144 Saleem et al.Archives of Insect Biochemistry and Physiology 39:144–154 (1998)

© 1998 Wiley-Liss, Inc.

Macromolecular and Enzymatic Abnormalities Induced bya Synthetic Pyrethroid, Ripcord (Cypermethrin), in Adult

Beetles of a Stored Grain Pest, Tribolium castaneum(Herbst.) (Coleoptera: Tenebrionidae)

Mushtaq A. Saleem,1 Abdul Rauf Shakoori,1* and David Mantle2

1Department of Zoology, University of the Punjab, New Campus, Lahore-20, Pakistan2Department of Neurochemistry, Newcastle General Hospital, Newcastle upon Tyne, United Kingdom

The effects of a synthetic pyrethroid insecticide, cypermethrin,administered as a formulation Ripcord 25EC (emulsified con-centrate), to adult beetles of a stored grain pest, Triboliumcastaneum, have been studied, with an objective to ascertainits toxicity on enzymes such as carbohydrases, phosphatases,dehydrogenases, aminotransferases, and concentration of vari-ous biochemical components such as monosaccharides, glyco-gen, cholesterol, nucleic acids, urea, total lipids, and totalproteins. Almost all the enzymes and biochemical componentswere sensitive to sublethal doses of Ripcord 25 EC and theseeffects were found to be dependent on the duration of treat-ment. All carbohydrate metabolizing enzymes (amylase, inver-tase, lactase, maltase, lactate dehydrogenase) were elevated,except for trehalase, which was also elevated up to day 3 butreturned to normal levels subsequently. Phosphatases (alka-line as well as acidic) were increased first and decreased there-after, while isocitrate dehydrogenase decreased throughout theexperimental period. Transaminases (aspartate aminotrans-ferase and alanine aminotransferase) showed a decreasingtrend. Of the other biochemical components tested, glucose con-tent decreased during the first 3 days but increased subse-quently. Fructose content showed an increase, while theglycogen content decreased throughout the study. Total lipidcontent was not disturbed up to day 3 but increased thereaf-ter. Cholesterol content was depleted by day 7. Total proteinsstarted decreasing from day 3 onwards, while soluble proteinswere not affected. DNA, RNA, and urea contents exhibited el-evated levels, while uric acid showed a decreasing trend. Sub-lethal doses of Ripcord, therefore, resulted in extensive enzymeinduction, and utilization of carbohydrates, proteins, and lip-ids, in the given order, perhaps to produce extra energy tocombat insecticidal stress. Arch. Insect Biochem. Physiol.39:144–154, 1998. © 1998 Wiley-Liss, Inc.

Abbreviations used: AcP = acid phosphatase; AkP = alkalinephosphatase; ALAT = alanine aminotransferase; ASAT = as-partate aminotransferase; ChE = cholinesterase; FAA = freeamino acids; ICDH = isocitrate dehydrogenase; LDH = lac-tate dehydrogenase.

*Correspondence to: Prof. Dr. A.R. Shakoori, Department ofZoology, University of the Punjab, New Campus, Lahore54590, Pakistan. E-mail: arshak@brain.net.pk

Received 15 May 1998; accepted 19 October 1998

Biochemical Effects of Ripcord in Tribolium 145

Key words: synthetic pyrethroid; Ripcord; Cypermethrin; enzyme activities;biochemical components; adult beetles of Tribolium castaneum

INTRODUCTIONSynthetic pyrethroids are chemical com-

pounds with an action similar to that of naturalpyrethrum. The major constituents of naturalpyrethrin (pyrethrin I and pyrethrin II) com-bined high insecticidal toxicity with low acutemammalian toxicity (Elliott and Janes, 1973).However, the problem of isolation on a largescale from Chrysanthemum cinerariaefolium lim-ited their availability. More important, their pho-tochemical instability rendered them totallyunsuitable for consideration as an agriculturalinsecticide. Permethrin was the first photostablesynthetic derivative reported by Elliott andJanes in 1973. It is structurally closely relatedto an earlier synthetic analogue, phenothrin, butthe dicholorovinyl side chain results in enhancedinsecticidal activity and much greater stability(Fig. 1). An outstanding characteristic of pyre-throids, which distinguishes them from otherlipophilic insecticides is their rapid biodegrad-ability. Cypermethrin is a synthetic pyrethroidof moderate mammalian toxicity (Breese and

Highwood, 1977). The introduction of an alpha-cyano group into the 3-phenoxybenzyl moiety, no-tably in Cypermethrin, Fenpropathrin, andDecamethrin, served to further enhance the bio-logical activity (Roberts, 1981). With the intro-duction of these chemical compounds, it wasuseful to know their biochemical fate in insectsas they are expected to supplement and possiblyreplace some of the conventional organochlo-rines, organophosphates, and carbamate insec-ticides in controlling resistant insect pests(El-Guindy et al., 1982).

