Mar Biotechnol
DOI 10.1007/s10126-010-9266-2
ORIGINAL ARTICLE
Ploidy Manipulation and Polyploid Detection in the White
Shrimp Litopenaeus vannamei (Boone 1931)
(Decapoda, Penaeidae)
Débora de Almeida Aloise & Francisco de Assis Maia-Lima &
Ruth Medeiros de Oliveira & Thiago de Melo Cabral & Wagner Franco Molina
Received: 12 May 2009 / Accepted: 27 December 2009
# Springer Science+Business Media, LLC 2010
Abstract Ploidy manipulation has been rarely used in the
genetic improvement of cultured marine shrimps. Although
polyploid induction has been proven to be successful in
Penaeids, including the species Litopenaeus vannamei, the
methodology still requires some improvements. In the
present work, different thermal shock treatments on ploidy
manipulation were tested and a protocol for detecting
polyploid individuals was also established. Fertilized eggs
were treated by cold (10°C) and heat (38°C) thermal shocks
for 8, 12, 15, 18, 20, and 22 min to induce polyploidy.
Nuclear measurements within distinct treatments revealed a
significant deviation in relation to the mean diameter of
nuclei in the control individuals. Triploid and tetraploid
metaphases were observed within treated individuals,
confirming the increase of interphasic nuclear diameter.
The cold thermal shock was more efficient than the hot
ones, besides leading to a higher and more homogeneous
hatchery rate. A mean number of three nucleoli per nucleus
were observed in diploid individuals, while treated samples
usually presented up to five nucleoli per nucleus. The
standardization of protocols to obtain and detect polyploid
products allows further utilization of such methods on a
commercial scale in order to evaluate the performance of
polyploid individuals in the genetic improvement of
L. vannamei.
D. de Almeida Aloise : R. M. de Oliveira : T. de Melo Cabral :
W. F. Molina (*)
Departamento de Biologia Celular e Genética, Centro de
Biociências Universidade Federal do Rio Grande do Norte,
59078-970 Natal, Rio Grande do Norte, Brazil
e-mail: [email protected]
F. de Assis Maia-Lima
Departamento de Biologia, Faculdade de Ciências,
Cultura e Extensão do Rio Grande do Norte–FACEX,
59080-020 Natal, Rio Grande do Norte, Brazil
Keywords Polyploidy . Crustacea . Litopenaeus vannamei .
Crustacean cytogenetics . Ag-NORs
Introduction
The increasing demand of the cultured shrimp market has
determined the development of genetic improvement
techniques focused on both higher productivity and
reduction of rearing costs.
Chromosomal manipulation, particularly polyploidization, has been extensively used to both experimental and
routine production in several aquatic organisms (Linhart et
al. 2001; Maclean et al. 1999; Teskered et al. 1993). The
triploid individuals obtained by this technique are usually
sterile, and once introduced into culture systems, they
potentially minimize the risks of accidental releases.
Sterility is also expected to cause a higher feeding
conversion ratio (FCR) and thereby faster growth rates
since triploid organisms would reduce the energy costs
related to gonad development, parental care, courtship, and
other reproductive aspects, thus directing them to the body
development (Foresti et al. 1996).
Shrimp ploidy manipulation was formerly obtained in
the penaeid Fenneropenaeus chinensis (Li et al. 2003). In
this species, 6-month-old tetraploid individuals presented a
growth rate 20% higher than that observed in diploids
(Xiang et al. 1992). Both high feeding costs (the most
expensive item within a rearing system) and the efficient
FCR reported in several manipulated organisms have
fostered further application of polyploidization techniques
in shrimp culture.
Polyploids have been obtained by exposing fertilized eggs
to physical or chemical agents. Among the chemical
inductors, cytochalasin B is indicated for mollusks, providing
Mar Biotechnol
high triploidy rates (Komaru et al. 1990; Gérard et al.
1994). However, this treatment may be disadvantageous
due to the low survival rate, as reported in Litopenaeus
vannamei and F. chinensis (Bao et al. 1994; Dumas and
Ramos 1999).
Previous triploidy induction in L. vannamei (Boone
1931) showed that the second polar body (PBII) was
released 15 min after spawning at a water temperature of
28°C. Thermal shocks applied between 10 and 12 min after
fertilization have been proven to be the most effective ones.
However, experiments involving thermal shocks for 14–
16 min after spawning have failed in inducing a triploidy
condition (Dumas and Ramos 1999). According to GarnicaRivera et al. (2004), when the water temperature reaches
29°C, the extrusion occurs between 16 and 20 min (usually
after 18 min).
