GSTF International Journal of BioSciences (JBio) Vol.1 No.2, July 2012
The Chromosomes of the Carambola Fruit Fly
Bactrocera carambolae (Diptera: Tephritidae):
Metaphase Karyotype and Polytene Genome
Farzana Yesmin, and Mahani Mansor Clyde
including cashew, mango, sugar palm, avocado, breadfruit,
jackfruit, guava, carambola, lemon, grapefruit, mandarin,
orange, tomato, sapodilla, West Indian cherry, tropical almond
and chilli pepper [6]. It is a serious pest of carambola, which
can be attacked while the fruit is still very young. This pest can
cause up to 100% damage in unprotected situations and tends
to predominate in orchard and urban areas and is rarely if ever
found in undisturbed rain forests [7].
The major means of control of the fly is based on chemical
insecticides. Recently, the controlling efforts have been
diverted to genetic control techniques [8]-[10]. The genetic
approach is hampered, however, by the absence of genetic and
cytological information on B. carambolae. Until now, this type
of data of B. carambolae was not available. The present report
provides a detailed description of the mitotic metaphase
chromosomes and their relation to the other known metaphase
karyotypes of Bactrocera species are discussed. In addition,
characteristic features of the polytene genome of salivary
gland cells are presented.
Abstract—The carambola fruit fly Bactrocera carambolae Drew
& Hancock (Diptera: Tephritidae) is a sibling member of the
Bactrocera dorsalis complex group. This species can cause serious
financial damage in fruits and vegetables in Malaysia. The mitotic
metaphase chromosomes from larval neural ganglia and polytene
genome from larval salivary gland cells of this species are
presented for the first time. Mitotic chromosomes consist of one
pair sex chromosomes (XY/XX) and five pairs of autosomes.
Morphometric characteristics of chromosomes, i.e. number,
centromeric index, morphology, relative length and arm ratio are
examined. In polytene genome, five banded chromosomes are
found which composed of a linear series of alternating bands and
interbands. The banding pattern is distinctive for each
chromosome. No polytenized sex chromosomes are observed,
indicating that five polytene chromosomes are corresponding to
five mitotic autosomes. This investigation shows that B.
carambolae has cytological material that may be very useful for
studying comprehensive genetic organization of the pest’s natural
populations and contributing towards its control.
Index Terms— carambola fruit fly, karyotype, salivary gland,
polytene chromosomes, novel control methods
II. MATERIALS AND METHODS
I. INTRODUCTION
A. Fly Stocks
Initial cultures of B. carambolae were collected from the
Malaysian Agricultural Research Development Institute
(MARDI), Serdang, Malaysia. The laboratory populations are
established and maintained generation wise in the laboratory of
Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor.
The adult food consists of yeast and sugar (1:3). Water is
supplied as soaked cottons. Fresh star fruits are used for
oviposition, 2 days/week. The rearing laboratory is maintained
at 25±1°C, 70±10% RH and a photoperiod of 14:10 (L: D).
The true fruit flies (Family Tephritidae) are ubiquitous and
found in all regions of the world [1]. Several of them possess
great potential to cause damage to agriculture and horticultural
production [2], [3]. In Malaysia, approximately a hundred
Bactrocera species are found, of which only about half have
been recorded [4]. Of these, the carambola fruit fly,
Bactrocera carambolae Drew & Hancock (Diptera:
Tephritidae) is a major agricultural pest [5]. This species has
been recorded on more than 151 kinds of fruits and vegetables
B. Mitotic Chromosome Preparations
Metaphase plates were made from neural ganglia of third
instar larvae, using the technique described in [11]. Larvae
were dissected in saline solution and the brain tissue to be
examined was cleaned as much as possible from unwanted
tissue. Dissected tissues were transferred immediately to a
hypotonic solution of sodium citrate (1%). The tissues were
fixed in methanol-acetic acid mixture (3:1) and then treated
with 60% acetic acid to prepare a cell suspension. A drop of
Manuscript received April 25, 2012. This work was supported by research
grant UKM-ST-06-FRGS0182-2010. Fellowship to Farzana Yesmin from
OWSDW and SIDA is thankfully acknowledged.