Pyrethroids are likely to cause extensivedamage to hemolymph and other systems at le-thal doses, but there are very few reports avail-able on the effects of sublethal doses of syntheticpyrethroids on the biochemical components of in-sects. This is more true of effects of synthetic pyre-throids at sublethal doses at the macromolecularlevel in the red flour beetle, Tribolium castaneum,which is considered one of the important pests ofstored grains in the world, specially in the UnitedStates, the United Kingdom, Australia, Sudan,Nigeria, Egypt, and Pakistan (Champ and Dyte,1977; Saleem and Shakoori, 1989).

Studies were undertaken in this laboratoryon the effects of synthetic pyrethroids at suble-thal doses on various enzyme activities and re-lated macromolecules of stored grain insect pestsin general and T. castaneum in particular (Saleem,1990; Saleem and Shakoori, 1987a,1993; Shakooriet al., 1993, 1994, 1995; Wilkins et al., 1995).Cypermethrin acts by interfering with the Na+

and K+ channels in the peripheral and central ner-vous systems of insects. Cypermethrin exposurecauses repetitive firings of the nerve endings aswell as increased and sustained membrane ac-tion potentials that overwhelm the muscular neu-rotransmitters resulting in paralysis and death(Casida et al., 1983; Elliott, 1989). In the presentpaper, we report the effects of synthetic pyre-throids at sublethal doses of Cypermethrin (Rip-cord 25 EC) on activities of several enzymes andlevels of various macromolecules related to gly-colytic pathway, Kreb’s Citric Acid Cycle, and oxi-dative phosphorylation in adult beetles of T.castaneum under laboratory conditions. Ripcordcaused significant elevation of all the carbohy-drases (except trehalase, which was also elevatedup to day 3 but normalised subsequently), which

Fig. 1. Chemical formulae of three synthetic pyrethroids;Phenothrin, Permethrin, and Cypermethrin.

146 Saleem et al.

depended on the duration of exposure. Increasedlevels of amylase, trehalase, invertase, lactase,and maltase indicated utilization of carbohydratesafter Ripcord exposure, and may be consideredas enzyme markers for insecticide treatment. Thiswork is expected to help in understanding thechemical control mechanisms of this stored graininsect pest in the field in order to plan effectivecontrol strategies.

MATERIALS AND METHODSBeetles

The methods for rearing beetles have been de-scribed elsewhere (Saleem and Shakoori, 1987a).The starter culture of T. castaneum was obtainedfrom the Food Storage Research Division of the Pa-kistan Agricultural Research Council, Malir Halt,Karachi, Pakistan. They were maintained in a tem-perature-controlled room at 30 ± 1°C with relativehumidity of 60 ± 5%. The insects were reared in300 ml glass jars covered with muslin cloth. Wholemeal wheat flour was used as the culture medium.The newly emerged adult beetles were aged for 10± 1 days and then used in the present study.

Toxicant Used

Commercial formulation of Cypermethrin[(RS)-alpha-cyano-3-phenoxybenzyl (IRS)-cis,trans-3-(2,2-dichloroviny1-2,2 dimethylcyclopro-panecarboxylate] a synthetic pyrethroid insecti-cide, Ripcord 25 EC, was obtained from PakistanBurmah Shell, The Mall, Lahore, Pakistan. Thecommercial formulation of Cypermethrin wasused for the present studies instead of technicalgrade because the chemical was available in thisform for use in the field. The formulation was,therefore, considered closer to existing applica-tion conditions and hence will produce comparableresults to those in the field.

Application of Toxicant

A pilot trial determined the range of toxicity(LC50) of Ripcord 25 EC against adult beetles un-der laboratory conditions. Following this, a suble-thal dose of 2 ppm, equivalent to LC20 dose levelfor adult beetles, was chosen for the experiments.This dose was calculated from a regression linedrawn between the dose-mortality relationship. Atthis dose, the mortality was lower, while physiologi-cal effects of the insecticide were expected to behigh, an observation based on previous studies inthe laboratory (Saleem, 1990; Saleem and Shakoori,1987a; Shakoori et al., 1993).