The difficulties in obtaining good metaphases in
newly hatched larvae is related to a low mitotic index
observed in these organisms during initial phases, as
detected in F. chinensis. The triploid larvae of that species
presented a metamorphosis period ranging from 4 to 8 h
longer than that observed in the diploid control (Li et al.
2003). Analyses of embryo development in polyploids of
Penaeus indicus indicated that the control group was in a
two-cell stage 40 min after fertilization, while most of
induced eggs remained undivided (Morelli 2003). It is still
unknown if such decreased cleavage rate can be extended
to other development stages of polyploid shrimps.
The failure of the early identification of polyploids
represents a shortcoming of polyploidization techniques
since they are phenotypically identical to diploid individuals and the available techniques are not totally effective
(Li et al. 2003). In fish, polyploidy specimens have been
successfully identified through erythrocyte measurements
(Maclean et al. 1999; Tiwary et al. 1999; Linhart et al.
2001; Teskered et al. 1993). Other methodologies such as
cytogenetic analyses, albeit laborious, have also been used
in finfish and mollusks (Linhart et al. 2001; Nomura et al.
2004). A simplified and practical variant to crustacean
cytogenetic analysis is counting the number of nucleoli
per nucleus, comprising an efficient and cost-saving way
to identify polyploid individuals (Linhart et al. 2001;
Okumura et al. 2001).
Few studies focused on the standardization of polyploidization methods have been carried out in Penaeidae. The lack
of a reliable and consistent method of polyploidy induction
represents the main obstacle for a large-scale application of
such technology of chromosomal manipulation in shrimp
culture. Besides, no reports about the early identification
of polyploidy individuals are currently available. In the
present work, an easy protocol for obtaining polyploid
stocks was established in the species L. vannamei by
comparing the efficiency of thermal shocks at both high
and low temperatures. In addition, methodologies for
polyploidy identification, involving chromosomal preparations, measurements of nuclear diameter, and nucleoli
count, are also presented.
Materials and Methods
The experiments of polyploidy induction were carried out in a
commercial shrimp larval culture facility in the state of Rio
Grande do Norte, Northeastern Brazil (25M 0267667; UTM
9321171). Chromosomal analyses and measurement of
interphasic nuclei were performed using a photomicroscope
(Olympus BX50) at ×1,000 enlargement, coupled with a highresolution digital image capture system DP72 Olympus, a
12.8-megapixel cooled digital color camera.
Controlled Spawning
In order to induce polyploidy, females of L. vannamei
(Boone 1931) bearing visible spermatophores were selected
from maturation tanks (20,000 L) and kept individually in
40-L manipulation baskets (MB). These baskets contained
a net (mesh size = 500 μm) placed inside another net
(100-μm mesh), which served as egg collectors. The
baskets were immersed in fiber tanks filled with 250 L
of seawater (30 ppt at 28±0.5°C). The females kept in
MB were monitored visually up to the moment of
releasing the eggs. After spawning, the internal MB net
(mesh size = 500 μm) was suspended to remove
spermatophores and excrement debris.
Chromosomal Manipulation
Thermal shocks were performed on eggs of L. vannamei,
10 min after spawning in distinct intervals, to produce
polyploids. Afterwards, the external net (mesh size =
100 μm) containing the collected eggs were suspended
and immersed in an expanded polystyrene box containing
100 L of seawater (30 ppt). The water temperature used in
the thermal shock was monitored by using a digital
thermometer under constant water aeration.
The efficacy of the thermal shocks was evaluated at
testing temperatures of 10°C and 38°C. L. vannamei eggs
were exposed to such temperatures for 8, 10, 12, 15, 18, 20,
and 22 min. All treatments, including the diploid control,
were performed three times. The treatments were named
CTS8 to CTS22, identifying the cold thermal shocks (10°C)
in intervals from 8 to 22 min, and similarly, the heat
thermal shock treatments at 38°C (8–22 min) were referred
as HTS8 to HTS22. After each exposure interval, the eggs
were transferred to a fiber tank (hatchery tank). This tank
was filled with water at the same temperature and salinity
Mar Biotechnol
Fig. 1 Distribution of mean
nuclear diameters (μm) in
L. vannamei nauplii after
distinct thermal shock treatments at 10°C and 38°C
from the spawning tanks. To optimize the hatchery rate, the
temperature was increased up to 31±0.5°C.
