Current Research journal of Biological Sciences 4(3): 301-305, 2012
ISSN: 2041-0778
© Maxwell Scientific Organization, 2012
Submitted: January 08, 2012
Accepted: February 09, 2012
Published: April 05, 2012
Ecological and Genetic Relationship of Chironomus circumdatus (Diptera,
Chironomidae) From Mitochondrial Cytochrome Oxidase Subunit I in
Chiang Mai Province, Northern Thailand
1
W. Roongruangwongse, 1C. Suwannapoom, 1N. Kamrin and 1,2S. Chomdej
1
Department of Biology, Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand
2
Materials Science Research Center, Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand
Abstract: This study provides means for more accurate identification of Chironomus circumdatus species for
environmental and ecological management by Population of C. circumdatus (86 individuals) and water samples
were collected from three different sites of Chiang Mai Province, Thailand. Specimens of Einfeldia sp. were
used as an initial out group. The genetic relationship of C. circumdatus specimens from these sources was
determined by using DNA sequence analysis of partial mtDNA gene’s Cytochrome Oxidase Subunit I (COI).
Analysis of genetic distance on the basis of sequence difference for COI mitochondrial gene showed very little
genetic difference and the data from phylogenetic analysis revealed a very large genetic difference among all
populations of this species for the COI gene sequences. In addition, NJ tree was related to with physicochemical parameters of the water samples.
Key words: Chironomus circumdatus, genetic distance, genetic diversity
Munstermann, 2002; de Brito et al., 2002). Inferences
about distribution are made based on the geographical
pattern of mitochondrial genetic variability, although this
pattern can be influenced by historical and/or ecological
events (Templeton et al., 1995). The relation of water
quality and phylogenetic relationship in C. circumdatus in
Chiang Mai province was observed as a main objective of
this study.
INTRODUCTION
Chironomidae is one of the most important families
of freshwater insects and are widely distributed in all
types of aquatic environments where they can be found in
various habitats and occupy a great variety of niches. Due
to these characteristics, chironomids can be used as a
source of information for river habitat classification and
water quality assessment (Marcello and Bambacigno,
1987). Chironomids are potentially useful as indicators of
water quality (Pinder, 1986) if identifications can be made
genus or species (Morris and Brooker, 1980). Chironomus
spp. (red blood worms) can be normally found inhabited
in heavy organic pollution and rich in nutrients (NO3-N
and PO4G3) areas (Roongruangwongse et al., 2005).
Chironomus consists of four subgenera: Chironomus,
Camptochironomus, Chaetolabis, and Lobochironomus,
which includes about 250 species (Guryev et al., 2001).
The majority of these species (>90%) belong to subgenus
Chironomus. The taxonomy of Chironomus is difficult to
distinguish except through an analysis of proteins. Large
complexes of cryptic species can be best recognized by
the banding pattern of the larval polytene chromosomes
(Martin, 1979). Mitochondrial DNA (mtDNA)
polymorphism is widely used in the study of molecular
markers for phylogenetic studies in animals, partly
because of its simple genomic structure (Avise, 2004;
Excoffier et al., 1992; Brower, 1994; Birungi and
MATERIALS AND METHODS
Study sites: During October 2008 to May 2009 we
obtained C. circumdatus larvae samples from three
populations that located in some area of Chiang Mai
Province, Thailand (in canal from Chiang Mai UniversityCMU, Maharaj Nakhon Chiang Mai Hospital-MN and
Maeka-Maeping-MK) (Table 1).
Water samples collection and physico-chemical
parameters analysis: The pH, electrical conductivity,
and Total Dissolved Solid (TDS) of the water were
measured in the field using a multi-meter (Consort C933).
The water velocity current was measured by a velocity
meter and the water temperature was measured by
thermometer (ºC).
Water samples for chemical parameter analysis were
collected at the three sites. The preservation and analysis
of Dissolved Oxygen (DO), Biological Oxygen Demand
Corresponding Author: S. Chomdej, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200,
Thailand
301
Curr. Res. J. Biol. Sci., 4(3): 301-305, 2012
Mk2
40
Mk4
32
Mk4
86
Mk6
Maeka Maeping
Mk7
CMU9
48
Chiang Mai university
CMU5
45
MN7
40
MN5
46
MN6
Maharaj Nokhon Chiang Mai hospital
Einfe Idia sp.