The insecticide was dissolved in acetone andits different concentrations were administeredto the insects by the residual deposit method.The acetone solution at each concentration (1.3ml) was applied to the bottom of a glass Petridish (130 cm2) with a micropipette and spreaduniformly by rotating the plate. The acetone wasevaporated and the dishes dried at room tem-perature before introducing adult beetles. Ineach Petri dish 100 healthy beetles were intro-duced. Four Petri dishes were used for each timepoint, both for the control and experimentalgroups. A total of seven sets, each with 4 Petriplates, were used for this study. Every day, for 7days, one set of 4 control and 4 treated Petriplates were used for biochemical analyses. Con-trol dishes were treated with acetone only. Allthe treatments were run parallel to each other.In the results, treated insects were comparedwith their respective controls (untreated andstarved). Beetles that had died were not includedin the biochemical tests, and the criterion formortality used here were those described byLloyd (1969), according to which the insects werejudged to be dead when the pressure from abrush failed to produce a response.

Biochemical Analyses

The methods for biochemical analyses usedwere the same as described elsewhere (Saleemand Shakoori, 1987a,b). About 100 adults, weigh-ing a total of 160–180 mg, in each replicate werecrushed in 4 ml of 0.15 M NaCl solution (at 4°C)with the help of a motor driven teflon-glasshomogenizer, cooled with ice. The homogenatewas centrifuged at 4,900g for 10 min at 4°C. Thesupernatant thus obtained was used for the esti-mation of acid phosphatase (AcP*), orthophospho-ric-monoester phosphohydrolase, acid optimum,(EC:3.1.3.2), and alkaline phosphatase (AkP, EC:3.1.3.1) activities according to Kind and King(1954); lactate dehydrogenase (LDH, L-lactate:NAD oxidoreductase, EC:1.1.1.27) activity by amethod based on Cabaud and Wroblewski (1958);isocitrate dehydrogenase (ICDH, threo-Ds isoci-trate: NADP oxidoreductase, EC:1.1.1.42) activ-ity by a procedure described by Bell and Baron(1960); aspartate aminotransferase (ASAT,Laspartate: 2-oxoglutarate aminotransferase,EC:2.6.1.1) and alanine aminotransferase (ALAT,L-alanine: 2-oxoglutarate aminotransferase, EC:2.6.1.2) activities according to Reitman andFrankel (1959); cholinesterase (ChE, acetylcho-line acetylhydrolase, EC:3.1.1.7) activity accord-

Biochemical Effects of Ripcord in Tribolium 147

ing to Rappaport et al. (1959); amylase (1,4-Dglucan glucanohydrolase, EC: 3.2.1.1) activity ac-cording to the procedure described in Wootton(1964); protease activity by the method of Ya-sunobu and McConn (1970); trehalase, invertase,maltase, and lactase activities according to theprocedure described by Dahlqvist (1966).

A brief account of the reaction mixtures usedfor the various enzymes mentioned above is givenbelow.

AkP activity. Ten milliliters of carbonatebuffer, pH 10, 1 ml 0.01 M disodium phenyl-phosphate + 0.l ml enzyme extract was incubatedfor 15 min. The reaction stopped with 0.8 mlNaOH (0.5 M), followed by addition of 1.2 ml of0.5 M sodium bicarbonate, l ml of aminoanti-pyrine (0.6%, vol/vol), and l ml of potassium fer-ricyanide (2.4%, wt/vol). Reddish brown colourread at 510 nm. Crystalline phenol (l mg/100 ml)was used as standard solution.

AcP activity. One milliliter of citrate buffer,pH 4.9, l ml of 0.01 M disodium phenylphosphate+ 0.2 ml enzyme extract was incubated for 1 h.The reaction stopped with l ml 0.5 M NaOH, fol-lowed by l ml of 0.5 M sodium bicarbonate and lml of aminoantipyrine (0.6%, vol/vol) and 1 ml ofpotassium ferricyanide (2.4%, wt/vol). Reddishbrown colour read at 510 nm. Crystalline phenol(l mg/100 ml) was used as standard solution.

ASAT activity. 100 mM L-aspartate (0.5 ml)and 2 mM 2-oxoglutarate in 100 mM phosphatebuffer + 0.2 ml of diluted extract was incubatedfor 30 min. The reaction was stopped by 5 ml of1.5 mM 2,4 dinitrophenylhydrazine and 0.4 MNaOH was used as diluent.

