications
have shown that for some unknown reason hor-
monal induction of sex reversal resulted in higher
(75%) mortality of fish bearing homogamous (XX
or ZZ) genotypes (see Pandian et al., '94).
Significant deviations from the 1:l sex ratio
among cichlid hybrids I:e.g., Pruginin et al., '75;
Pandian and Varadaraj, '87) have stimulated in-
terest in discovering the underlying mechanisms
of sex determination. Simple models for sex de-
termination involving :KY and WZ sex chromo-
somes have proved inadequate to explain certain
aberrant sex ratios associated with particular hy-
brid crosses (e.g., Kornfield, '84). Other models of
sex determination such as the autosomal balance
hypothesis involving two loci (Avtalion, '82) and a
single locus-multiple allele hypothesis (Kornfield,
'84) have been put forward t o explain the imbal-
ance of sex ratio in some cichlid populations.
Though appreciable amounts of work has been
carried out in regard t o genetics (e.g., Kornfield,
'84) and sex manipulation (see Pullin and Lowe-
McConnell, '82) in cichlids, the genetic mecha-
nisms of sex determination and differentiation still
remain far too complicated, demanding further
experimental studies. Hence, the present study
was carried out in the zebra cichlid, Cichlasoma
nigrofasciatum, with the objectives of (1)optimiz-
ing the treatment parameters for production of
monosex male and female populations, and (2) de-
termining the heterosomal system of sex deter-
mination through progeny testing.
MATERIALS AND METHODS
A batch of C. nigrofasciatum was bought from
an ornamental fish dealer and bred in the labora-
tory for 5 generations. Fry (4-day posthatchling)
were pooled from two to six different breeding par-
ents. Two series of experiments were carried out,
Received November 29, 1995; accepted in revised form March 25,
1996.
Address reprint requests to Prof. T.J. Pandian, Department of Ge-
netics, School of Biological Sciences, Madurai Kamaraj University,
Madurai, 625 021, India.
T.George's present address is School of Applied Life Sciences, Ma-
hatma Gandhi University, Kerala 689 645, India.

0 1996 WILN-LISS, INC.