Farzana Yesmin is with the School of Environmental and Natural
Resource Sciences, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor DE, Malaysia; on
study leave from the Institute of Food and Radiation Biology (IFRB),
Bangladesh Atomic Energy Commission, E-12/A, Agargaon, Sher-E-Bangla
Nagar, Dhaka- 1207, Bangladesh (e-mail:[email protected]).
Mahani Mansor Clyde is with the School of Environmental and Natural
Resource Sciences, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor DE, Malaysia.
DOI: 10.5176/2251-3140_1.2.9
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© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.1 No.2, July 2012
the cell suspension was put on heated clean glass slides and the
slides were dried at 40-50°C on a hot plate. After that the
slides were stained for 30 min in 5% Giemsa in 10mM
phosphate buffer (pH 6.8). Suitable preparations were
photographed with an Olympus BX41 microscope. The
nomenclature for chromosome morphology and the
centromeric index are based upon work of [12]. The
centromeric index and the relative length of chromosomes
were calculated from the mean of 30 measured metaphase
preparations.
[12], the chromosomes can be grouped into two pairs of
submetacentric (numbers 2 and 3), two pairs of acrocentric
(numbers 5 and 6) and two pairs of metacentric (X
chromosome and number 4) (Fig. 1-3 and Table 1).
C. Polytene Chromosome Preparations
Third instar larvae (7-8 days old) were used for the salivary
gland polytene chromosome preparations following the
method described in [13]. Salivary glands of larvae were
dissected in 45% glacial acetic acid and the tissues were fixed
in 3 N HCl for 3-5 min, then removed and placed in a drop of
lactoacetic acid (80% lactic acid- 60% acetic acid, 1:2) for
about 5 min, subsequently stained with lactoacetic orcein for
about 30-60 min. Excess stain was removed by washing the
glands two or three times in a drop of lactoacetic acid.
Squashing was effected by tapping gently and patiently to
spread the chromosomes. Well spread nuclei were examined
and photographed.
III. RESULTS AND DISCUSSION
A. Mitotic Chromosomes
The metaphase karyotypes of B. carambolae are presented
in Fig. 1(A)-(C). It consists of five pairs of somatically paired
autosomes and a XY/XX sex chromosome pair. Chromosomes
were numbered according to the system used by [8] for med
fly Ceratitis capitata. Most of the fruit fly cytogeneticists used
this system in karyological studies. It labels the sex pair as the
first and the autosomes from 2 to 6 in descending size. The
five autosomes are more lightly stained than the sex
chromosomes and show chromatid separation. The sex
chromosomes are well-differentiated. The Y chromosome is
dot-like, totally heterochromatic and the most deeply stained
component of the set. One arm of the X chromosome is also
highly heterochromatic (Fig. 1A,C, indicated by block arrows),
another arm stained more lightly and does not reveal
chromatid separation as seen in the autosomes (also see Figs. 2
and 3). This situation suggests that this arm of X chromosome
has characteristics of both euchromatin and heterochromatin.
Table I summarizes the results of morphometric chromosome
characteristics of the karyotype. Calculations of relative
chromosome length show chromosome 2 to stand out as the
longest autosome among the complements. Chromosome 2 and
3 are very close to each other in terms of arm ratio and relative
length. The remaining autosomes fall into two pairs of close
relative length, suggesting that distinguishing them by length
in visual inspection would be very difficult. The X
chromosome is the shortest pair among the karyotype. The Y
chromosome is dot-like and could not be measured. Some of
the photographs showing sex chromosomes are presented in
Fig. 2 and 3. According to the arm ratio and centromeric index
Fig. 1. Mitotic chromosomes of B. carambolae from neural ganglia of third
instar larvae. Homologous chromosomes show somatic pairing (indicated by
arrow lines at both ends). Block arrows show the heterochromatic arm of X
chromosome. Single arrows indicate the centromere. (A, B) Female
karyotype. (C) Male karyotype.