ALAT activity. 100 mM (0.5 ml) DL-alanineand 2 mM 2-oxoglutarate in 100 mM phosphatebuffer + 0.l ml of diluted extract was incubatedfor 30 min. The reaction was stopped by 0.5 mlof 1.5 mM 2,4 dinitrophenylhydrazine and 0.4 MNaOH was used as diluent.

LDH activity. One milliliter of 7.84 × 10–1

M sodium pyruvate in 9.6 × 10–2 M phosphatebuffer, pH 7.5 containing 1.3 × 10–3 M NADH +0.1 ml of extract was incubated for 30 min. Thereaction was stopped by 1 ml of 1 × 10–3 M 2,4dinitrophenylhydrazine (in 1 M HCI) and 4 × 10–1

M NaOH was used as diluent.ICDH activity. 0.2 ml of 10 µM DL-isoci-

trate trisodium in phosphate buffer, pH 7.8 con-taining 0.l ml NADP (3 mg/ml) + 0.l ml of extractwas incubated for 30 min. The reaction wasstopped by 0.5 ml of 1.5 mM 2,4 dinitrophenyl-hydrazine and 0.5 M NaOH was used as diluent.

Amylase activity. 1.0 ml of 0.2 g/1 amylose+ 15 mM NaCl in 20 mM phosphate buffer, pH 7.1+ 0.01 ml extract was incubated for 15 min. 0.008N iodine solution was used as colour reagent.

ChE activity. 0.1 ml extract was incubatedwith 1.0 ml of 5.3 mM m-nitrophenol in phosphatebuffer pH 7.8, 0.1 ml of 0.15 M NaCl and 0.l ml of0.83 M acetylcholine chloride for 30 min and yel-low colour read immediately at 420 nm againstblank.

Trehalase activity. Enzyme extract (0.3 ml)was mixed with 0.5 ml of 0.02 M, trehalose and0.6 ml of 0.1 M sodium citrate-citric acid bufferpH 5.6 and incubated for 15 min. The reactionwas stopped by addition of l ml of Ba(OH)2 and lml of ZnSO4 made up to 10 ml, stirred and cen-trifuged for 3,000g for 10 min. Aliquots of the su-pernatant were tested for reducing sugar byO-toluidine method of Hartel et al. (1969).

Invertase, lactase, and maltase activi-ties. Enzyme extract (0.05 ml) was mixed with0.l ml of appropriate buffered substrate solution(0.056 M solution of appropriate disaccharide in0.1 M sodium maleate buffer, pH 6.0) and incu-bated for 60 min. After that 0.5 ml water wasadded and the extract was immersed in boilingwater bath for 2 min to inactivate the disaccha-ridase. The extract was then cooled with tapwater. A blank was prepared with the same com-position, which was boiled immediately. Aliquotswere tested for reducing sugar by O-toluidinemethod of Hartel et al. (1969).

Protease activity. A 2% casein solution in0.1 M phosphate buffer, pH 7.5, was incubatedwith 0.l ml extract for 30 min at 37°C. The reac-tion was stopped by adding 10% PCA. The amountof hydrolytic products in the supernatant weremeasured by the method of Lowry et al. (1951).

The various enzymatic activities have beenexpressed as units per mg wet weight, while bio-chemical components have been expressed interms of content/mg wet weight content. The defi-nitions used for various enzyme units are as fol-lows: IU, international units, the amount ofenzyme, which under defined assay conditions,will catalyze the conversion of 1 µmol of substrateper minute; RU, Rappaport units, the amount ofenzyme which will liberate 1 µmol of acetic acidfrom acetylcholine in 30 min at 25°C at pH 7.8under the conditions of this test; SU, Somogyiunits, the amount of enzyme digesting 5 mg ofstarch in the experimental conditions used here;Sigma units, the amount of enzyme in 1 ml ex-tract that will produce 1 nmol of NADPH in 1 h

148 Saleem et al.

at 25°C, pH 7.5, under the conditions of this test(see Saleem and Shakoori, 1987a,b).

The supernatant was also analysed for solu-ble protein content by the method of Lowry et al.(1951), free amino acids (FAA) content accordingto Moore and Stein (1954), glucose content by theo-toluidine method of Hartel et al. (1969), fruc-tose content according to the procedure describedby Consolazio and Iacono (1963), trehalose con-tent by the anthrone method of Carroll et al.(1956) as modified by Roe and Dailey (1966) andSteele and Paul (1985), urea content according tothe diacetyl monoxime method as described byNatelson et al. (1951), and uric acid content ac-cording to the procedure of Bauer et al. (1974).