Effectiveness of Polyploidy Induction
The effectiveness of each of the 15 treatments (seven for each
tested temperature plus a control group) was evaluated by
chromosomal counts, nuclear measurements, and number of
nucleoli per interphasic nuclei. The hatching rate in the most
effective treatment was also evaluated.
Chromosomal Analysis—Protocol for Obtaining Mitotic
Chromosomes
Developing eggs (3 h after spawning) and nauplii (15 h
after spawning) were transferred to 15-mL conic tubes. The
tubes were centrifuged at 1,000 rpm for 5 min and the
supernatant removed. Then, a 0.025% colchicine solution
was added and eggs and nauplii were maintained in such
solution for 80 and 45–60 min, respectively. Afterwards,
the tubes were centrifuged at 1,000 rpm for 5 min and
the supernatant removed. Eggs and nauplii were then
kept for 90 min in a hypotonic solution (0.075 M KCl).
After another centrifugation step (1,000 rpm, 5 min), the
supernatant was removed and Carnoy’s fixative was
added (methanol/acetic acid 3:1), followed by storage
in microtubes at 4°C.
About 50 μL of 50% acetic acid solution was added to a
watch glass containing eggs or nauplii previously fixed
(300 μL). This material was fragmented with a glass pestle
and the cell suspension was placed onto a preheated slide at
65°C. The slides were stained with a solution of 10%
Giemsa diluted in phosphate buffer (pH 6.8) for 20 min.
Nuclear Analysis—Nuclear Measurements and Nucleoli
Counts
The measurements were carried out in interphasic nuclei
obtained from the cell suspension of newly hatched nauplii,
previously fixed in methanol: acetic acid (3:1) and stained
as described for the chromosomal preparation. In order to
standardize the protocol, the largest nuclei of each slide,
displaying a good perimeter definition, were selected. Each
nucleus was digitally photographed using the software
QCapture Pro 32. Nuclei images were exported to the
program Image Tool 3.0 for Windows to establish their
diameter in micrometers. A hundred nuclei were analyzed
per treatment and the diameter was initially determined by
the mean measurements of the biggest and smallest axis.
Based on these measurements, the total mean diameter of
Table 1 ANOVA among mean nuclear diameters within control
group and thermal shock treatments at 10°C and 38°C
Parameters
X (μm)
Thermal shock at 10°C
2n
37.2
CTS8a
51.2
CTS10a
55.3
a
CTS12
58.7
CTS15a
65.0
CTS18a
76.4
CTS20a
67.8
CTS22a
45.4
Thermal shock at 38°C
2n
37.2
HTS8a
45.7
HTS10a
46.9
HTS12a
47.3
HTS15a
HTS18a
HTS20a
HTS22
49.3
46.9
46.1
37.7
Variance
SD
SE
9.317
27.553
59.342
57.650
99.278
95.970
116.939
62.603
3.051
5.249
7.703
7.593
9.964
9.796
10.814
7.912
0.261
0.519
0.646
0.708
0.864
0.883
1.087
0.650
9.307
101.684
78.308
85.910
3.051
10.084
8.849
9.269
0.261
0.884
0.776
0.868
77.675
103.761
57.005
32.640
8.813
10.186
7.550
5.713
0.666
0.832
0.707
0.574
a
High significant differences between the mean nuclear diameter in the
control group and that of nuclei exposed to distinct times of thermal shocks
at 10°C and 38°C (P<0.001)
Mar Biotechnol
Fig. 2 Effectiveness of polyploidy induction in eggs of
L. vannamei after cold and heat
thermal shocks. The polyploidy
induction was indicated by the
presence of nuclei with more
than 55 μm of diameter
each treatment was determined. Significance tests using
ANOVA were performed to verify the differences between
diameters of interphasic nuclei in control diploid samples
and in thermal shock treatments for polyploidy induction.
In order to assure the reliability of the data, only those
nuclei presenting a diameter 10% larger than the largest
diploid diameter value (50 μm) were considered polyploid.
Therefore, the effectiveness of each treatment was indicated
by the relative frequency of such nuclei within samples.
The identification and counting of nucleoli was
performed according to the silver nitrate staining (Howell
and Black 1980).