 HORMONAL SEX REVERSAL IN CICHLASOMA 375

one of them using estradiol-P, and the other us- The fish were weighed individually once ev-
ing 17a-methyltestosterone (Sigma, St. Louis, ery 3 months to monitor growth, using a mono-
MO). Each series consisted of three groups, which pan weighing balance (Stanton, U.K.). The
were treated for 10, 20 or 30 days. Each estra- morphology of gonads was studied after dissect-
diol-p group consisted of six batches of 40 fry each, ing the animals. Gonads were weighed to calcu-
receiving 25, 50, 100,200,300 or 400 mg hormone late the gonadosomatic index (GSI).
per kg feed. Each group of the 17a-methyltest-
osterone-treated series consisted of nine batches RESULTS
of 40 fry each, receiving 5 , 10, 25, 50, 75, 100, Feminization
200, 400 or 800 mg hormone per kg diet. Briefly,
18 batches of the 4-day posthatchling received Optimum treatment and survival
estradiol-a and one batch served as control. An- Oral administration of 200 and 300 mg estra-
other 27 batches of the posthatchling received diol-P/kg diet for 20 or 30 days resulted in the
17a-methyltestosterone; the 28th batch served production of all-female populations of C. nigro-
as control. fasciaturn (Table 1).Clearly, this cichlid is also
After termination of hormone treatment, the amenable for 100%feminization. Fry treated with
fish were separately reared until sexual maturity the optimum dose of 200 mg estradiol-P/kg diet
(4months). Males were identified by the absence for 20 days registered a survival of 75% after ter-
of reddish-orange color on the lateral sides of the mination of treatment and 50% at sexual matu-
abdominal region and by the presence of more rity. Increase in the dose and treatment duration
pointed fins and extended dorsal fin (Petrowicky, resulted in decreased survival after treatment as
'89). Sexes of progeny F1 and F2 at juvenile stage well as at sexual maturity. The 30-day treatment
were identified by squash preparation of the go- at 400 mgkg diet led t o the total mortality of the
nads (Guerrero and Shelton, '74). tested individuals on or before sexual maturity.
Progeny testing was carried out in randomly se-
lected individuals from the all-female batches to Progeny testing
identify the homogametic and heterogametic fe- After sexual maturity, 20 randomly selected fe-
males. Genotypes of the treated individuals (Tr) males from the estradiol series were subjected to
were inferred from the sex ratio of the F1progeny progeny testing. To mate these females, six males
obtained after mating such females with normal were also randomly selected from the control se-
genetic males. Figure 1 shows the experimental ries. The females 24 and 28 were mated by the
procedure for the production of homogamous (YY) same male ZM4; they sired progenies, whose sex
male (at Fl), as identified from the sex ratio of F2 ratio was about 0.5 P : 0.5 6. But the ratio of
progenies. progenies sired by the Z9 0 and ZM4 6 deviated
significantly from the expected 0.5 : 0.5 ratio
(Table 2). Likewise, that sired by the pair 214 9
and ZM3 6 was the expected one, but that by 220
0 , which was also mated by the same ZM3 6,
xx xv
deviated significantly. Therefore, these deviations
I Dtctary odminirtmtion of 2Wmg
cstrodiol-0 lor 20 days observed in the sex ratio among the progenies of
29 and 220 may solely be due to the genotype of
these females. Only 3 out of the 20 sex-reversed
females produced F1progenies, whose sex ratio is
1 0 : 3 6 6,when a female carrying XY genotype
is crossed with a male having XY chromosomes.
Hence, the results suggest that these sex-reversed
FI females were heterogamous, and that the xx/xy
mechanism of sex determination is in operation
in C. nigrofasciutum. 'Ityo observations must be
Fig. 1. Diagrammatic sketch of experimental procedure recorded here: firstly, the sex ratios observed for
for the production of YY males in C. nigrofasciutum. From all three broods of the pair 220 0 x ZM3 6 and
the 1 0 :3 6 6 (i.e., 1 XX: 2 XY :1 YY) sex ratio obtained in
F1, the Tr females were characterized with the genotype XX
that of the second brood of the pair Z10 0 x ZM2
or X Y . Time required for hormone treatment and progeny (F1 6 were about 1 9 : 4 66 and suggest that auto-
and F2) testing is indicated by a vertical scale on the right. soma1 genes also play a role in sex determina-
376 T. GEORGE AND T.J. PANDIAN
T M L E 1. Effect o f dietary administration of estradiol-,8on the 4-dayposthatchling C. nigrofasciatum'
Treatment Survival (%) at
Duration Dose Termination Sexual Sex distribution
(day) (mgkg) of treatment maturity P I1 d
Control 0 97 95 0.53 0.00 0.47
10 25 92 85 0.45 0.00 0.55
50 87 65 0.52 0.00 0.48
100 85 67 0.60 0.00 0 40
200 77 60 0.64 0.00 0.36
300 75 57 0.70 0.00 0.30
400 60 42 0.70 0.00 0.30
20 25 90 65 0.70a2 0.00 0.30
50 90 55 0.64 0.00 0.36
100 82 57 0.80a 0.00 0.20
200 75 50 1.00a 0.00 0.00
300 60 50 1.00a 0.00 0.00
400 50 45 0.85a 0.15 0.00
30 25 80 65 0.70a 0.00 0.30
50 77 65 0.80a 0.00 0.20
100 75 60 0.80a 0.00 0.20
200 62 50 1.00a 0.00 0.00
300 50 45 0.90a 0.00 0.10
400 40 0 - - -
'I, Intersex.
'x2 test: a indicates significant deviation from the sex ratio of 0.5 P : 0.5 d a t the level of P = 0.05.

