Acute effect of tmof against op polinators not transformed

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Acute effect of tmof against op polinators not transformed

  1. 1. Acute Effect of TMOF (WP), TMOF (RH), MPOB Ecobac-1(EC), Against Oil Palm Pollinators, Elaeidobius kamerunicus Ramlah Ali A S, Najib M, Mazmira M M and Basri M W AbstractsTrypsin Modulating Oostatic Factor (TMOF) is a decapeptide hormone originallypurified from ovaries of females Aedes aegypti inhibits the biosynthesis of trypsin andchymotrypsin like enzymes in midgut epithelial cell of female and larval mosquitoes by atranslational control mechanism. The lack of free amino acids liberated from the bloodmeal in adult females or larval gut causes inhibition of egg development (sterility) toadult, anorexia and death of larval mosquitoes. Like MPOB Bt1, Ecobac-1(EC),TMOF(WP) is delivered in water based solution by spraying while the TMOF(RH)granules are spread evenly in waterlogged area for control of mosquitoes.Screening for acute effect of TMOF against oil palm pollinators was conducted byspraying oil palm spikelets from anthesizing male flower containing adult pollinatingweevils and grubs with solution of TMOF(WP), TMOF(RH), MPOB BT1 Ecobac-1(EC),water as untreated control and sprayable chemical, cypermethrin. The pollens on thespikelets were directly exposed to the different treatments prior to feeding by the weevilfor one week. The bioassay was conducted for three different populations of Elaeidobiuskamerunicus. Results indicated that like MPOB Bt1 Ecobac-1(EC), TMOF (WP) andTMOF (RH) did not reveal any significant acute effect as compared to untreated control.On the contrary cypermethrin is absolutely lethal to the oil palm pollinators. IntroductionMosquitoes are vectors for major diseases such as malaria, dengue, encephalitis and arenuisance in the temperate zones (Borovsky et al., 2010). An active ingredient known asTrypsin Modulating Oostatic Factor (TMOF) is used against mosquito larvae in aquaticenvironments. TMOF is a small protein containing 10 amino acids that interferes withdigestion in mosquito larvae. TMOF is a decapeptide hormone originally purified fromthe ovaries of female Aedes aegypti (Borovsky et al, 1990). It inhibits the biosynthesis oftrypsin and chymotrypsin like enzymes in the midgut epithelial cells of female and larvalmosquitoes by a translational control mechanism (Borovsky et al., 1996; Borovsky et al.,2006). The lack of free amino acids liberated from the blood meal in adult females orfrom digested proteins liberated in the larval gut causes inhibition of egg development 1
  2. 2. (sterility) to adults and anorexia and death to larval mosquitoes, respectively (Borovskyet al., 2003; Borovsky et al., 2006). TMOF also inhibits de novo biosynthesis of trypsin inthe midgut cells and this will lead to the insect starvation (Danuta et al., 1998). Oogenesisin insects is a well-studied and complex process. Because the target tissue of the hormoneis the mosquito midgut and not the ovary or the brain, the hormone was named “TrypsinModulating Oostatic Factor” (TMOF).Two oostatic factors called Aea-TMOF was isolated from yellow fever mosquito Aedesaegypti (Borovsky et al., 1991) and Neb-TMOF found in fleshfly, Neobellieria bullata(Bylemans et al., 1994) have been identified. Both are Dipteran and are anautogenousspecies, i.e., they need a protein meal in order to be able to produce mature eggs.Digestion of blood or meat provides amino acids that are required for vitellogeninproduction by fat body. The vitellogenins are then selectively taken up by the oocyctes ina process called vitellogenesis. At the end of vitellogenesis the ovaries release oostaticfactor (TMOFs) which inhibits denovo biosynthesis of trypsin in the midguts (Danuta etal., 1998)TMOF has been used against larvae of mosquitoe larvae of Anopheles Mosquito(transmitter malaria), Aedes aegypti (aedes) and Aedes albopictus (Chikungunya). It canbe applied in mosquito larvae habitat such as ponds, streams, ditches, puddles, and othersources of standing water. TMOF can be applied directly to water or any mosquito larvabreeding site. TMOF protein is broken down quickly in the human gut and doesnt havethe opportunity to inhibit trypsin synthesis (TMOF fact sheet, US EPA)Investigation on TMOF has been carried out by several researchers for almost 20 years.One of the report was conducted by Dany et al. (1994) in sequencing and characterizationof trypsin modulating oostatic factor (TMOF) from the ovaries of the grey fleshfly,Neobellieria (Sarcophaga) bullata instead of Aedes aegypti. There are also study ontrypsin modulating oostatic factor (Neb-TMOF) and its analogs reported by Danuta et al.(1998) and on Aea-TMOF reported by Borovsky and Hamdaoui (2008) Recent study onthe synergistic of TMOF and Bacillus thuringiensis -endotoxin was reported by 2