Hatching Rate
About 15 h after spawning, six egg aliquots (20 mL each)
from each treatment were removed. The rate of hatched
eggs able to reach the first larval stage was calculated by
establishing the total number of eggs produced per female
and the relative frequencies of hatched eggs (total of
nauplii) and non-hatched eggs in the distinct thermal shock
treatments.
diameter increased according to the duration of treatment
(r2 =0.92, P≤0.001), being the maximum mean diameter
equal to 76.4±9.8 μm after 18 min of exposure. The
ANOVA test showed that the values observed in nuclei
from control and cold thermal shocks differed significantly
(P<0.001). After long exposure times, such as CTS22, the
nuclear diameters were more homogeneous (Fig. 1), but
significantly different in relation to the control group (P<
0.001; Table 1).
As for the heat thermal shock, the mean nuclear diameter
after each treatment has also increased (HTS8-20 min: r2 =
0.61, P≤0.01), although slighter than that observed in the
cold thermal shocks. The highest nuclear mean was
detected in the treatment HTS15 (49.3±8.8 μm), which
was significantly different from the average in the control
group (Table 1). The averages in the heat shock treatments
were remarkably similar (Fig. 1), but all treatments
evidenced significant differences to the mean nuclei
diameter in the diploid control, excepting HTS22 (P=
Results
The procedure performed to obtain mitotic chromosomes in
the white shrimp suggested that the most suitable development stage for chromosomal analyses would be 3 h after
spawning. The cytogenetic analysis (data not shown in the
present work) revealed controls presenting 2n=88 and
chromosomal numbers ranging from 132 to 176 in the
inducted samples. These data confirmed the presence of
both triploid and tetraploid individuals.
The nuclear measurements revealed a mean value of
37.2±3.05 μm for the diploid nuclei with a modal value of
37/38 μm. After cold thermal shocks, the mean nuclear
Fig. 3 Frequency of nucleoli per interphasic nucleus within the
diploid control sample of L. vannamei. Interphasic nuclei bearing
different numbers of nucleoli are shown above each column (1–5)
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Fig. 4 Induction of polyploidy
in relation to the counts of
nucleoli per interphasic nucleus
of embryos after thermal shocks
at 10°C and 38°C. These values
refer to frequencies equal to or
higher than five or more
nucleolus/nucleus
0.59). Therefore, the frequency of these nuclei was used as
an indicator of the effectiveness of the polyploidy induction
after each treatment. Based on these results, we verified a
positive correlation between the treatment efficacy, as
shown by the increasing frequency of polyploid nuclei
and cold thermal shocks at 10°C within 8–20 min of
exposure (r2 =0.86, P≤0.01), reaching its highest frequency
in CTS18 (all nuclei were polyploid). The treatments CTS15
and CTS20 were also efficient, presenting, respectively,
92.5% and 92.1% of nuclei bearing polyploidy features. After
22 min at 10°C, the effectiveness was reduced (7.4% of
polyploidy nuclei), thus representing the temporal limit for a
successful induction (Fig. 2).
On the other hand, heat thermal shocks determined
relatively lower rates of polyploidy induction, lacking any
correlation between treatments (8–20 min) and the frequency of polyploidy nuclei (r2 =0.10, P=0.27). Using the same
procedure aforementioned, the treatment HTS15 proved to
be the most effective one, reaching 26.3% of polyploidy
nuclei. The efficacy of induction was severely reduced in
HTS22, which produced 3% of polyploidy nuclei (Fig. 2).
This condition would be possibly associated with a severe
restriction to cell division.
The present results showed that diploid individuals
presented up to five nucleoli per interphasic nucleus in
their early ontogenetic development, with modal values of
Fig. 5 Relationship between the
induction and hatchery rates in
thermal shocks at 10°C
two to three silver nitrate marks (Fig. 3). Based on the
diploid pattern of ribosomal DNA expression, the nuclei
bearing a higher frequency of nucleoli in relation to diploid
individuals were regarded as polyploid.
The frequency of nucleoli ranged from one to nine in cold
thermal shocks, with a prevalence of five marks in the
treatments CTS12 to CTS20. The effectiveness of cold
thermal shocks at 10°C based on the nucleoli frequency has
also shown a positive correlation for the CTS8-20 treatments
(r2 =0.61, P≤0.05).
The maximum value of nucleoli number was observed
in CTS15, with 59% of polyploidy nuclei (Fig. 4).
However, in the heat thermal shocks, these nuclei were
observed at a lower frequency, reaching up to six marks.
There was no correlation between the number of nucleoli
and increasing exposure periods at 38°C (r2 =0.18, P=
0.20). The most effective heat thermal shock treatment
(HTS15) presented only 18.5% of polyploid nuclei, with
decreasing values of 16% and 8.9% in HTS18 and HTS20,
respectively. These data corroborate the information
provided by nuclear measurements, indicating a lower
effectiveness of heat thermal shocks in relation to the cold
shock induction.