TABLE 2. Results ofprogeny testing among the two batches (200 and 300 mglkgj of (Tr)females treated with estradiol-p
(200 i n p i k d for 20 days and mated with normal males of C. nierofasciatuml
F1 Sex distribution
Male Progeny of F1 progeny Genotype of
TR? used (no.) No. sexed 0 8 Tr?
Series I : Group 2 : Batch IV : 200 mgkg diet
Control ZM1 83 50 0.56 0.44 xx
135 50 0.54 0.46
0.55 0.45
ZI to Zil ZM1 to ZM5 88 39 0.51 0.49 xx
136 52 0.52 0.48
215 50 0.49 0.51
0.51 0.49
z9 ZM4 63 30 0.27 0.73 XY
110 50 0.32 0.68
174 50 0.23 0.77
0.29 0.71
z10 ZM2 73 35 0.35 0.65 XY
107 50 0.16 0.84
181 50 0.30 0.70
0.26 0.74
Batch V : 300 mg/kg diet
Control zM1 83 50 0.56 0.44 xx
135 50 0.54 0.46
0.55 0.45
Z l l to Z19l ZM1 to ZM6 84 40 0.51 0.49 xx
50 0.56 0.44
0.53 0.47
220 ZM3 59 35 0.18 0.82 XY
103 50 0.20 0.80
148 50 0.19 0.81
'Mean values of 8 or 9 females, which are likely to be homogamous.
 HORMONAL SEX REVERSAL IN C Z C H U S O a 377

tion. Secondly, the observed sex ratio of the months, the differences in body weight of females
progenies of 220 0 x ZM3 8 were arrived at from a and males treated with 400 mgkg for 20 days
sample of only 135 individuals, which survived out compared with the respective control fish aver-
of 310 progenies; hence, it is possible that prog- aged about 3% and 17%, respectively; at 12
enies suffered sex-dependent mortality. months, the differences in body weight were about
Progeny testing was also carried out among F1 2%in females but 25% in males. Clearly, genetic
males, for example, XM5a, XM5b, XM5c, etc., from males, which resisted the estradiol-P treatment,
brood I of Tr female Z5, which are expected t o suffered significant (P < 0.05) growth retardation.
possess either XY or YY genotype in the ratio of Briefly, males suffering treatment at doses of 100
2:l (see Fig. 1).In order to identify the genotype mgkg and above exhibited significantly ( P < 0.05)
of these males, five individuals were randomly stunted growth.
selected from each of the first and second broods
Reproductive performance
produced by the Tr female Z9 and Z10 (Table
3). Surprisingly, none of the 20 broods produced Fecundity of sex-reversed females (Tr) was sig-
by the F1 males was found t o sire an all-malenificantly less than that of genetic females. Table
F, progeny; the highest proportion of males 3 also shows equally higher mortality and lower
(0.33 0 0 : 0.66 6 6 )was sired by the pair X1fecundity suffered by F2 progenies, whose grand-
0 and XM5d 0. These data suggest that YY in- parent (Tr) was subjected to estradiol treatment.
It is not clear how the negative effects of hor-
dividuals were perhaps inviable but do not rule
monal treatment of Tr is transmitted to F1 and
out the possibility that all 20 males tested could
be XY by chance. F2progenies, which did not suffer hormone treat-
ment at all.
Growth Table 4 presents a summary of the reproduc-
tive performance of the control, treated and sex-
Growth was retarded in fish (Tr)treated at reversed females. The following conclusions can
doses higher than 100 mg estradiol-Pikg. At 9 be made: (1)interspawning period was prolonged