  3. 3. Borovsky et al. (2010). This study is conducted to investigate the acute effect of TMOFas compared to MPOB BT1 Ecobac-1(EC) and chemical on three populations of oil palmpollinators. MATERIALS AND METHODSource of E. kamerunicusThree populations of pollinating weevil, E. kamerunicus were obtained from a week oldanthesizing male inflorescences of oil palms. The three populations of oil palmpollinators used in the study were collected from the MPOB Research Station at Bangi,Selangor, Teluk Intan Perak and Kluang Johore.Experimental designThe acute tests were conducted for the three populations of pollinators using fivetreatments. Each treatment was conducted in replicates of three. For each replicate fourspikelets containing both living weevils and grubs of the pollinators were subjected to thetreatments. Total of twelve spikelets were exposed to the different treatments.TreatmentsFive different treatments namely, Trypsin Modulating Oostatic Factor (TMOF) wettablepowder, TMOF rice husk, Bt product, Ecobac-1 (EC), chemical control, Cypermethrinand untreated were prepared. TMOF is a small mosquito-derived peptide expressedwithin Pichia pastoris yeast cells. TMOF has synergistic effect with strain of B.thuringiensis israelensis, therefore, can be formulated together with this protein. TheEcobac-1 (EC) is an emulsified concentrate of MPOB Bt1 indigenous isolate propagatedand formulated at MPOB Microbial Technology and Engineering Center (MICROTEC). 3
  4. 4. Preparation of test pollinating weevils and feedAdult weevils and grubs in the four spikelets were transferred into clean sterilizedcylinder measured 25 cm in height with diameter of 13.5 cm. The cylinder containingtest oil palm pollinators were sealed using cheese cloth with rubber bands. The cylindersand their contents were dried under bright sunlight for 30 minutes and pretreated with 2% antifungi. The pollens on each of the four spikelets per cylinder were kept intacted onas feed for the adult weevils.BioassayTMOF wettable powder solution was prepared by weighing 10g of TMOF wettablepowder mixed with 1 L of distilled water. As for the TMOF rice husk, the sameprocedure was repeated. The dosage of Ecobac-1 (EC) applied was 5.2 x 109 cfu/ml.Twelve spikelets were treated with recommended concentrations of each TMOF wettablepowder, TMOF rice husk and Ecobac-1 (EC). Each of the four spikelets containing grubsfor each replicate were thoroughly sprayed with the products in the fumehood using 2ml/spikelets of the product using a hand held sprayer. For chemical control, the spikeletswere sprayed with 5% Cypermethrin at 2 ml/spikelet. For the untreated control, theweevils were sprayed with only distilled water at 2ml/spikelet. Spraying of weevils wasdone in the fume cupboard using a 1L hand held sprayer.Each group of the four treated spikelets containing the grubs and the adult weevils weretransferred into a 500 ml sterilized cylinder (measured 25 cm in height with diameter of13.5 cm.) labelled according to the treatments and replicates. The cylinders were sealedusing cheese cloths and dried under the bright sunlight for 30 minutes prior to incubationin the indoor insectory at temperature 24oC to 28oC and relative humidity 50%. 4
  5. 5. Data recordingThe mortality of E. kamerunicus population from Bangi was recorded at 7 days aftertreatment (DAT). At the end of 7 days the spikelets were dissected and the numbers oflive and dead weevils and grubs were recorded. The dissected spikelets containing liveand dead grubs were photographed using dissecting microsope with a camera attached.Data AnalysisData on survival and mortality was calculated for each replicate and treatments. Themortality was then converted to corrected mortality using the Abbot formula, thenanalyzed in one-way ANOVA using SPSS software version 11.5. The means wereanalyzed by the Least Significant Difference (LSD) test using the same software. RESULTS AND DISCUSSIONTable 1 shows that the exposure of E.kamerunicus population from Bangi, to TMOFWP, TMOF RH, MPOB Ecobac-1(EC) for 1week resulted in no significant difference inmortality at P< 0.05 as compared to untreated control. The average mortality at 7 daysafter treatments (DAT) was 13.8%, 16.1%, 12.4% and 10.8 % for TMOF WP, TMOFRH, MPOB Ecobac-1(EC) and untreated control, respectively. This implies that bothforms of TMOF and Ecobac-1(EC) have no acute effect on the oil palm pollinatingweevils. The contrasting effect was noted for chemical insecticide, in this casecypermethrin which resulted in 100% mortality of E.kamerunicus (Table 1)The corrected mortality of E. kamerunicus population from Bangi subjected to thevarious treatments as mentioned above indicated the same effect as seen in Figure 1.Minimal corrected mortality of E. kamerunicus of 3.1%, 5.9% and 1.7% 5