TABLE I
MORPHOMETRIC CHARACTERISTICS OF THE CHROMOSOMES OF BACTROCERA
CARAMBOLAE BASED ON THIRTY METAPHASE PREPARATIONS
Pair
No
2
3
4
5
6
XX
Centromeric
Index
Relative
Length
Mean ± SE
Arm Ratio
Mean± SE
31.58±0.48
33.07±0.41
46.20±0.43
24.12±0.48
23.13±0.57
43.20±0.69
9.52 ± 0.13
9.38 ± 0.22
8.78 ± 0.31
7.72 ± 0.23
7.28 ± 0.18
6.32 ± 0.23
2.19 ± 0.05
2.04 ± 0.04
1.18 ± 0.02
3.03 ± 0.08
2.88 ± 0.09
1.33 ± 0.04
Chromosome
Morphology
Submetacentric
Submetacentric
Metacentric
Acrocentric
Acrocentric
Metacentric
The diploid chromosome number of B. carambolae is 12
(n=6), including an XY/XX sex chromosome pair, in accord
with the earlier report of the med fly, Ceratitis capitata [10],
[14]-[15], the melon fly, Bactrocera cucurbitae [11], [13],
[16], the orientel fruit fly, Bactrocera dorsalis [17], the
Queensland fruit fly, Bactrocera tryoni [18], the olive fruit fly,
Bactrocera oleae [19], the Mexican fruit fly, Anastrepha
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GSTF International Journal of BioSciences (JBio) Vol.1 No.2, July 2012
Fig. 2. Metaphase karyotypes of B. carambolae showing intimate association of homologous chromosomes.
Female karyotype = a, d, n. Male karyotype = b, c, e-m, o-q. Arrows indicate the sex chromosomes, X and Y.
Fig. 3. Metaphase karyotype of B. carambolae. Arrows indicate the location of sex chromosomes in the karyotypes.
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© 2012 GSTF
GSTF International Journal of BioSciences (JBio) Vol.1 No.2, July 2012
Fig. 4. Polytene genome of B. carambolae showing chromosome arms. C-Centromere; L-Left arm; R- Right arm; W- Weak points; P- Prominent puffs.
H- Heterochromatic mass.
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GSTF International Journal of BioSciences (JBio) Vol.1 No.2, July 2012
ludens [20] and in the sheep blow fly, Lucilia cuprina
(Calliphoridae) [21]. The two smallest pairs i.e. chromosome 5
and 6, as described in results, are acrocentric. This
corresponds to the situation in Bactrocera tryoni [18]. The
somatic counts of mitotic chromosomes revealed that the
individual
chromosomes
can
be
distinguished
morphologically. The sex chromosomes are particularly well
differentiated (Fig. 1-3). In X chromosome, one arm is deeply
stained and heterochromatic (as indicated by block arrows in
Fig. 1). The staining of other arm is lighter and bears features
of both euchromatin and heterochromatin. This supports the
previous cytological studies of other Bactrocera species [11],
[13], [17]. It has been suggested that the homologous
chromosomes examined in all somatic cells are found in
intimate association, a common feature of the Diptera [22].
The results presented here are similar with this idea (Fig. 1c
and Fig. 2). Few exceptions to this feature are found only in
case of sex chromosomes, sometimes positions were far away
from each other (Fig. 1a and 3a-d) and dot-like Y chromosome
was seen in between of two autosomes (Fig. 3e,f). In Thailand,
some works on cytotaxonomy of the Bactrocera dorsalis
complex have been performed. The study mainly focused on
distinctive patterns of heterochromatin in autosomes and sex
chromosomes [23]-[26].
are regarded as a probable centromere site. The interesting
features of the polytene genome of B. carambolae are the
absence of any polytenized sex chromosomes in the cells and
numerous weak points in the chromosome 2. These correspond
well with the situation in C. capitata [14], [27], [28], B.
cucurbitae [11], B. dorsalis [17] and B. tryoni [18]. Numerous
puffed regions are found in the polytene genome of B.
carambolae. These can differ between individuals and the
possibility of puffing patterns varying with developmental
stage [28]. This has not been investigated and only the most
prominent puffs are noted here (Fig. 4).