For the quantitative analyses of lipid andcholesterol, about 30 insects in each replicatewere crushed in ethanol and left overnight be-fore centrifugation at 1,300g for 15 min. The etha-nol extract was used for the estimation of lipidcontent according to the method of Zollner andKirsch described by Henry and Henry (1974).

Glycogen content was determined via theanthrone method of Consolazio and Iacono (1963)for which 8 adult beetles (weighing 11–14 mg inall) were used in each replicate.

Nucleic acids were extracted from about 30 in-sects according to the method reported by Shakooriand Ahmad (1973). DNA and RNA contents wereestimated as mentioned by Schneider (1957). Totalprotein was analyzed from the pellet left after theextraction of nucleic acids. The pellet was digestedin 0.5 N NaOH and then used for colorimetric esti-mation according to Lowry et al. (1951).

Keeping in view the fact that Tribolium spe-cies possess ethyl and methly 1-4 benzoquinones,which could inhibit certain enzyme activities, bio-chemical analyses were conducted simultaneouly.

RESULTSBody Weight

Adult beetles at eclosion weighed 1.89 ± 0.03mg. After treatment with insecticide, the bodyweight decreased 20, 24, 33, and 34% after 1, 2,3, and 4 days of insecticide exposure.

Effect of Ripcord 25EC on Enzymatic Activitiesin Tribolium

Effects of a sublethal dose of Ripcord 25 ECon some carbohydrases (such as amylase, tre-halase, invertase, lactase, and maltase) and otherenzymes (such as AkP, AcP, LDH, ICDH, ASAT,ALAT, ChE, and proteases) of adult beetles of T.

castaneum are shown in Table 1, while Figure 2shows percent increase or decrease in various en-zymes of treated beetles compared with their cor-responding controls.

Of the carbohydrases, amylase activity in-creased significantly from day 2 through day 7(112% on day 7), invertase was elevated from day3 through day 7 (90% on day 6), whereas lactasewas raised from day 4 through day 7 (55% on day6). Trehalase activity showed elevation during thefirst 3 days (102% on day 3), and normalised there-after. Maltase activity was, however, significantlydepleted on day 1 and day 2, re-adjusted on day 3and manifested augmentation subsequently ondays 4 through 7 (118% on day 7).

The phosphatases responded alike to thesublethal dose of Ripcord. They increased duringthe initial 3 days of treatment (AkP 34% on day2, AcP 24% on day 3) followed by significant deple-tion during the subsequent period (AkP 62% onday 6, AcP 42% on day 6).

The LDH activity was significantly elevated(140% on day 6), whereas ICDH activity was con-versely significantly decreased (35–60%) through-out the treatment period.

Transaminases were not altered on day 1but showed significant reduction subsequently.ASAT activity was reduced 38% on day 2 afterinsecticide treatment but increased 34% on day4. The ALAT activity was likewise decreased(24% on day 2), but increased afterwards, show-ing an increase of 24% after 6 days of insecti-cide treatment.

ChE activity showed continuous elevationfrom day 2 through day 7 (71% on day 7). On thecontrary, protease activity did not show any no-ticeable deviation from that of control insects.

Carbohydrates and Lipids

Table 2 and Figure 3 show the effect of asublethal dose of Ripcord 25 EC on glucose, fruc-tose, glycogen, lipid, cholesterol, proteins, uricacid, urea, FAA, and nucleic acids contents of T.castaneum adult beetles. Glucose was decreased(up to 58%) during the first 3 days of treatmentfollowed by significant elevation (up to 97%) af-ter 7 days of insecticide treatment. On the otherhand, fructose content was significantly elevatedfrom day 1 through day 7 (65% on day 6), whileglycogen content was reduced 58% (day 7). Lipidcontent was not altered until day 3 but showedsignificantly raised levels thereafter. Cholesterolcontent was reduced (51%) on 7th day of treat-ment only.