Cold or heat thermal shocks can lead to an unstable
development pattern and produce unviable individuals.
Thus, once the highest polyploidy induction efficiency
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was verified in cold thermal shocks, the rate of viable eggs
after such treatment was also evaluated.
The inviability of eggs after cold thermal shocks was
directly related to the duration of the thermal induction (r2 =
0.91, P ≤0.001). Therefore, shorter treatments (CTS8,
CTS10, and CTS12) presented a higher survival (hatchery)
rate, ranging from 63% to 70%, close to that observed in
the diploid control sample (±80%). In longer treatments
(CTS15, CTS18, and CTS20), the survival index decreased
(41–43%), representing a reduction of 50% when compared
to the control group (Fig. 5). An overall analysis of the
present data revealed that despite some reduction in the
hatchery rate, the treatments CTS15, CTS18, and CTS20
were the most effective ones by presenting 92% to 100% of
polyploidy nuclei based on the nuclear measurements and
47 to 59% of nuclei bearing five nucleoli or more after
silver nitrate staining.
Discussion
In the present work, the thermal shock was performed 10 min
after spawning at an initial water temperature of 28°C. This
period was apparently suitable for the polyploidy induction
since triploid and even tetraploid individuals were detected
through chromosomal counts. In L. vannamei, when the
water temperature reaches 28°C, the extrusion of the first
polar body (PBI) takes place 8 min after spawning (Dumas
and Ramos 1999). Once the whole spawning process lasts
about 3 min, a temporal difference between the first and
the last fertilized eggs when the thermal shock is applied
10 min after spawning could explain the presence of
nuclei two to fivefold larger than the diploid ones (±74–
92 μm). Therefore, the first fertilized eggs could be still
exposed to the thermal shock after PBII extrusion and
prior to the first cleavage, thereby giving rise to
tetraploid organisms through cytokinesis inhibition. In
the case of late fertilized eggs, the shock might have
taken place during the first meiosis, i.e., before the PBI
extrusion, thus resulting in pentaploid individuals.
Therefore, a wider range of polyploid nuclei was
observed in cold thermal shocks (7–100%) when compared
to heat thermal shocks (3–26%), showing clearly that the
former is more efficient in inducing polyploidization than
the latter. Similar results were previously detected in a
study of chromosomal manipulation in L. vannamei using
thermal shocks from 9°C to 40°C, indicating that low
temperatures would be more efficient and able to determine
higher hatchery rates than heat thermal shocks (GarnicaRivera et al. 2004). The best performance of cold thermal
shocks could be related to the adaptability of L. vannamei
to warm waters since this species naturally inhabits tropical
areas. This hypothesis is corroborated by the inverse
condition observed in F. chinensis, a typical cold water
species where the rate of polyploidy induction was above
90% after heat thermal shocks (Li et al. 2003).
Within the cold shock treatments, we observed that the rate
of polyploid individuals was mostly affected by the exposure
time once the survival index decreased as the thermal shock
lasted longer. A remarkable reduction in the polyploidy
frequency was observed in the CTS22 treatment, as detected
by both reduction in the diameter of the interphasic nuclei,
when compared to CTS20, and lower nucleoli frequency.
Although the reasons for this behavior remain poorly
understood, it might putatively indicate that such exposure
time would be over the cryptic survival threshold of the
induced polyploid individuals. The remaining individuals in
the CTS22 treatment would comprise those free from
induction or the few polyploids that managed to survive. A
lower survival rate in the polyploid products is thought to be
related to the disruption in the spindle assembly, thus leading
to abnormal chromosomal segregation and giving rise to
aneuploid individuals (Wang et al. 1999; Sakao et al. 2006).
A comparative analysis among the most efficient
treatments (CTS15, CTS18, and CTS20) in relation to
the number of polyploidy nuclei indicates that CTS18
provided the best conditions and the highest effectiveness
in the induction of triploid and tetraploid stocks of
L. vannamei, producing no diploid individuals. Further and
continuous studies about the performance of polyploidy
stocks under rearing conditions are important to determine
the applicability of this methodology on shrimp farms to
significantly improve the production of commercially-reared
L. vannamei specimens.
Acknowledgments The authors are grateful to the National Counsel
of Technological and Scientific Development (CNPq) and FINEP for
the financial support. We would also like to thank the staff from
commercial farms for supporting field work.
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