0.01
Fig. 1: Phylogenetic tree based on the sequences of cytochrome oxidase subunit I gene of C. circumdatus. The tree was constructed
by the neighbor-joining method with 2,000 bootstrapped replications. Numbers above are bootstrap percentages
Table 1: Sampling locations and geographic coordinates of populations
of C. circumdatus analysed in this study
Locality
Code
Latitude/longitude Accession No.
Maeka-maeping
MK6
18º47!/98º57!
JQ287743
JQ287744
MK3
18º47!/98º57!
JQ287745
MK7
18º47!/98º57!
JQ287746
MK2
18º47!/98º57!
JQ287747
Maharaj nakhon
MN5
18º47!/98º58!
JQ287748
chiang mai hospital
MN6
18º47!/98º58!
JQ287749
MN7
18º47!/98º58!
JQ287750
Chiang mai university CMU9 18º44!/98º58!
JQ287751
CMU5 18º44!/98º58!
three sites using pond net by kicking sampling for 10 min
at each site. The larvae were sorted, counted and
preserved in 75% ethanol. Specimens were mounted on
microscope slides and identified in the laboratory by using
Wiederholm (1983), Armitage et al. (1995) and Cranston
(1995). The results of chironomid larvae identification are
shown in Fig. 1.
DNA extraction: Total genomic DNA was extracted from
the samples and preserved in 75% ethanol using modified
proteinase K digestion following the phenol/chloroform
extraction protocol (Sambrook et al., 1989).
Table 2: Physico-chemical parameters of water samples at the three
study sites
Site
---------------------------------------------------Parameters
CMU
MN
MK
DO (mg/L)
2.80
3.50
6.0
5.0
5.5
8.5
BOD5 (mg/L)
Conductivity (ms/cm)
348
208
485
TDS (mg/L)
185
108
258
pH
6.81
7.60
6.33
Water Temperature (°C) 25.0
29.0
26.5
Air Temperature (°C)
29.5
31.0
31.5
Velocity (m/s)
0.72
0.52
0.05
5.0
20.0
10.0
NO-3-N (mg/L)
9.10
4.90
12.8
NH3-N (mg/L)
0.70
4.60
2.20
PO4-3 (mg/L)
Water quality category
3-5
3-5
3-5
PCR primer amplification and sequencing: The
polymerase chain reaction (PCR) was used to amplify a
region of the mitochondrial (COI) gene using the primers
C O I 9 1 1 ( 5 '- T T A A C T T C A G G G TGA C C A A
AAAATCA-3'), COI 912 (5'-TTACTA CCAATCATA
AAGATATTGG-3') (Salman et al., 2009). Every 25 :L
reaction volume consisted of 50 ng extracted DNA, 1X
PCR buffer, 0.2 :M of each forward and primers, 0.2 :M
dNTP, 5 mM MgCl2 and 1 unit Taq DNA polymerase
(Vivantis, Malaysia). Amplification was conducted under
the following conditions: 1 cycle at 94ºC for 5 min; 35
cycles at 94ºC for 30 s, 55ºC for 30 s; and 72ºC for 30
min and at 72ºC for 5 min. All PCR products were
purified using the Qiagen PCR Purification Kit (Qiagen,
Germany) and finally sequenced (Macrogen, Korea).
(BOD) and alkalinity were measured according to APHA
et al. (1992). Nitrate nitrogen (NO3-N), ammonia nitrogen
(NH3-N) and reactive phosphorus (PO4-3) were measured
(DR 2100, HACH Company). These physico-chemical
parameters are shown in Table 2.
Relationship analysis: Sequences were aligned using the
computer programs ClustalX v1.64 (Thompson et al.,
1994; Thompson et al., 1997). Genetic relationships of
C. circumdatus were analyzed with neighbor joining (NJ)
Chironomid larvae collection and identification:
Chironomid larvae (Chironomidae) were collected at all
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Curr. Res. J. Biol. Sci., 4(3): 301-305, 2012
Table 3: Pairwise genetic distance kamura 2-parameter for the COI gene sequence
MK6
MK3
MK7
MK2
MK4
MN5
MN6
MN7
CMU9
CMU5
MK6
MK3
0.002
MK7
0.000
0.002
MK2
0.002
0.003
0.002
MK4
0.002
0.003
0.002
0.003
MN5
0.005
0.006
0.005
0.006
0.006
MN6
0.005
0.006
0.005
0.006
0.006
0.003
MN7
0.008
0.010*
0.008
0.010*
0.010*
0.006
0.006
CMU9
0.013
0.014
0.013
0.014
0.014
0.011
0.011
0.014
CMU5
0.014*
0.016*
0.014*
0.016*
0.016*
0.013*
0.013* 0.016*
0.018
Marks of significance: * p<0.05 analyses were conducted using the Kumar method in MEGA5 (Nei and Kumar, 2000; Tamura et al., 2011)
Table 4: Mean genetic distance between tree populations
MN
CMU
MK
MN
0.007
CMU
0.015
0.013
C. circumdatus indicates that the COI gene sequence
shows larger distance when compared to the COI gene
sequence of (Diptera, Sarcophagidae) species in India
(Bajpai and Tewari, 2010). The Tajima test statistic
(Tajima, 1989) was estimated by using MEGA 5.0
(Tamura et al., 2011). All positions containing gaps and
missing data were eliminated from the dataset (complete
deletion option). The overall Nucleotide diversity (B) for
the three populations was 0.0301. Mean diversity within
subpopulation (±SE) was 0.008 (±0.002) and coefficient
of differentiation was 0.015 (±0.182) for the
polymorphism statistics for the 635 bp region of the
mitochondrial COI gene in all populations of C.