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TABLE 1. Effect of Cypermethrin (Ripcord 25 E) on Carbohydrases, Phosphatases, Dehydrogenases, Transaminases, Cholinesterases, andProteases of Adults of Tribolium castaneum†

Day 1 Day 3 Day 5 Day 7Control Treated Control Treated Control Treated Control Treated

Parameters (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4)

Amylase (SoU/mg) 5.76 ± 0.08 5.73 ± 0.01 5.76 ± 0.09 6.60 ± 0.08* 5.73 ± 0.06 9.74 ± 0.44*** 7.09 ± 0.15 15.03 ± 1.13Trehalase (IU/mg) 0.15 ± 0.007 0.19 ± 0.007** 0.12 ± 0.007 0.23 ± 0.01*** 0.12 ± 0.01 0.20 ± 0.01 0.21 ± 0.01 0.19 ± 009Invertase (IU/mg) 0.19 ± 0.006 0.18 ± 0.004 0.18 ± 0.004 0.21 ± 0.002* 0.14 ± 0.01 0.26 ± 0.009*** 0.1 ± 0.007 0.28 ± 0.009***Lactase (IU/mg) 0.20 ± 0.006 0.20 ± 0.006 0.19 ± 0.006 0.22 ± 0.006 0.23 ± 0.003 0.33 ± 0.008*** 0.25 ± 0.003 0.38 ± 0.008***Maltase (IU/mg) 0.21 ± 0.01 0.18 ± 0.002* 0.19 ± 0.008 0.19 ± 0.01 0.17 ± 0.008 0.32 ± 0.005*** 0.15 ± 0.007 0.34 ± 0.03***AkP (IU/mg) 0.23 ± 0.02 0.30 ± 0.01* 0.13 ± 0.02 0.16 ± 0.02 0.17 ± 0.007 0.11 ± 0.008*** 0.09 ± 0.003 0.05 ± 0.02***AcP (IU/mg) 4.91 ± 0.04 5.46 ± 0.04** 0.48 ± 0.03 6.06 ± 0.14 4.29 ± 0.30 3.31 ± 0.19* 3.80 ± 0.09 2.30 ± 0.30**LDH (IU/mg) 1.56 ± 0.17 1.77 ± 0.17 1.29 ± 0.10 2.05 ± 0.10** 1.54 ± 0.17 2.80 ± 0.04*** 1.21 ± 0.07 2.07 ± 0.03***ICHD (SiU/mg) 47.02 ± 0.65 30.5 ± 1.9*** 41.18 ± 2.25 16.00 ± 2.97*** 41.66 ± 4.72 16.48 ± 2.75 32.99 ± 0.14 15.31 ± 4.68**ASAT (IU/mg) 8.51 ± 0.37 8.06 ± 0.19 8.29 ± 0.52 6.64 ± 0.07* 11.37 ± 0.35 11.86 ± 0.34 12.95 ± 1.47 15.03 ± 0.76ALAT (IU/mg) 4.31 ± 0.28 3.55 ± 0.44 3.51 ± 0.13 2.80 ± 0.19* 3.69 ± 0.11 3.90 ± 0.06 3.48 ± 0.11 4.16 ± 0.12**ChE (RU/mg) 1.81 ± 0.01 1.79 ± 0.005 1.71 ± 0.06 2.07 ± 0.06** 1.64 ± 0.003 2.73 ± 0.19*** 1.86 ± 0.01 3.18 ± 0.071Protease (IU/mg) 0.17 ± 0.004 0.16 ± 0.009 0.17 ± 0.003 0.14 ± 0.003** 0.51 ± 0.06 0.42 ± 0.03 0.35 ± 0.02 0.40 ± 0.02†Values represent the mean ± SEM; Student’s t-test.*P < 0.05.**P < 0.01.***P < 0.001.

150 Saleem et al.

Nucleic Acids, Proteins, and Some MetabolitesOf the other macromolecules determined in

this study, total protein and uric acid contentshowed a depressed level in almost all the 7 daysof treatment. On the other hand, FAA, urea,DNA, and RNA content were increased during 7days of Ripcord treatment. This increase was39% for FAA on day 7, 228% for urea on day 6,77% for DNA on day 6, and 50% for RNA on day5 (Table 2, Fig. 3).

DISCUSSION

The various monosaccharides (i.e., glucose)decreased significantly during the first 3 days oftreatment but increased subsequently. On theother hand, fructose increased, and glycogen de-creased throughout the experimentation. This in-dicated that glucose as well as trehalose wereutilized as energy sources under the initial stressof insecticide treatment, while fructose was not.Utilisation of glycogen increased with increasingduration of treatment. With the increasing mobi-lization of glycogen, total lipid content adoptedan inverse route. Cholesterol content, however,decreased particularly during the final days oftreatment. It appears that utilization of disaccha-rides and polysaccharides was simultaneously in-

duced as a protective measure to prepare thebeetles for further vicissitudes, in case the presentstress conditions were to persist.

