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Differential expression of glutathione s-transferase enzyme in different life stages of various insecticide-resistant strains of Anopheles stephensi: a malaria vector.

INTRODUCTION

Anopheles stephensi Liston (Diptera: Culicidae) is the primary vector of urban malaria in the Indian subcontinent with distribution range extending from southern China to the Red Sea coast (1-2). This species accounts for 15% of the total malaria incidences in India (3). Efforts to control malaria have become more intricate because malarial parasites have become drug resistant and mosquitoes have become resistant to insecticides (4). Mosquitoes have developed resistance to all major groups of insecticides, including biocides (5). Genetics and intensive application of insecticides are responsible for the rapid development of resistance in many insects (6).

Glutathione s-transferases (GSTs) (GSTs; E.C. 2.5.1.18) belong to family of protein that are involved in the detoxification of a wide range of xenobiotics, protection from oxidative damage, intracellular transport of hormones, endogenous metabolites, and exogenous chemicals including insecticides (7-8). They can metabolize insecticides by facilitating their reductive dehydrochlorination or by conjugating glutathione to xenobiotic compounds with electrophilic centers (e.g. drugs, herbicides and insecticides), converting them from reactive lipophilic molecules into water-soluble non-reactive conjugates that may easily be excreted (9-10). The conjugation of glutathione to insecticides results in their detoxification via two distinct pathways. O-dealkylation pathway where, glutathione is conjugated with the alkyl portion of the insecticide, e.g. the demethylation of the tetrachlorvinphos in resistant houseflies (11) and O-dearylation pathway, where glutathione reacts with the leaving group, e.g. the detoxification of parathion and methyl parathion in the diamondback moth Plutella xylostella (12). In addition, they contribute to the removal of toxic oxygen free radical species produced through the action of pesticides (13). GSTs are expressed at high levels in multiple isoenzyme forms and in different patterns at various insect development stages (14). Different insect GST forms are responsible for different insecticide specificities (15). The objective of this study was to compare biochemical characterizations of GST activities expressed among different insecticide resistant strains of An. stephensi.

MATERIAL & METHODS

Mosquito rearing

Ten insecticide resistant strains of An. stephensi derived from different classes of insecticides maintained in the laboratory were used for the study. These strains were maintained at 25 [+ or -] 1[degrees]C and 75 [+ or -] 5% relative humidity with 14 h photoperiods according to the procedure of Shetty (16). The adults were fed on 10% sucrose in 8 x 8 x 8 inch iron cages covered with cotton net cloth. Plastic cup (33" diam) containing clean water lined with filter paper was placed inside the cage for oviposition. The eggs were kept for 72 h to ensure complete hatching. The hatched larvae were transferred to enamel tray and reared. Powdered mixture of fish feed and dog biscuits were given as larval diet. The early IV instar larvae were subjected to insecticide susceptibility test using the diagnostic dose as recommended by WHO (17-18) at each generation so as to maintain its resistance and susceptibility status.

Development of insecticide resistant strains

Laboratory-induced resistant strains of An. stephensi were used in this study (Table 1). The said resistant strains have been established after continuous selection and inbreeding for several generations. WHO diagnostic dosages (Table 1) were selected and resistance tests were carried out according to the procedure of WHO (17-18). The III instar larvae from the isofemales of resistant strains were exposed to their respective diagnostic doses in two replicates for 24 h. Larval diet was added to ensure none of the larval mortality occurs due to lack of feed. Mortality was recorded after 24 h moribund larvae (presenting weak, rigidity or mobility to reach water surface on touch, being in the state of inactivity or dying) were considered as dead. The surviving larvae after the treatment were maintained separately. The process of selective inbreeding was repeated until cent percent survival was reported at given diagnostic doses. Generation taken to attain cent percent survivability is listed in Table 1.

Susceptible batch of larvae which showed 100% mortality when exposed to diagnostic dose of insecticides was selected as control for the study. This susceptible/ control was also obtained after several generation of inbreeding and selection.

Enzyme preparation

About 100 larvae, pupae, adult males, adult females and eggs (100 [micro]g in total weight) were collected and used for the experiment. The extracts were prepared from each of the insecticide-resistant strains and control. The samples were weighed as required and homogenized in 0.02 M phosphate buffer, pH 7.0. Homogenates were then centrifuged at 10,000 x g for 5 min at 5[degrees]C, and the supernatant was used for enzyme analysis.