TABLE 3. Sex distribution of F2 progenies of C. nigrofasciatum to identify the genotype of randomly selected F1 male siblings'
F2 F2 Sex distribution of
TR P F1 S progeny mortality F2 progeny'
XY F1 brood tested 0 used (no.) (%I 0 S
z9 I XM5a x1 61 28 0.38 0.62
XM5b x2 78 13 0.40 0.60
xM5c x5 73 19 0.48 0.52
XM5d x1 65 18 0.33 0.67
XM5e x4 75 20 0.53 0.47
Mean 70 20 0.42 0.58
I1 XM5f x3 86 13 0.53 0.47
XM5g x4 81 11 0.45 0.55
XM5h X6 76 9 0.55 0.45
XM5i x2 80 13 0.48 0.52
XM5j x3 70 37 0.45 0.55
Mean 79 17 0.49 0.51
z10 I XM8a X6 81 37 0.43 0.57
XM8b x7 64 60 0.45 0.55
XM8C X8 72 22 0.55 0.45
XM8d x7 59 31 0.50 0.50
XM8e x11 61 34 0.45 0.55
Mean 67 37 0.48 0.52
I1 XM8f x9 71 21 0.50 0.50
x10 82 28 0.40 0.60
XM8h X6 73 45 0.53 0.47
XM8i X8 68 19 0.43 0.57
xM8j x9 79 27 0.48 0.52
Mean 48 28 0.47 0.53
'Note that F1 male siblings were produced by the sex-reversed Tr females (xy)and were mated with normal females.
'From each Fz brood, sex was determined for 40 individuals.
378 T.GEORGE AND T.J.PANDIAN
TABLE 4. Comparison of reproductive performance of untreated control (AX.., treated (X3J
and sex-reuersed m)
females of C . nigrofasciatum'
Sex distribution
F1 progeny (no.) 1.S.P.' Mortality of F1progeny
Type of 0 Genocype I I1 111 (day) (%) 0 6
Untreated xx 89 2 4
'
* 143 Iga 277 Iloa 15 +- 1' loa 0.53 0.47
(n = 3)
Treated XX 88+9' 12717~ 21518~ 18 +- 3b ISb 0.51 0.49
(n = 3)
Treated sex reversed XI? 68 f 6b 108 I3' 177 I14' 22 +- 1
' 24' 0.24 0.76
(n = 3)
'The means for a t least three fish in each group have been compared.
'I.S.P., interspawning period.
*SNK test: Values superscribed t 8 ydifferent alphabets within the same column are significantly (P < 0.05) different.

significantly ( P c 0.05) from 15 days in the con-
trol t o 22 days in the sex-reversed (XY) female,
(2) the number of eggs produced by the sex-re-
versed ( X Y ) female decreased from 89 to 68, 143
to 108, and 277 to 177 in the I, I1 and I11 broods,
respectively, and (3) mortality increased to 15%
and 24% among the F, progenies sired by hor-
monally treated XX and XY females, respectively,
from the 10% suffered by the control.
The ovary size and GSI values (1.34) of the fe-
males treated with the optimum dose (200 mg/
kg) were comparable to that of the control (1.56),
but females treated with 400 mgkg diet had sig-
nificantly (P < 0.01) lowcir values (0.74). Likewise,
the males (Tr)which failed t o reverse the sex,
when treated with doses of 100 and 400 mg es-
tradiol-P for 20 or 30 days, showed lower GSI, in
comparison to that of an untreated male.
Masculinization
Effect of 17a-methylteetosterone
Dietary administration of methyltestosterone at
doses ranging from 5 to 800 mgkg for 10, 20 or
30 days was ineffective in producing an all-male
C. nigrofasciatum population. A dose of 200 mg
methyltestosterone adiministered t o the 4-day
posthatchling for 20 days produced a maximum
of 82% males (Table 5 ) . The treatment resulted
in 55% survival at sexual maturity. Higher dose
or treatment duration resulted in higher mortal-
ity and the 800 mg dose proved lethal, as all the
individuals succumbed prior t o sexual maturity.
have the xx/xy system, the sex-reversed male
(genetic female) should be XX and produce an
all-female population, when mated with a normal
XX female. Obviously, autosomal factors appear
t o have modified the sex ratio determined by the
XOXY sex chromosomes.
Growth
Growth trends observed for the methyltestoster-
one-treated series were more or less similar to
that observed for the estradiol-a-treated ones. In-
dividuals treated with the highest doses of 400
and 800 mgkg diet showed stunted growth.
Reproductive performance
The males produced in the batches receiving 200
mg 17a-methyltestosterone showed reduced GSI
(0.34) compared to the control (1.03). Some sur-
viving males, which received 200 or 400 mg/kg,
could not mate when paired with an untreated
female of suitable size. These stunted males had
poorly developed or filiform testes. The M12 male
was less aggressive in territorial defense, and
when mated with a normal XX female (A5), it
sired fewer young ones in three consecutive broods
(71,118 and 167)than the control, which produced
84, 143 and 284 fry in the I, I1 and I11 broods,
respectively (Table 7). The interspawning period
of a female was also extended to about 24 days,
when the male M12 was allowed to mate her.
Thus, a sex-reversed (XX) male was not function-
ally equivalent to a genetic male (=>.