  6. 6. TABLE 1. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobiuskamerunicus SAMPLED FROM MPOB STATION IN BANGI SELANGOR AT 7DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS______________________________________________________________________Treatments dead weevil alive (weevils & grubs)_____________________________________________________________________Control 10.8±2.4 a 89.2±2.4 aTMOF WP 13.8±2.9 a 86.2±2.9 aTMOF RH 16.1±5.3 a 83.9±5.3 aEcobac-1(EC) 12.4±1.5 a 87.6±1.5 aCypermethrin 100+0.0 b 0+0.0 bThe numbers are average percentage grubs and weevils for three replicates each with four spikelets.Numbers with the different letters are significantly different at P<0.05.was ascribed by TMOF WP, TMOF RH, MPOB Ecobac-1(EC) as compared to 100% forcypermethrin. Unlike chemical, TMOF WP, TMOF RH, MPOB Ecobac-1(EC) are safefor the oil palm pollinating weevils and grubs found in the florets (Figure 2).Figure 1. Average percentage corrected mortality of Elaeidobius kamerunicus sampledfrom MPOB Station in Bangi Selangor at 7 days after exposure to different treatmentsSpraying of cypermethrin was detrimental to both the weevil and grubs because itresulted in 100% mortality of both stages of the pollinator. The rest of the treatmentsincluding untreated resulted in almost 100% survival of the grubs and majority of theweevils (Figure 2). 6
  7. 7. a b c dFigure 2. Microscopy of E.kamerunicus with magnification 40x. Showing live adultsweevil (a) and grubs (b) of E.kamerunicus subjected to TMOF and dead weevil treatedwith cypermethrin outside (c) and within the spikelet (d).Table 2 shows that the exposure of E.kamerunicus originated from MPOB Station inTeluk Intan Perak, to TMOF WP, TMOF RH, for 1week resulted in no significantTABLE 2. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobiuskamerunicus SAMPLED FROM MPOB STATION IN TELUK INTAN PERAK AT7 DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS_____________________________________________________________________Treatments dead weevils alive (weevils & grubs)Control 32.4±7.5 a 67.6±7.5 aTMOF WP 45.6±5.3 a 54.4±5.3 aTMOF RH 32.4±3.5 a 67.6±3.5 aEcobac-1(EC) 22.9±11.1 ab 77.1±11.1 abCypermethrin 100+0.0 c 0+0.0 cThe numbers are average percentage grubs and weevils for three replicates each with four spikelets.Numbers with the different letters are significantly different at P<0.05. 7
  8. 8. difference in mortality at P< 0.05 as compared to untreated control. However, MPOBEcobac-1 (EC) resulted in much lower mortality, 22.9% than TMOF WP, TMOF RHand untreated control at 7 DAT. The average mortality at 7 days after treatments (DAT)for TMOF WP, TMOF RH and untreated control was 45.6%, 32.4% and 32.4%,respectively. This implies that both forms of TMOF and Ecobac-1 (EC) have no acuteeffect on the oil palm pollinating weevils. The contrast effect was noted for chemicalinsecticide, cypermethrin which resulted in 100% mortality of E.kamerunicus (Table 2).The corrected mortality of E.kamerunicus sampled from MPOB Station in Teluk IntanPerak, subjected to TMOF WP, TMOF RH, Ecobac-1 (EC) for 7days was 16 %, 0 % ,0% significantly much safer as compared to 100% for chemical, cypermethrin (Figure 3).Figure 3. Average percentage corrected mortality of Elaeidobius kamerunicus sampledfrom MPOB Station in Teluk Intan Perak at 7 days after exposure to different treatmentsDissection of the spikelets at 7 DAT revealed the same result as in the case for pollinatorsfrom MPOB Station in Bangi. Unlike cypermethrin which resulted in 100% mortality ofboth weevils and grubs of E.kamerunicus, TMOF WP, TMOF RH, MPOB Ecobac-1(EC)did not affect the grubs.The same senerio was observed for both population of oil palm pollinators sampled fromMPOB Stations in Perak and Kluang (Table 3 and Figure 4). Table 3 shows that the 8