Phosphatases (AkP and AcP) first increasedand then decreased after Ripcord treatment. Ofdehydrogenases, LDH activity was increased,while ICDH activity was decreased throughoutthe experimental period. Transaminases (ASATand ALAT) manifested a decreasing trend,whereas ChE adopted an increasing trend. Phos-phatases are the generalised enzymes involvedin dephosphorylation and release of energy.Aminotransferases catalyze utilization/conversionof amino acids into keto acids, which then laterenter into the Kreb’s cycle. Dehydrogenases areinvolved in oxidation/reduction processes inanaerobic glycolysis (LDH) and in the Kreb’s cycle(ICDH). All these enzymes are indicators of vari-ous aspects of intermediary metabolism and hencethrow light on the array of biochemical reactionsthat are involved during insecticidal stress con-ditions. Esterases are the detoxication enzymes,which are usually induced after toxic chemicaladministration (Saleem and Shakoori, 1987b;Terriere, 1984). The increased activity of ChE ac-tivity after insecticide treatment from day 2 on-wards also supported the above contention.

Increased activities of enzymes such as car-

Fig. 2. Changes (in %) in various enzymes of adults ofTribolium castaneum exposed to sublethal dose of Ripcord

25 EC. The percentage change has been calculated with ref-erence to untreated insects.

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TABLE 2. Effect of Cypermethrin (Ripcord 25 E) on Biochemical Components of Adult Beetles of Tribolium castaneum†

Day 1 Day 3 Day 5 Day 7Parameters Control Treated Control Treated Control Treated Control Treated(µg/mg) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4) (n = 4)

Glucose 0.04 ± 0.001 0.02 ± 0.001*** 0.03 ± 0.003 0.01 ± 0.002*** 0.03 ± 0.001 0.05 ± 0.003*** 0.03 ± 0.001 0.06 ± 0.003***Fructose 0.04 ± 0.001 0.05 ± 0.001*** 0.04 ± 0.001 0.06 ± 0.001*** 0.04 ± 0.002 0.06 ± 0.003*** 0.04 ± 0.002 0.06 ± 0.003***Glycogen 39.40 ± 1.75 29.81 ± 1.22** 23.46 ± 2.65 15.06 ± 0.73** 28.60 ± 3.57 13.05 ± 0.41** 17.13 ± 2.20 7.37 ± 0.71***Lipds 110.50 ± 1.88 124.18 ± 9.72 122.52 ± 4.41 129.07 ± 7.86 120.17 ± 3.69 154.29 ± 8.08** 115.19 ± 3.39 159.10 ± 0.53***Cholesterol 0.13 ± 0.04 0.12 ± 0.02 0.19 ± 0.006 0.24 ± 0.01* 0.14 ± 0.01 0.15 ± 0.02 0.19 ± 0.006 0.10 ± 0.01***Total proteins 329.86 ± 2.11 324.85 ± 4.43 302.66 ± 4.10 280.52 ± 4.51* 281.00 ± 3.80 260.30 ± 3.84* 233.33 ± 2.70 206.06 ± 4.38*FAA 0.09 ± 0.002 0.09 ± 0.002 0.11 ± 0.003 0.12 ± 0.005 0.11 ± 0.002 0.15 ± 0.01** 0.13 ± 0.003 0.18 ± 0.009**Uric acid 0.51 ± 0.02 0.37 ± 0.01 0.32 ± 0.002 0.29 ± 0.007 0.54 ± 0.021 0.45 ± 0.1 0.62 ± 0.12 0.30 ± 0.02*Urea 0.21 ± 0.02 0.24 ± 0.02 0.28 ± 0.02 0.36 ± 0.004** 0.15 ± 0.004 0.37 ± 0.02*** 0.13 ± 0.02 0.40 ± 0.01***DNA 2.53 ± 0.03 2.58 ± 0.03 2.86 ± 0.04 2.8 ± 0.05*** 2.86 ± 0.05 3.99 ± 0.05** 3.03 ± 0.05 4.90 ± 0.04**RNA 9.75 ± 0.31 11.00 ± 0.29 8.96 ± 0.23 9.91 ± 0.31* 9.70 ± 0.04 14.59 ± 0.05*** 10.87 ± 0.12 13.83 ± 0.19**†Values represent the mean ± SEM; Student’s t-test.*P < 0.05.**P < 0.01.***P < 0.001.