Protein content assay

Protein contents of the enzyme homogenate were determined according to the method of Lowry et al (27) using bovine serum albumin as the standard. The measurement was performed with the wave length of 660 nm. Five replicates for each insecticide-resistant strains from each life stage were used for the assay and compared to control.

GST activities

Glutathione s-transferase activities were determined using the model substrates 1-chloro-2, 4-dinitrobenzene (CDNB) and reduced GSH as substrates according to Habig et al (28) with slight modifications. The non-enzymatic reaction of CDNB with GSH measured without homogenate served as control. The change in absorbance was measured continuously for 5 min at 340 nm and 37[degrees]C in a Jenway UV/Visible (UK) spectrophotometer. Five replicates for each insecticide-resistant strain and controls for each life stage were used for the assay. Changes in absorbance per min were converted into nmol CDNB conjugated/min/mg protein using the extinction coefficient of the resulting 2,4-dinitrophenyl-glutathione: 9.6 nM/cm at 340 nm. GST activities among the resistant strains were observed at the same time against susceptible/controls.

Data analysis

Means of protein quantity, GST activities and specific GST activities were subjected to one-way ANOVA using Dunnett test in GraphPad Prism version 5.00 Windows, GraphPad software, San Diego California, USA.

RESULTS & DISCUSSION

Comparative protein content assay

Average [micro]g protein/mg body weight in different life stages of insecticide-resistant strains is presented in Table 2. Maximum expressions of protein/mg weight in all the life stages of insecticide-resistant strains were compared to that of susceptible control. Eggs from propoxur resistant strain of An. stephensi showed highest expression 57.95 [+ or -] 0.12 [micro]g protein/mg weight in average, whereas least protein concentration was observed in the eggs of bifenthrin-resistant strains with 40.90 [+ or -] 0.21 [micro]g protein/ mg weight. Protein expression in larval stages was found to be more in cyfluthrin-resistant strains with an average of 134.20 [+ or -] 0.56 [micro]g protein/mg weight. Larvae of chloropyrifos strain showed comparatively lower mean protein content of 96.30 [+ or -] 1.60 [micro]g protein/mg weight among all the insecticide-resistant strains. Among pupa highest mean protein concentration was observed in alphamethrin-resistant strain with 103.60 [+ or -] 0.49 [micro]g protein/mg weight, followed by the least in pupa of bifenthrin-resistant with 77.74 [+ or -] 0.57 [micro]g protein/mg weight. Adult males of carbofuran-resistant strain showed highest expression of protein at an average of 123.15 [+ or -] 0.78 [micro]g protein/mg weight. Least protein content was observed in temephos resistant strains with 97.64 [+ or -] 1.27 [micro]g protein/mg weight. Approximately, 147.59 [+ or -] 0.989 [micro]g protein/mg weight was the maximum observed in adult females of alphamethrin-resistant strain and least protein expression of 112.26 [+ or -] 0.64 [micro]g protein/ mg weight in adult females of bifenthrin-resistant strains. Level of proteins in all the life stages was less in susceptible control strains of An. stephensi.

Comparative assay of GST activities

Glutathione s-transferase activity per mg weight and its specific activity were examined using CDNB as GST substrate for different life stages of different insecticideresistant strains and susceptible control. Activity and specific activity of GSTs in different life stages of 10 insecticide-resistant strains are presented in Table 3. GST activity in the eggs of insecticide resistant strains ranged from maximum of 0.045 nmol CDNB conjugated/min/mg protein in deltamethrin-resistant strain to the least of 0.138 nmol CDNB conjugated/min/mg protein, in DDT- resistant strains. Although marginal variations in activity of GST were observed in eggs of insecticide-resistant strains when compared to that of susceptible control, it was found to be statistically significant (F = 43.65, p <0.05, df = 9, 150).

Overall range of GST activities in the larval stages showed maximum of0.1365 nmol CDNB conjugated/min/ mg protein in DDT resistant strains followed by the larvae of cyfuthrin-resistant strains with 0.1033 nmol CDNB conjugated/min/mg protein. Larvae-resistant to organophosphates namely, chloropyrifos and temephos showed comparatively less activity of GST with 0.0544 nmol CDNB conjugated/min/mg protein and 0.0586 nmol CDNB conjugated/min/mg protein, respectively. Among larvae-resistant to carbamate group of insecticides, propoxur-resistant strains showed more GST activity of 0.0834 nmol CDNB conjugated/min/mg protein. Pooled data among larvae of insecticide-resistant strains showed significant difference in activities of GST (F = 27.12, p <0.05, df = 9, 150).