Progeny testing DISCUSSION

Out of 18 males surviving at maturity, 12 were Feminization
subjected t o progeny testing. One male (M12) pro- The long-term experiments (15 months) involv-
duced consecutive broods with a sex ratio of 0.81 ing progeny testing of the treated (TY)individu-
P : 0.19 6 (Table 6). If C. nigrofasciatum should als and their F1 and Fz progenies (e.g., Table 3)
 HORMONAL SEX REVERSAL IN CICHIASOMA 379

TABLE 5. Effect of dietary administration of 17a-methyltestosterone on the 4-day posthatchling of C . nigrofasciatum'

Survival (%I at
Treatment Termination of Sex ratio
Duration (day) Dose (mgkg) treatment Sexual maturity P d

Control 0 95 92 0.51 0.49

10 5 95 87 0.45 0.55
10 92 82 0.39 0.61
25 90 75 0.37 0.63
50 90 72 0.31 0.69a'
75 85 65 0.31 0.69a
100 82 62 0.28 0.72a
200 77 60 0.27 0.73a
400 75 15 0.32 0.68
800 50 0 - -
20 5 90 82 0.49 0.51
10 87 80 0.44 0.56
25 82 75 0.40 0.60
50 82 72 0.35 0.65
75 77 67 0.26 0.74a
100 75 67 0.23 0.77a
200 67 55 0.18 0.82a
400 52 5 - -
800 35 0 - -
30 5 90 82 0.48 0.52
10 82 77 0.45 0.55
25 82 70 0.36 0.64
50 75 67 0.34 0.66
75 72 65 0.31 0.69a
100 67 57 0.22 0.78a
200 60 45 0.25 0.75a
400 40 0 - -
800 30 0 - -

'x2 test: a indicates significant deviation from the sex ratio of 0.5 P : 0.5 d at the level of P = 0.05.

have clearly shown that C. nigrofasciatum males the production of an all-female population in the
are heterogametic and the fish is amenable to hor- ornamental cichlid, C. nigrofasciatum using es-
monal induction of feminization. The present tradiol-p at a dose of 200 mgkg diet for 20 days.
work is perhaps the first to report a protocol for Previously all-female populations have been pro-

TABLE 6. Results of progeny testing among !& males treated with 17a-methyltestosterone (200 mglkg) for 20 days
and mated with normal females o f C. nimofasciatuml