Study of the genetic system of economically important
species needs the characterization and recognition of the
individual chromosomes of the pest [8]. In this study, we
described the metaphase karyotype and polytene chromosomes
of B. carambolae, attempting to determine the chromosomal
characteristics of this species. The information should give
great impetus to both applied research aimed at novel control
of this pest and to work of fundamental scientific interest
dealing with the population structure of the pest.
ACKNOWLEDGMENT
Helpful comments on the chromosomes by Prof. Dr.
Antigone Zacharopoulou (Greece) are greatly appreciated.
Sincere thanks to Dr. Jorge Hendrichs (IAEA, Austria) and Dr.
A.R. Clarke (QUT, Australia). Special thanks to MARDI,
Malaysia for providing pupae of the carambola fruit fly.
B. The Polytene Genome
In salivary gland cells, five banded autosomal polytene
chromosomes are found. The chromosomes are numbered
from 2 to 6 as it is commonly done for the tephritid fruit fly
species. The centromere position has been determined by two
criteria described for the medfly Ceratitis capitata, familyTephritidae [10]. Some of these are constrictions located in
regions of heterochromatic mass (Fig. 4b). The longer part of
each chromosome arm is assigned as left arm (L) and the
shorter part as right arm (R). Some photographs of polytene
nuclei are shown in Fig. 4(a)-(c). The tips of each chromosome
arm were identified according to the standard procedure
reported for C. capitata [15] and Bactrocera spp. [11], [17].
The characteristic features, centromeric regions, presence of
prominent puffs as well as band patterns are presented in Fig.
4(a)-(c).
Generally polytene nuclei of Bactrocera species are
characterized by (1) the presence of weak points, which
usually break during slide preparation, (2) the great length of
chromosome arms, (3) the inter- and intra-chromosomal
connections, or ectopic pairing, (4) surface adhesions and (5)
asynapsis observed in the polytene genome [27]. In this study,
high levels of ectopic pairing with many constrictions and
presence of weak points were observed in the polytene nuclei
Fig. 4(a)-(b). The polytene genome of Bactrocera dorsalis s.s
(B. carambolae is a sibling species of this group) revealed the
same number of chromosomes in polytene cells [17]. They
reported a total number of five long chromosomes with two
arms each. Our findings are similar with the above report. Also
the centromeric regions in B. dorsalis have similar
characteristics [17], so these types of region in B. carambolae
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
14
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Farzana Yesmin received the B.Sc. (Honors) in
Zoology (1997) and M.Sc. (1999) in Entomology
from University of Dhaka, Bangladesh.
She joined Bangladesh
Atomic Energy
Commission as Scientific Officer in 2000 and is
currently serving as Principal Scientific Officer in the
Radiation Entomology Division focusing on Insect
Pest Control and Management. At present, she is
pursuing her Ph.D. in Universiti Kebangsaan Malaysia in the field of Insect
Cytogenetics. She has published 17 articles in national and international
journals.
Ms.Farzana is the recipient of OWSDW fellowship (2010-2013) and she
was IAEA Fellow in 2007. She is a life member of the Asiatic Society of
Bangladesh and Zoological Society of Bangladesh. She was a member of the
Australian Entomological Society during 2002 to 2005.
Professor Dr. Mahani Mansor Clyde
graduated from University of Queensland,
Australia with a BSc (First Class Honours)
degree in 1974 and a PhD in Genetics in
1978 from the same university under a
Commonwealth Postgraduate Scholarship.
Her academic career in Universiti
Kebangsaan Malaysia (UKM) began in 1979
as Lecturer, then Assoc. Prof. (1984) and
Professor (1994) in the Department of Genetics, Faculty of Life Sciences,
UKM. Her area of specialization is Cytogenetics. She has published 120
articles in journals and conference proceedings. Prof. Mahani is Past
President (2002-2002) of the Genetics Society of Malaysia, member of the
Malaysian Society of Applied Biology and the International Society for
Horticultural Science, and is on the Editorial Board of Pertanika Journal of
Tropical Agricultural Science.
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