GSTs assayed among the pupae of various insecticide-resistant strains also showed significant difference in activity level (F = 6.35, p <0.05, df = 9, 150). Pupae of carbofuran resistant strain showed higher GST activity of 0.1443 nmol CDNB conjugated/min/mg protein followed by that of deltamethrin, cyfluthrin and DDT with GST activity of 0.1295, 0.1274 and 0.1178 nmol CDNB conjugated/min/mg protein, respectively. GST activity at an average of 0.09 nmol CDNB conjugated/min/mg protein was observed in pupae of chloropyrifos, temephos, alphamethrin, bifenthrin and neem-resistant strains.

Among the males of insecticide-resistant strains significant differences were observed in the GST activity level ranging from 0.0304 to 0.0763 nmol CDNB conjugated/min/mg protein (F = 346.51, p <0.05, df = 9, 150). Males of neem-resistant strains showed higher GST activity of 0.0763 nmol CDNB conjugated/min/mg protein followed by the males of mosquitoes resistant to carbamate group, i.e carbofuran-resistant strains with GST activity of 0.0675 nmol CDNB conjugated/min/mg protein. Mosquitoes resistant to insecticides belonging to pyrethroid showed least GST activity with males of alphamethrin-resistant strains showing 0.0304 nmol CDNB conjugated/min/mg protein and deltamethrinresistant strains showing 0.0345 nmol CDNB conjugated/ min/mg protein. GST activities ranged from 0.0144 to 0.0637 nmol CDNB conjugated/min/mg protein in females of DDT and chloropyrifos-resistant strains. GST activity of 0.037 nmol CDNB conjugated/min/mg protein was the average activity recorded among the females of various insecticide-resistant strains with statistically significant difference (F = 434.26, p <0.05, df = 9, 150).

Insect GSTs have been implicated in resistance to insecticides through direct metabolism of the insecticide (29), sequestration (30) or by protecting against secondary toxic effects, such as increase in lipid peroxidation, induced by insecticide exposure (31). In this study, we compared quantitative expression of GST isozyme activity levels of different insecticide-resistant strains. Results showed that, although eggs, larvae, pupae and adults from insecticideresistant strains presented higher activity of GST compared to that of susceptible control, this difference was more accentuated in larvae of insecticide-resistant strains. Since these insecticides act in larval stages and the selection process for resistance was based on larval exposure to this chemical, it is natural that higher expression of detoxifying enzymes is found at this life stage. Interestingly, in the present study, adults of bifenthrin, temephos and chloropyrifos-resistant strains were also reported with elevated activity for GST compared to that of larvae. Thus, higher GST activity in these insecticide-resistant adults possibly reflects natural differences in the expression of GST enzymes in different life stages (32).

The involvement of GSTs in resistance to insecticides other than DDT has been reported in the houseflies (15). GSTs are also known as DDT hydrochlorinases because of their role in DDT metabolism (33). GST activity levels observed in the present study in all the different life stages of insecticide-resistant strains of An. stephensi were found to be comparatively higher. Similar observations were also reported in DDT-resistant strains of the African malaria vector, An. gambiae (34). In Ae. aegypti, elevated expression of GST-2, caused by a mutation in a transacting factor was found to be associated with insecticide resistance (35).

It is evident from earlier studies that pyrethroids do not serve as substrates for GST (32). Conversely, we have reported elevated level of GST activity in the present study for An. stephensi resistant to pyrethroid insecticides namely, cyfluthrin, deltamethrin, bifenthrin and alphamethrin. Adult life stages expressed more of this detoxifying enzyme in alphamethrin and bifenthrin resistance, whereas, An. stephensi resistant to deltamethrin and cyfuthrin showed higher expression of GST in the larval stages. Elevated levels of GSTs have been found to bind molecules of many pyrethroid insecticides compromising effectiveness and toxicity by a sequestering mechanism in diamond black moth P. xylostella (L.) and coleopteran Tenebrio molitor (30,36). However, these reports on pyrethroid resistance suggest that the role of GST in insecticide resistance is as an antioxidant defense agent or binding protein (30-31,37). There is an example of direct metabolism of the pyrethroid tetramethrin by a non-insect GST (38).