F1 Sex Distribution
Female P=ogenY of F1 progeny Genotype of
Trs used (no.) No. sexed P 6 Trd
Series I1 : Group 2 : Batch VII
Control A1 81 40 0.55 0.45 XY
156 50 0.49 0.51
293 50 0.52 0.48
0.53 0.47
M1 to M l l ' A1 to A6 78 36 0.51 0.49 XY
118 46 0.51 0.49
184 50 0.50 0.50
0.51 0.49
M12 A54 71 35 0.80 0.20 xx
118 40 0.83 0.17
167 50 0.84 0.16
0.82 0.18
'Mean values of 11 males, which are likely to be heterogamous.
380 T. GEORGE AND T.J. PANDIAN
T D L E 7. Comparison of reproductive performance of untreated control @?J,treated
and sex-reversed Ory) males of C. nigrofasciatum'
F1 progeny (no.)
Type of d Genotype I I1
Untreated XY 84 2 4'* 143 k loa
(n = 3)
Treated xy 76 2 7b 110 f 13b
(n= 3)
Treated xx 71 118
sex-reversed
'The mean for at least three fish have been compared.
'I.S.P., interspawning period.
*SNK test: Values superscribed by different alphabets within the same column are significantly ( P i0.05) different.
duced in many cichlidr; belonging t o the genus
Oreochrornis. The commonly used hormones for
feminization were estr,adiol-P (e.g., Mair et al.,
'871, ethynylestradiol with methallibure (e.g.,
Mair et al., '87) or without methallibure (e.g.,
Nakamura and Takahashi, '73) and diethylstil-
bestrol (e.g., Tayamen and Shelton, '78). The ef-
fective dose ranged from 50 mg ethynyl-estradioy
kg diet in 0. rnossarnbicus (Nakamura and Taka-
hashi, '73) to 100 mgkg in 0. niloticus (Tayamen
and Shelton, '78).
Masculinization
However, the treatment of the 4-day post-
hatchling C. nigrofusciutum using selected com-
binations of hormone dojje and treatment duration
significantly ( P < 0.01) increased the percentage
of males to 82% (Table 5). Yet, l7a-methyltest-
osterone was ineffective in producing 100%males,
though it is a relatively more potent and com-
monly used androgen (Pandian and Sheela, '95).
One hundred percent masculinization has been
achieved using a minimim dose for a short period
in cichlids (e.g., 5 mg 17amethyltestosterone/kgdiet
for 11days in 0. rnossanzbicus; Pandian and Vara-
daraj, '90). However, a few authors have previously
reported unsuccessful attempts to produce an all-
male cichlid population. For instance, Guerrero
('76), who administered 50 mg ethynyltestoster-
onekg diet for 40 days t o Tilapia zilli, reported
failure; neither could he induce 100% masculin-
ization when he administered 200 mg methyltes-
tosteronekg diet for 20 days. However, Woiwode
('77) achieved 100% masculinization following ad-
ministration of 50 mg niethyltestosteronekg diet
for 45 days posthatching. Therefore, usage of a
more potent hormone such as ethynyltestosterone
(Yamamoto, '69) and/or a longer treatment period
a)
Sex distribution
1.S.P.' Mortality 0fF1 Progeny
111 (day) (%I 0 6
284 f loa 15 2 1' 8' 0.53 0.47
184 k 31b 19 2 3b loa 0.50 0.50
167 24 20 0.82 0.18
may lead t o the production of an all-male C. ni-
grofasciaturn population.
Survival of the hormone-treated C. nigrofascia-
turn was less than that of control at the termina-
tion of hormone treatment as well as at sexual
maturity. Most unexpectedly, a vast majority of
the treated and sex-reversed XY females and XX
males succumbed to death before attaining sexual
maturity. A striking observation from the prog-
eny testing of C. nigrofasciatum was the sex-de-
pendent mortality of the treated individuals.
There are indications in the literature for this kind
of sex-dependent mortality (Kavumpurath and
Pandian, '92). Very few studies have adopted prog-
eny testing to identify the genotype of the sex-
reversed males and females, and t o assess the
sex-dependent mortality. As the progeny testing
involved only the sexually matured individuals,
and as these individuals were subjected to more
or less the same experimental conditions, a
greater mortality of, for example, the sex reversed
females at any time from the treatment period
until breeding, would reflect the stress suffered
by the genetic male (XY),which had been reversed
to perform the function of a female. Table 8 sum-
marizes the available information on the genotype
composition of fish, which were subjected t o hor-
monal induction of sex reversal and subsequently
to progeny testing. The genotype composition val-
ues are 8%for XX males and 20% for X Y females
of C. nigrofasciaturn; these values are far lower
than those thus far reported.
Sex determination
Male C. nigrofasciatum is heterogamous and
autosomes appear to modify the sex ratio deter-
mined by XY sex chromosomes. Our results pro-
vide evidence for the autosomal balance hypothesis
 HORMONAL SEX REVERSAL IN CICHLAS0AZ.A 381

TABLE 8. Percentage genotype composition of fish which were subjected to sex reversal'
Effective minimum dose Genotype composition (%)
Species Steroid (m&g diet) of treated individuals
Masculinization XY dd xx 66
B. splendens 19-nor-ET2 8 80 20
17a-ET2 15 70 30
And.2 90 62 38
~I-KT~ 60 80 -
0. mykiss 17a-MT2 1 85 15
I? reticulata 19-nor-ET 300 83 17
17a-ET 500 90 10
And. 200 58 42
C. nigrofasciatum 17a-MT 200 92 8
Mean sex composition (%) 76 24
Feminization xx 9 9 xY99
B. splendens P-E2 125 56 44
P-E 50 70 30
DES~ 20 63 37
0. mossambicus DES 100 45 55
r! reticulata P-E 400 69 31
P-E 200 61 39
DES 300 56 44
C. nigrofasciatum
I .
O-E 200 80 20
Mean sex composition (%) __
63 37
_.