152 Saleem et al.

bohydrases, phosphatases, LDH, and ChE afterRipcord treatment indicate (1) higher concentra-tion of enzymes due to decreased body weight, (2)increased synthesis of particular enzymes to de-fend against insecticide stress conditions, (3) in-creased phosphatases levels to increase the sourceof energy production through breakdown of phos-phate bonds of energy rich compounds, and/or (4)raised carbohydrases to supplement energy produc-tion through catabolism of their respective reservecarbohydrates. Induced activities of various en-zymes after insecticide treatments have also beenreported from other laboratories (Kacew andSinghal, 1973). Likewise, Terriere (1984) has de-scribed the induction of several detoxication en-zymes such as esterases, mixed function oxidases,and glutathionestransferases in insects. Such in-creases in enzyme activities have been shown toprotect insects from insecticide poisoning as partof defense mechanism. The decreased ICDH ac-tivity and raised LDH activity indicated a pos-sible non-oxidative metabolism of insecticide,ultimately leading to accumulation of lactic acid,

causing death of insects. Likewise Shakoori et al.(1994) have reported induction of LDH and deple-tion of ICDH in Bifenthrin-treated adult beetlesof the PAK strain of T. castaneum. The results ofthe present study tally with the data already re-ported from this laboratory.

The level of total lipids in insecticide-treatedbeetles was not disturbed up to day 3 and increasedsignificantly thereafter, whereas cholesterol con-tent was depleted after insecticide treatment. Theconcentration of total lipids showed an inverse re-lationship with those of di- and polysaccharides.In a similar study, Orr and Downer (1982) de-scribed depleted levels of glycogen and trehalose,an elevation of fat body acyl glycerol reserves, andan associated decrease of hemolymph free fattyacid levels in the American cockroach, Periplan-eta americana, following lindane treatment. Suchan inverse relationship between levels of lipids andcarbohydrates has also been reported by Singh(1986) in Locusta migratoria exposed to bio-resmethrin treatment. This pyrethroid insecticidecaused depletion of hemolymph carbohydrate

Fig. 3. Changes (in %) in various biochemical componentsof Tribolium castaneum exposed to sublethal dose of Ripcord

25 EC. The percentage change has been calculated with ref-erence to untreated controls.

Biochemical Effects of Ripcord in Tribolium 153

(hypoglycemia) and elevation in blood lipid (hyper-lipemia). Singh (1986) suggested that biores-methrin-induced elevation in hemolymph lipidresults from the release of hyperlipemic hor-mone(s). The depletion of hemolymph carbohy-drate, however, may be due to their increasedutilisation in response to the hyperactivity causedby pyrethroid treatment. Hence the results of thepresent study are in accordance with the findingsof Singh (1986) and Bounias et al. (1985).

Of the remaining metabolites analysed inthis study, total protein depleted from day 3 on-wards, while soluble protein was not affected. FAAcontents were elevated from day 4 onwards. Like-wise DNA, RNA, and urea content manifested sig-nificant elevations, while uric acid decreased.Increased RNA content could occur because ofRNA synthesis under insecticide stress conditions.Ripcord, therefore, may have enhanced the syn-thesis of nucleic acids. The higher soluble pro-tein content, FAA, and enzyme activities areexplainable by insecticide-induced syntheses. De-creased total protein content may be related tothe utilization of carbohydrates for production ofenergy when insecticide exposure was continuedand extended after day 3 onwards. SimilarlySubba (1984) reported increased proteolyticactivity and decreased FAA in hemolymph of cock-roach after pyrethroid and OP insecticide treat-ment. Rajender (1984) has also reported a decreasein total carbohydrates, glucose, and glycogen anda simultaneous increase in proteins, FAA, and to-tal RNA in cockroach after OP administration.These are the trends, which are very similar tothose seen in Tribolium beetles after Ripcordtreatment.

The results of the present study also re-vealed that almost all enzymes and biochemicalcomponents analysed here were sensitive mark-ers to Ripcord at sublethal concentrations. Suchmacromolecular abnormalities were clearly evi-dent on the final (day 7) stage of treatment. It is,therefore, concluded that the survivors of fieldapplications of Ripcord may develop severe mac-romolecular abnormalities, which could play animportant role in the pest control programs.

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