GSTs have a more supportive or facilitating role against the pyrethroids and organophosphates (39-40). We have shown elevated GST activity in temephos resistant strains of An. stephensi. Higher level of altered activity was found to be associated with temephos resistance in Ae. aegypti from Brazil (41). Increased levels of GSTs were observed in Latin American population of Ae. aegyptiresistant to deltamethrin, temephos, chloropyrifos and cyfluthrin (42). Few studies have also suggested the involvement of GSTs in temephos-resistance may be due to a cross-resistance to pyrethroids from a previous exposure to this insecticide (43-44). This can be attributed to the possible synergistic effect of insecticides (45) or due to over production of esterases (40). However, as the matter of fact, GST's tend to play a significant role in organophosphate resistance (46-48). Strains of housefly-resistant to parathion, diazon and diazoxon were reported with increase in GST activity via de-ethylation of these insecticides (49-50). Similarly, increase in GST activity via demethylation of tetrachlorvinphos was pragmatic in tetrachlorvinphos resistant houseflies (11, 51). Glutathione conjugation was a major resistance mechanism for parathion and methyl parathion in diamondback moth (52) and Lygus lineolaris with resistance to malathion had significantly higher (1.5 fold) GST activity (53). GST gene transcript has also been found to be elevated in resistant strain by 1.3 fold (54). The role of GSTs as a secondary resistance mechanism in detoxication of the oxon analog of fenitrothion was reported in An. subpictus (55). A higher level of GST has been associated with organophosphate detoxification in several other insect species (56).

The present study also reports increase in activity of GST with subsequent life stages in An. stephensi in most of the insecticide-resistant strains used with prominent observable in chloropyrifos and carbofuran resistant strains. Maximum activity of GSTs in larval and pupal stages followed by that in adult stages of An. stephensi was observed in propoxur-resistant strains in the present study. Higher GST activity was marked in An. subpictus resistant to carbamate insecticide propoxur (57). Reports of GST in neem resistant mosquito being scarce, we have reported in our study elevated level of GST in larval and pupal stages of An. stephensi resistant to the plant extract neem (botanical insecticide). One factor that influences the expression levels of enzyme is the number of alleles of a resistance gene present. Large enzyme families have a degree of redundancy or overlap substrate specificities and thus it may be expected that metabolic mechanisms of insecticide resistance against a particular insecticide would differ between different populations of the same species (10). In addition, regulation of GST expression is subject to a complex set of developmental, sex, and tissue-specific factors, as well as environmental and dietary parameters (9).

Insecticide resistance is an important man-made example of natural selection, and the factors governing the origin and spread of resistance-associated mutations are both of academic and of applied importance (58-59). Distribution of GSTs is known to be widespread in nature and there is no question about the importance of these enzyme systems for they may play critical role in explaining selective (60-61) as well as non-selective (62) toxicity and resistance mechanism among various organisms (63). The detoxification function of these enzymes may achieve a particular significance in the insect world by contributing to the development of resistance to insecticides by catalyzing their degradation (14). The biosynthesis of these enzymes seems to reflect a direct response to xenobiotics (60,64). Present study signifies the verity that GST activity is closely associated with insecticide resistance among An. stephensi. Our study strengthens the fact that one of the mechanisms associated with insecticide resistance found in many other insects includes an increase of GST activity, probably as a result of gene amplification.

In conclusion, the results presented here provide the first report of comparative GST activity in An. stephensi resistant to insecticides belonging to pyrethroid, organophosphate, organochlorine, carbamate and biocide group. The data amply demonstrate a predominant role of GSTs in conferring resistance in An. stephensi. This basic knowledge of GST activity may serve as a useful database and will be beneficial in unraveling the prevailing resistance mechanisms which in turn may pave way for the development of molecular marker for resistance detection. This may have an important implication in resistance management in the field and may vastly contribute in implementing effective mosquito control programmes in India.

ACKNOWLEDGEMENTS

DS and VS are grateful to the Poornaprajna Institute of Scientific Research (PPISR), Bengaluru, for research fellowships. The authors also acknowledge the Department of Science and Technology (DST) and University Grants Commission (UGC), Govt. of India, New Delhi for financial assistance.