circumdatus according to Tajima's Neutrality Test for 11
sequences. The pairwise genetic distance among sample
of C. circumdatus ranges from 0.000-0.018 and mean
genetic distance = 0.032 (±0.003) for the mitochondrial
COI genes (Table 3) and mean genetic distance between
tree populations (Table 4). Analysis of genetic distances
on the basis of sequence difference for the COI
mitochondrial gene shows very little genetic difference. A
phylogenetic tree was constructed by the neighbor-joining
analysis with 2,000 bootstrapped replications in MEGA
5.0 (Fig. 1). The discrepancy in the phylogenetic trees
based on individual genes may be due to the fact that
these genes are evolving at different rates.
as implemented by the program MEGA 5.0 (Tamura
et al., 2011) on the basis of the amino acid alignment
using both an uncorrected p and more sophisticated
distances (Jones et al., 1992). Tree robustness was
evaluated by bootstrapping (Felsenstein, 1985) with 2,000
replicates. The average interspecific genetic divergence
was calculated with the Kimura 2-parameter model
(Kimura, 1980) using the MEGA 5.0 program.
RESULTS AND DISCUSSION
Water quality at the study sites: The physicochemical
parameters of water samples from all three sites,
especially DO, BOD5, NOG3-N, and NH3-N, were rated
according to the classification of water quality as defined
by the Surface Water Quality Standard of Thailand (The
National Environment Committee Announcement No. 8,
B.E. 2537). The revealed water quality at all sites was
classed in the third to fifth (3-5) categories. The overall
water quality assessment indicated that the water was rich
of in NOG3-N and PO4G3 which is caused by agriculture,
industry and transportation. These, nutrients also promote
the growth of moss and attach algae at the bottom of
water channel which provides habitat and much food for
Chironomus spp.
Phylogenetic relationship: The genetic relationship
using the neighbor-joining (NJ) method with 2,000
bootstrapped replications. It was found that the groups
from Chiang Mai University (CMU) and Maharaj Nakhon
Chiang Mai Hospital (MN) can be grouped as one and
Maeka-Maeping (MK) had a clear genetic difference (Fig.
1) The NJ tree was found to be related to the physicochemical parameters of the water samples. Our work
could be used not only as a guideline for further study on
genetic diversity but also as parameters of water samples
of the C. circumdatus in other population groups.
Genetic diversity: The sequences obtained in the present
study were merged with previously published sequences
(accession numbers JQ287743-JQ287751) from three
locations in Chiang Mai Province, Thailand. COI
amplified, were 635bp in C. circumdatus. The
mitochondrial COI genes of C. circumdatus samples
analyzed correspond to the positions 635 for COI genes of
C. circumdatus. The average nucleotide composition
across all C. circumdatus was A = 26.2%, T = 40.1%, C
= 17.4% and G = 16.3%. A smaller transition/transversion
ratio has been ascribed to the increase of distances among
diptera (Segura et al., 2006). The average
transition/transversion ratio of 15.42 for COI in
CONCLUSION
This study has shown that genetic relationships
between C. circumdatus obtained from cytochrome
oxidase subunit I gene in mtDNA and water parameters
are related. The COI mtDNA gene has proven useful in
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ACKNOWLEDGMENT
We wish to thank the National Research University
Project under Thailand's Office of the Higher Education
Commission for financial support, The Graduate school,
Chiang Mai University which supported this work and we
thank the Animal Genetic Laboratory, Department of
Biology, Faculty of Science, Chiang Mai University, for
assistance.
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