'Genotype of the sex-reversed fish was identified by progeny testing (from Pandian et al., '94; modified).
'19-nor-ET. 19-nor-ethvnvltestosterone: 17a-ET. 17a-ethvnvltestosterone,
" I " " And., Androtenedione; DES, Diethylstilbestrol; ll-KT, 11-
ketotestosterone; 17a-MT, 17a-methyltestosteronef B-E, Estradiol-P.

of Avtalion ('82). Accordingly, a cross between XY potency, the sex-reversed male failed to evoke the
female C. nigrofasciatum and XY male was ex- normal response from a responding female. Sur-
pected t o produce approximately one homogametic prisingly, F1 and F2 progenies sired by hormon-
(XX) female, two heterogametic (XY) males and ally treated (Tr)males and females suffered
one homogametic (YY) male; the homogametic significantly higher mortality. This observation is
male could be distinguished from the heteroga- the first of its kind; hence, it is difficult at present
metic male by the sex ratio of F2 progenies. Such to envisage how the negative effects of the ad-
a long-term (15 months) study clearly showed the ministered hormone in treated individuals are
total absence of YY males. YY males are known transmitted on to F1 and F2progenies.
for their inviability (e.g., Betta splendens, George Though estradiol-P treatment resulted in 100%
feminization of C. nigrofasciatum, the sex-reversed
et al., '94). Yet the viability of homogametic males
(e.g., Oryzias latipes, Yamamoto, '55; 0. mossam- females suffered from protracted interspawning
bicus, Varadaraj and Pandian, '89; 0. niloticus, Scott
period and lower fecundity. This observation con-
et al., '89) and homogametic females (Poecilia re- firms the previous report of Kavumpurath and
ticulata YY, Kavumpurath and Pandian, '93; I? Pandian ('92); they observed that the treated B.
sphenops ZZ, George and Pandian, '95a) has been splendens females carrying XX or X Y genotypes
established. For instance, Pandian et al. ('94) suffered from delayed puberty and reduced fecun-
showed that in selected fish, mortality of F, malesdity. Similar findings have also been reported in
carrying XY and YY genotypes was 11%and 89%, the viviparous molly Poecilia sphenops (George
respectively. Not surprisingly, hardly one or two and Pandian, '95b).
heterogamous females and homogamous males of Gonadosomatic indices of C . nigrofasciatum
C. nigrofasciatum were recorded t o be alive. which were amenable and not amenable for sex
reversal showed lower values than the untreated
Reproductive potential males and females. In a recent study, George and
The present study has confirmed that the ad- Pandian ('95b) estimated the sperm counts of €?
ministered steroid impaired the gonadal develop- sphenops, which were subjected t o l7a-methylt-
ment, fecundity and interspawning period in estosterone; irrespective of whether the fish was
female C. nigrofasciatum. Due t o its inherent im- amenable t o endocrine sex reversal or not, the
382 T. GEORGE AND T.J. PANDIAN
treatment significantly reduced the sperm count.
Similarly, studies on salmonids have clearly indi-
cated that due t o “inherent impotency,” the sex-
reversed males do not produce normal sperm
counts (Baker et al., ’88) and are not so aggres-
sive to elicit the normal output of eggs in the fe-
male partner. Clemens et al. (’66) also reported
that the methyltestosterone-treated male guppy
I? reticulutu failed to mate with the female part-
ner, despite displaying full male coloration, and
yielding viable sperm on stripping. Hence, hor-
mone treatment affects both males and females.
ACKNOWLEDGMENTS
We are grateful to Prof. R. Kucherlapati and
an anonymous referee, who have helped us to im-
prove the manuscript. We are thankful to Ms.
Marian Fernandez for help rendered during this
work. Financial support by the Department of Bio-
technology, Government of India, is gratefully ac-
knowledged.
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