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D. Sanil [1,2], V. Shetty [1,3] & N.J. Shetty [1]

[1] Centre for Applied Genetics, Bangalore University, Bengaluru; [2] Yenepoya Research Centre, Yenepoya University, Mangalore; [3] Department of Biological Sciences, Poornaprajna Institute of Scientific Research, Bengaluru, India.

Correspondence to: Dr N.J. Shetty (Professor Emeritus), Centre for Applied Genetics, Bangalore Universtiy, J.B. Campus, Bengaluru-560 056, India.

E-mail: shetty_nj@yahoo.co.in

Received: 13 July 2013

Accepted in revised form: 5 April 2014
Table 1. Insecticide resistant strains of An. stephensi
used in the study

S.    Insecticide resistant strains   Diagnostic   Generation taken
No.   of An. stephensi                dose mg/l    to attain 100%
                                      (ppm)        survivability

      Pyrethroids

1.    Cyfluthrin-resistant              0.005             26
      strain (CYF-R) *

2.    Deltamethrin-resistant            0.004             20
      strain (DLM-R) (19)

3.    Alphamethrin-resistant             0.12             27
      strain (AM-R) (20)

4.    Bifenthrin-resistant strain        0.06             27
      (BIF-R) (21)
      Organophosphates

5.    Temephos-resistant                 0.02             21
      strain (TR-R) (22)

6.    Chlorpyrifos-resistant             0.2              23
      strain (CPF-R) (23)
      Carbamates

7.    Propoxur-resistant                 0.01             16
      strain (PR-R) (24)

8.    Carbofuran-resistant               0.5              17
      strain (CBF-R) *
      Organochlorine

9.    DDT-resistant                       3               19
      strain (DDT-R) (25)
      Plant extract

10.   Neem-resistant                     0.43             36
      strain (NM-R) (26)

* Unpublished data.

Table 2. Average protein ([micro]g protein/mg weight) level in the
different life stages of insecticide-resistant strains of An.
stephensi

S.    Insecticide-             Eggs                    Larvae
NO.     resistant          ([micro]g/mg)           ([micro]g/mg)
        strains

1.    Susceptible       30.80 [+ or -] 1.50    86.25 [+ or -] 1
      control
      [CTRL]
      Pyrethroids

2.    CYF-R            46.23 [+ or -] 0.50 *   134.20 [+ or -] 0.56 *

3.    DLM-R            52.26 [+ or -] 0.88 *   109.26 [+ or -] 0.65 *

4.    AM-R             45.12 [+ or -] 0.35 *   102.36 [+ or -] 0.59 *

5.    BIF-R            40.90 [+ or -] 0.21 *   132.36 [+ or -] 0.17 *

      Organophos-
        phates

6.    TR-R             48.32 [+ or -] 0.59 *   98.23 [+ or -] 0.49 *

7.    CPF-R            46.30 [+ or -] 0.70 *   96.30 [+ or -] 1.60 *

      Carbamates

8.    PR-R             57.95 [+ or -] 0.12 *   127.57 [+ or -] 0.80 *

9.    CBF-R            45.90 [+ or -] 1.10 *   103.65 [+ or -] 1.20 *

      Organochlorine

10.   DDT-R            52.32 [+ or -] 0.95 *   112.21 [+ or -] 1.02 *

      Plant extract

11.   NM-R             55.14 [+ or -] 0.54 *   119.89 [+ or -] 0.91 *

S.    Insecticide-             Pupae                 Adult [male]
NO.     resistant          ([micro]g/mg)            ([micro]g/mg)
        strains

