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STUDY OF PESTICIDE INDUCED PUFFS IN CHIRONOMUS STRIA TIPENNIS

DAMA.

Patii M.S., Pawar K.R and Jawale C.S.*

Department of Zoology, KSKW Art Science and Commerce College, Nasik-422008, (M.S.), India.
* Department of Zoology, H.P.T. Arts and R.Y.K. Science College, Nashik-422005, (M.S.), India.
(E-mail: mayuraspatil@gmail.com)
ABSTRACT
Pesticide has an ample application on account of its efficiency against a wide variety of insect pest. its entry affect
aquatic flora and fauna. The present study deals with the acute toxicity testing of endosulfan to IVth instar larvae of
Chironomus stratipennis, a most common and widely occuring Indian species. The information on the biology, band
maps and puffing activity of species inhabiting in Maharashtra and especially Nasik region is meager. Hence the
present study was undertaken to study normal puffing of fourth instar larvae and induction of puffs after pesticide
treatment in vivo and in vitro in Chironomus striatipennis from Nasik region. A result was endosulfan-induced puff
in 13A and 14C region of III chromosome arm in vivo and in 14C region of III chromosome arm in vitro._________
KEY WORDS: Chironomus stratipennis, endosulfan, IV instar, in vivo.
INTRODUCTION
Polytene chromosomes have been reported from many Dipterans like Chironomous. These are present in the nuclei by
successive chromosomal duplication inside the intact cell nucleus without cell division. The cells of salivary glands;
Malpighian tubule tracheae, fat body, midgut and hindgut of dipteran larvae show the presence of polytene
chromosomes. Chironomous adults are commonly known as midges. They look like mosquito except proboscis. Midges
belong to the suborder Nematocera of order Diptera. These are all non biting midges. Chironomid midges exhibit
unique life cycle. The adults have short lifespan of 3 -4 days swarming of adults occur near water bodies & female lays
eggs in gelatinous mass numbering 650-700. They are found in fresh water bodies. Larvae feed on diatoms, algae and
organic detritus material. The red color of larvae is because of presence of hemoglobin like respiratory pigment hence
known as blood worm.These larvae are mostly predated by fishes. Polytene chromosome occurs in tissues which are
engaged in vigorous metabolic activities these cells may be secretory or nonsecretory in function. The nuclear
membrane & nucleoli remain intact throughout replication cycle. Thus, the nucleus containing the haploid number of
giant chromosome each composed of 1000-2000 DNA strands giving multistranded cable like appearance. The
peculiar characteristic feature of polytene chromosome is presence of puffs, bulbs and balbiani rings, which are formed
at certain loci and are formed at certain loci and are transient features.
The chironomids are important components in aquatic food chain and have been presently classified as bio-indicators
of pollution (Pinder, 1986; Armitage et al., 1994). Fourth instar larva salivary gland polytene chromosome
preparations before and after exposure of larvae (In vivo) to acute concentration of endosulfan was observed under
microscope so as to study the endosulfan sensitive gene loci after acute stress. Chironomus stratipennis provides an
ideal material to study polytene chromosome band maps and puffing activity on them. In salivary gland cells, DNA
replicates in intact nucleus i.e. endomitosis. Thus, changes in chromosomal structure such as deletions, transpositions,
duplications, etc can be visually varified using the band maps prepared by Gupta and Kumar (1991).
MATERIALS AND METHODS
Test animal: fresh water Chironomus striatipennis
Test Chemicals: -Commercially available pesticide endosulfan 35 EC.
Collection and rearing of common Chironomus species found in Nasik CIDCO area. In vivo studies after toxicant
treatment for 30 minutes, the glands transferred to normal culture medium for recovery for 30 minutes. In vitro studies:
cultured IV instar larvae dissected and salivary glands put into culture medium. After culturing for 1-2 hours, same
pesticide dose (LC50) is put in culture and allow owing its toxicity effect. After toxicant treatment for 30 minutes, the
glands transferred to normal culture medium for recovery for 30 minutes. Then the glands were fixed, stained, squashed
the squashed preparations were made according to method of French et.al (1962) except the use of dissecting fluid.
Carnoys fluid was used as fixative, insect saline (0.68 % saline) was used as dissecting fluid, Aceto -orcein was used as
chromosomal stain And squash preparations were scanned for puff induction. Thus, changes in chromosomal structure
can be visually verified using the band maps prepared by Gupta and Kumar (1991).
RESULTS AND DISCUSSIONS
Well-spread and highly polytenized chromosomal arms were selected for observations. About 200 preparations from
the IV instar larvae were observed for chromosomal puffs. Observation of all four chromosomal arms revealed the
presence of puffs at various loci on each arm. These puff loci are as under:

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Chromosome I: -this chromosome arm showed large puff in 6E region.
Chromosome II: - large puff on 10C and 10D region.
Chromosome III: - No puff.
Chromosome IV: - puff on 16A, 17B and 18A in region.
Thus, in normal chromosome arm I and II has one puff each. Chromosome III has no puff and Chromosome IVth has
three puffs.
LC50 dose for the Chironomus striatipennis
Analysis of the static bioassay carried out on IVth instar larvae of this species showed 0.00195ppb as LC50 value.
Endosulphan induced puffs
In vivo Exposure of IVth instar larvae to 0.00195ppb endosulfan solution resulted in induction of puffs in 13A and 14C
region of chromosome III arm.
Endosulphan induced puffs
In vitro Exposure of salivary glands to endosulphan (LC50. conc.) in insect saline i.e. In vitro also caused the induction
of puff on 14C region only on Chromosome III arm. Thus, it appears that two gene loci In vivo and one gene locus In
vitro are sensitive to endosulphan are present in polytenic genome of Chironomus striatipennis. (Endosulphan induced
- puff).
The karyotype of C. striatipennis comprises of four pairs of chromosomes (2n = 8) in which the sex chromosomes, like
other Chironomus species are not distinguished from the autosomes The nucleolar organizer region is located in 17C
region of chromosome IV.Nucleolar organizer contains the gene loci coding for ribosomal RNA. It is proved that,
nucleolar organizer is an excellent cytological marker for species identification of species with great karyotic similarity
that are found together in natural habitat. Comparison of location of nucleolar organizer in other Chironomus species
revealed that it is located on 2D region of chromosome I in C circumdatus, 17C/18C of chromosome IV in C.
striatipennis, 6B of chromosome I in C ramosus. These suggest that every species has its own characteristic location of
nucleolar organizer regionHowever, the position of centromeres, telomeres and nucleoler organizer can be revealed by
C-banding analysis will confirm the position. Study of polytene chromosome band maps in Chironomus species as
C.ramosus; C. striatipennis, and C. circumdatus reveal clear-cut differences among themselves Bower and Godbole
(1985) reported that the exposure of IV instar larvae to sumithion organochlorine induced puff on locus (93D) of
chromosomal arm of the III chromosome in Anopheles stephensi indicating that on gene locus (93D) is sensitive to
pesticide sumithion. The puff might be synthesizing a protein which in turn act as enzyme that might be neutralizing
the toxic / stress effects of sumithion. In the present study 0.00195ppb (LC50) endosulphan treatment in vivo resulted in
induction of 2 puffs viz. 13A and 14C, while In vitro resulted in induction of puff in 14C region on chromosome arm
III, indicating that these gene loci are endosulphan sensitive.
The chromosome IV shows characteristic feature of inversion of nucleoler organizer of 18C segment into 17C segment
indicating that this species is prone to rearrangements in chromosomal arms. In vivo and In vitro studies revealed the
same gene locus (14C) present on chromosome III arm are sensitive to endosulphan. Whether they are present under
the influence of hormones or not. The 13A puff observed in vivo only indicating that this gene locus is sensitive to
endosulphan in presence of body hormones or any other such factors. The loci showing puffing activity both In vivo
and In vitro situations, indicate that they have some common function under both the conditions. The exact factor
which triggers the induction of puffs in In vivo culturing of salivary glands is not known. (Figur 1 to 4). The
chromosome IV shows characteristic feature of inversion of nucleoler organizer of 18C segment into 17C segment
indicating that this species is prone to rearrangements in chromosomal arms.
Table 1: Percent mortality of Chironomus striatipennis larvae after exposure to endosulfan
Number of
Larvae

Concentration of
Endosulfan In ppb

Number of Larvae Dead After

24 hrs.

Percent mortality

10
10
10
10
10
10
10

0.000244
0.000488
0.0009765
0.00195
0.00391
0.0078125
0.0156

0
0
3
5
6
7
9

0%
0%
30%
50%
60%
70%
90%

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Trends in Biotechnology Reseorch

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Figure 1: I chromosome band map.

Figure 2: II chromosome band map

Figure 3: III chromosome band map with in vivo.

DAMA

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Xvvs'it>Yi of ia '

x * 5 '0 '

Figure 4: IV chromosome band map with observed inversion of nucleolar region.

REFERENCES
Balbiani E. G. (1881). Sur la structure du noyau des cellules salivaires chez les larves de Chironomus. Zool. Anz. 4
:637-641.
Beerman W. and Clever U. (1964). Chromosome puffs. Scientific American. 210:50-58.
Bower S. and Godbole N. N. (1985). Sumithion induced puffs in Anopheles stephensi. Euro. J. Biol. 5:75-77
Bridges C.B. (1935b). Salivary chromosome maps. J. Heredity. 26:60-64.
Burdette W. J. and Anderson R. (1965). Conditioned response of the salivary gland chromosomes of Drosophila
melanogaster to ecdysone. Genetics. 51:625- 633.
Ellgaard E.G. and Clever U. (1971). RNA metabolism during puff induction in Drosophila melanogaster.
Chromosoma (Berlin.). 36:60-78.
Gupta Jai Pal and Kumar Ajay (1991). Chromosomal characterization of chironomous stratipennis. Keiff (Dipteran
chironomidae) Zool. Sci. 8:959-965
Kumar A. and Gupta J. P. (1990). Cytogenetic studies of chironomous circumdates from India (Dipteran
Chironomidae). Genetica. 82:157-163.
Nath B.B. and Godbole N.N. (1997). Chromosomal characterization of a tropical midge. Cytobios 91:25-31.
Oliver D. R. (1971). Life history of chironomidae. Rev. Entomol. 16:211-230.