1.    Susceptible      58.33 [+ or -] 0.68      89.67 [+ or -] 0.75
      control
      [CTRL]
      Pyrethroids

2.    CYF-R            91 [+ or -] 0.40 *       112.69 [+ or -] 0.94 *

3.    DLM-R            79.23 [+ or -] 0.48 *    99.67 [+ or -] 0.89 *

4.    AM-R             103.60 [+ or -] 0.49 *   98.65 [+ or -] 0.85 *

5.    BIF-R            77.74 [+ or -] 0.57 *    105.98 [+ or -] 1.40 *

      Organophos-
        phates

6.    TR-R             86.25 [+ or -] 0.98 *    97.64 [+ or -] 1.27 *

7.    CPF-R            87.90 [+ or -] 0.16 *    116.75 [+ or -] 0.65 *

      Carbamates

8.    PR-R             97.95 [+ or -] 0.26 *    102.81 [+ or -] 0.95 *

9.    CBF-R            91.10 [+ or -] 1.20 *    123.15 [+ or -] 0.78 *

      Organochlorine

10.   DDT-R            96.65 [+ or -] 0.98 *    119.68 [+ or -] 0.85 *

      Plant extract

11.   NM-R             97.30 [+ or -] 0.62 *    111.25 [+ or -] 0.90 *

S.    Insecticide-     Adult [female]
NO.     resistant      ([micro]g/mg)
        strains

1.    Susceptible      97  [+ or -] 0.50
      control
      [CTRL]
      Pyrethroids

2.    CYF-R            124.80 [+ or -] 0.95 *

3.    DLM-R            136.42 [+ or -] 1.27 *

4.    AM-R             147.59 [+ or -] 0.98 *

5.    BIF-R            112.26 [+ or -] 0.64 *

      Organophos-
        phates

6.    TR-R             136.87 [+ or -] 1.36 *

7.    CPF-R            118.69 [+ or -] 1.75 *

      Carbamates

8.    PR-R             112.36 [+ or -] 2 *

9.    CBF-R            124.80 [+ or -] 1 *

      Organochlorine

10.   DDT-R            127.54 [+ or -] 0.99 *

      Plant extract

11.   NM-R             119.95 [+ or -] 1.50 *

* Non-significant to control at p <0.05.

Table 3. Comparison of the mean and specific GSTs activity in
different life stages of diverse insecticide-resistant strains
(mean [+ or -] S.D.) of An. stephensi

Insecticide-                    Mean [+ or -] S.D
resistant
strains                                Eggs

                          Activity of            Activity of
                             GSTs                   GSTs
                          (nmol/min)           (nmol/min/mg pro)
Pyrethroids

  CYF-R            1.78 [+ or -] (0.0167)     0.117 [+ or -] (0.0011)
  Control          2.66 [+ or -] (0.025)      0.111 [+ or -] (0.0007)

  DLM-R            1.67 [+ or -] (0.0165)     0.138 [+ or -] (0.0006)
  Control          2.11 [+ or -] (0.0104)     0.109 [+ or -] (0.0010)

  AM-R             1.74 [+ or -] (0.0166)     0.114 [+ or -] (0.0010)
  Control          2.02 [+ or -] (0.0159)     0.142 [+ or -] (0.0015)

  BIF-R            1.75 [+ or -] (0.0171)     0.114 [+ or -] (0.0011)
  Control          2.22 [+ or -] (0.0099)     0.145 [+ or -] (0.0006)

Organophosphates

  TR-R             1.29 [+ or -] (0.0078)     0.084 [+ or -] (0.0005)
  Control          2.05 [+ or -] (0.0200)     0.147 [+ or -] (0.0010)

  CPF-R            1.19 [+ or -] (0.0079)     0.078 [+ or -] (0.0005)
  Control          1.93 [+ or -] (0.0175)     0.174 [+ or -] (0.0016)

Carbamates

  PR-R             1.29 [+ or -] (0.0068)     0.085 [+ or -] (0.0004)
  Control          2.16 [+ or -] (0.0231)     0.134 [+ or -] (0.0013)

  CBF-R            1.14 [+ or -] (0.0034)     0.075 [+ or -] (0.0002)
  Control          1.70 [+ or -] (0.0125)     0.132 [+ or -] (0.0010)

Organochlorine

  DDT-R            0.68 [+ or -] (0.0046) *   0.045 [+ or -] (0.0003)
  Control          1 [+ or -] (0.008)         0.065 [+ or -] (0.0005)

Plant extract

  NM-R             1.60 [+ or -] (0.0222)     0.105 [+ or -] (0.0014)
  Control          2.25 [+ or -] (0.0155)     0.126 [+ or -] (0.0011)

Insecticide-                            Larvae
resistant
strains                  Activity of             Specific activity
                             GSTs                     of GSTs
                          (nmol/min)             (nmol/min/mg pro)
Pyrethroids

  CYF-R            4.48 [+ or -] (0.0509)     0.103 [+ or -] (0.0011)
  Control          3 [+ or -] (0.036)         0.069 [+ or -] (0.0008)

  DLM-R            3.42 [+ or -] (0.0278)     0.078 [+ or -] (0.0006)
  Control          2 [+ or -] (0.0193)        0.062 [+ or -] (0.0005)

  AM-R             2.65 [+ or -] (0.0236)     0.061 [+ or -] (0.0005)
  Control          2.43 [+ or -] (0.0262)     0.057 [+ or -] (0.0003)

  BIF-R            2.36 [+ or -] (0.0241)     0.054 [+ or -] (0.0005)
  Control          1.86 [+ or -] (0.0035)     0.024 [+ or -] (0.0005)

Organophosphates

  TR-R             2.55 [+ or -] (0.017)      0.058 [+ or -] (0.0004)
  Control          1 [+ or -] (0.0182)        0.023 [+ or -] (0.0005)

  CPF-R            2.37 [+ or -] (0.0029)     0.054 [+ or -] (0)
  Control          1.07 [+ or -] (0.0247)     0.023 [+ or -] (0.0004)

Carbamates

  PR-R             3.62 [+ or -] (0.0923)     0.083 [+ or -] (0.0021)
  Control          1.04 [+ or -] (0.0229)     0.056 [+ or -] (0.0005)

  CBF-R            2.58 [+ or -] (0.0229)     0.059 [+ or -] (0.0005)
  Control          2.50 [+ or -] (0.0163)     0.045 [+ or -] (0.0004)

Organochlorine

  DDT-R            5.92 [+ or -] (0.1259) *   0.136 [+ or -] (0.0029)
  Control          2.79 [+ or -] (0.0301)     0.064 [+ or -] (0.0006)

Plant extract

  NM-R             4.07 [+ or -] (0.0384)     0.093 [+ or -] (0.0009)
  Control          2.73 [+ or -] (0.0218)     0.042 [+ or -] (0)

Insecticide-                          Pupae
resistant
strains                  Activity of          Activity of
                             GSTs             GSTs
                          (nmol/min)          (nmol/min/mg pro)

Pyrethroids

  CYF-R            3.20 [+ or -] (0.0763)     0.127 [+ or -] (0.0013)
  Control          2.78 [+ or -] (0.0086)     0.066 [+ or -] (0.0004)

  DLM-R            4.62 [+ or -] (0.1272)     0.129 [+ or -] (0.0035)
  Control          3.36 [+ or -] (0.0141)     0.094 [+ or -] (0.0004)

  AM-R             2.35 [+ or -] (0.0293)     0.089 [+ or -] (0.0021)
  Control          1.89 [+ or -] (0.0251)     0.078 [+ or -] (0.0002)

  BIF-R            2.46 [+ or -] (0.0219)     0.069 [+ or -] (0.0006)
  Control          1.94 [+ or -] (0.0170)     0.054 [+ or -] (0.0004)

Organophosphates

  TR-R             3.39 [+ or -] (0.0366)     0.091 [+ or -] (0.0014)
  Control          2.38 [+ or -] (0.0176)     0.056 [+ or -] (0.0004)

  CPF-R            3.18 [+ or -] (0.0396)     0.089 [+ or -] (0.0011)
  Control          2.27 [+ or -] (0.0135)     0.065 [+ or -] (0.0007)

Carbamates

  PR-R             4.20 [+ or -] (0.1293)     0.065 [+ or -] (0.0008)
  Control          2.41 [+ or -] (0.0264)     0.053 [+ or -] (0.0006)

  CBF-R            3.26 [+ or -] (0.0528)     0.144 [+ or -] (0.0055)
  Control          2.02 [+ or -] (0.0166)     0.067 [+ or -] (0.0007)

Organochlorine

  DDT-R            5.15 [+ or -] (0.1994) *   0.117 [+ or -] (0.0036)
  Control          2.16 [+ or -] (0.0199)     0.063 [+ or -] (0.0003)

Plant extract

  NM-R             4.54 [+ or -] (0.0468)     0.095 [+ or -] (0.0010)
  Control          2.32 [+ or -] (0.0259)     0.060 [+ or -] (0.0005)

Insecticide-                            Adult males
resistant
strains                   Activity of              Activity of
                             GSTs                    GSTs
                          (nmol/min)          (nmol/min/mg pro)

Pyrethroids

  CYF-R            1.72 [+ or -] (0.0044)     0.050 [+ or -] (0)
  Control          0.54 [+ or -] (0.011)      0.010 [+ or -] (0.0002)

  DLM-R            1.96 [+ or -] (0.0059)     0.034 [+ or -] (0.0001)
  Control          0.60 [+ or -] (0.0087)     0.008 [+ or -] (0.0002)

  AM-R             2.30 [+ or -] (0.0059)     0.030 [+ or -] (0.0001)
  Control          1.73 [+ or -] (0.0054)     0.034 [+ or -] (0.0001)

  BIF-R            3.14 [+ or -] (0.0113)     0.055 [+ or -] (0.0001)
  Control          1.95 [+ or -] (0.0111)     0.010 [+ or -] (0.0001)

Organophosphates

  TR-R             4.35 [+ or -] (0.0216)     0.057 [+ or -] (0.0002)
  Control          2.02 [+ or -] (0.0131)     0.009 [+ or -] (0.0002)

  CPF-R            3.76 [+ or -] (0.0151)     0.066 [+ or -] (0.0002)
  Control          1.98 [+ or -] (0.0055)     0.030 [+ or -] (0)

Carbamates

  PR-R             3.10 [+ or -] (0.0118)     0.040 [+ or -] (0.0001)
  Control          1.92 [+ or -] (0.0120)     0.034 [+ or -] (0)

  CBF-R            3.28 [+ or -] (0.0123)     0.067 [+ or -] (0.0001)
  Control          0.61 [+ or -] (0.0115)     0.030 [+ or -] (0)

Organochlorine

  DDT-R            3.85 [+ or -] (0.0090) *   0.054 [+ or -] (0.0001)
  Control          0.47 [+ or -] (0.0132)     0.035 [+ or -] (0.0002)

Plant extract

  NM-R             2.86 [+ or -] (0.0032)     0.076 [+ or -] (0.0004)
  Control          1.71 [+ or -] (0.0037)     0.033 [+ or -] (0.0002)

Insecticide-                         Adult females
resistant
strains                  Activity of            Activity of
                             GSTs                  GSTs
                          (nmol/min)           (nmol/min/mg pro)

Pyrethroids

CYF-R              3.01 [+ or -] (0.0058)     0.034 [+ or -] (0)
Control            1.95 [+ or -] (0.0117)     0.015 [+ or -] (0)

DLM-R              1.29 [+ or -] (0.0158)     0.017 [+ or -] (0.0002)
Control            1.09 [+ or -] (0.0039)     0.015 [+ or -] (0.0001)

AM-R               2.19 [+ or -] (0.016)      0.041 [+ or -] (0)
Control            1.85 [+ or -] (0.0112)     0.030 [+ or -] (0)

BIF-R              3.05 [+ or -] (0.0072)     0.041 [+ or -] (0.0001)
Control            1.16 [+ or -] (0.0145)     0.034 [+ or -] (0.0002)

Organophosphates

TR-R               3.92 [+ or -] (0.0202)     0.025 [+ or -] (0.0002)
Control            2.02 [+ or -] (0.0118)     0.016 [+ or -] (0.0002)

CPF-R              4.66 [+ or -] (0.0155)     0.063 [+ or -] (0.0002)
Control            1.87 [+ or -] (0.0126)     0.030 [+ or -] (0)

Carbamates

PR-R               1.05 [+ or -] (0.0151)     0.030 [+ or -] (0.0002)
Control            0.94 [+ or -] (0.0115)     0.015 [+ or -] (0.0001)

CBF-R              1.86 [+ or -] (0.0222)     0.056 [+ or -] (0.0001)
Control            1.11 [+ or -] (0.0146)     0.035 [+ or -] (0.0002)

Organochlorine

DDT-R              4.11 [+ or -] (0.0123) *   0.014 [+ or -] (0.0002)
Control            1.96 [+ or -] (0.0084)     0.013 [+ or -] (0.0001)

NM-R               2.55 [+ or -] (0.0028)     0.053 [+ or -] (0.0002)
Control            1.13 [+ or -] (0.0093)     0.033 [+ or -] (0.0002)

* Significant difference compared to control (p <0.05).
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Author:Sanil, D.; Shetty, V.; Shetty, N.J.
Publication:Journal of Vector Borne Diseases
Article Type:Report
Date:Jun 1, 2014
Words:6477
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