  9. 9. TABLE 3. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobiuskamerunicus SAMPLED FROM MPOB STATION IN KLUANG, JOHORE AT 7DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS____________________________________________________________________Treatments dead weevils alive (weevils & grubs)__________________________________________________________________Control 34.0±3.4 a 66.0±3.4 aTMOF WP 34.4±0.8 a 65.6±0.8 aTMOF RH 44.9±3.5 a 55.1±3.5 aEcobac-1(EC) 33.9±7.4 a 66.1±7.4 aCypermethrin 100+0.0 b 0+0.0 bThe numbers are average percentage grubs and weevils for three replicates each with four spikelets.Numbers with the different letters are significantly different at P<0.05.exposure of E.kamerunicus from MPOB Station in Kluang to TMOF WP, TMOF RH,MPOB Ecobac-1(EC) for 1week resulted in no significant difference in mortality at P<0.05 as compared to untreated control. The average mortality at 7 days after treatments(DAT) was 34.4%, 44.9%, 33.9% and 34.0% for TMOF WP, TMOF RH, MPOB b a a aFigure 4. Average percentage corrected mortality of Elaeidobius kamerunicus sampledfrom MPOB Station in Kluang Johore at 7 days after exposure to different treatmentsEcobac-1(EC) and untreated control, respectively. This implies that TMOF and Ecobac-1(EC) have no acute effect on the oil palm pollinating weevils. On the contrary,chemical insecticide, cypermethrin gave 100% mortality of E.kamerunicus (Table 3).Figure 4 shows that the corrected mortality of E. kamerunicus sampled from MPOB 9
  10. 10. Station in Kluang, Johore subjected to the various treatments showed minimal correctedmortality of E. kamerunicus of 0%, 16% and 0% for TMOF WP, TMOF RH, MPOBEcobac-1(EC) ) as compared to 100% for cypermethrin (Figure 4). ConclusionsLike other biological insecticide such as MPOB Ecobac-1 (EC), TMOF WP and TMOFRH are safe for beneficial insects such as the oil palm pollinating E.kamerunicus, asproven by the acute toxicity trials. Unlike chemical, the use of these biologicalinsecticides, MPOB Ecobac-1 (EC), TMOF WP and TMOF(RH) did not detriment thepollinators particularly the grubs. AcknowledgementsThe authors would like to thanks Y Bhg Datuk Choo Yuen May, Director General ofMPOB, Y Bhg Dato Seri Utama Shahrir Abdul Samad, the Chairman of MPOB, MPOBBoard Members for their permission to undertake this joint project between MPOB andEntoGenex Sdn Bhd. The authors are very grateful for the continuous support given bythe subordinates or supporting staff of Microbial Technology Research Group of MPOB. ReferencesBorovsky D., Carlson D.A., Hunt F., Mosquito oostatic hormone a trypsin modulatingoostatic factor, (Menn J.J., Kelly T.J., Masler E.P., Eds.), ACS Symposium Series 453,1991, pp. 133-142.Borovsky D, Carlson DA, Griffin PR, Shabanowitz R, Hunt DF. Mosquito oostaticfactor: a novel decapeptide modulating trypsin like enzyme biosynthesis in the midgut.FASEB J 1990; 4: 3015- 20.Borovsky D. and Hamdaoui A. 2008. Binding of Aedes aegypti Trypsin ModulatingOostatic Factor (Aea-TMOF) to its receptor stimulates phosphorylation and proteaseprocessing of gut-membrane proteins. Pestycydy (1-2): 13-25.Borovsky D, Janssen I, Vanden Broeck J, et al. Molecularsequencing and modeling ofNeobellieria bullata trypsin: Evidence explain why TMOF was not detected by mass 10
  11. 11. spectrometry: for translational control with Neb TMOF. Eur J Biochem 1996; 237: 279-87.Borovsky D. Trypsin Modulating Oostatic Factor: A Potential New Larvicide forMosquito Control. J Exptl Biol 2003; 206: 3869-75.Borovsky D, Rabindran S, Dawson WO, et al. Expression of Aedes TMOF on the virionof TMV: A potential larvicide. Proc Nat Acad Sci USA 2006; 103: 18963-8.Borovsky D, Khasdan V, Nauwelaer S, Theunis C, Bertieor L Bendov E and Zaritsky A.2010.Synergy between Aedes aegyti Trypsin Modulating Oostatic Factor and delta-endotoxins. The Open Toxinology Journal. 3,116-125.Bylemans , D., Borovsky, D., Hunt, D. F., Grauwels , L., and DeLoof , A. 1994.Sequencing and characterization of oostatic factor (TMOF) from the ovaries of the greyfleshfly Neobellieria ( Sarcophaga ) bullata . Regulatory Peptides 50: 61-72.Danuta K., Hubert B.B., Mariola K., Grzegorz R., Ine J., DeLoof , A. 1998. Insect trypsinmodulating oostatic factor (Neb-TMOF) and its analogs: Preliminary structure/biologicalfunction relationship studies Letters in Peptide Science 5: 391-393.Dany B., Borovsky V., Donald F. H., Jeffrey S., Luc G. and Loof A.D. 1994. Sequencingand characterization of trypsin modulating oostatic factor (TMOF) from the ovaries of thegrey fleshfly, Neobellieria (Sarcophaga) bullata. Regulatory Peptides 50, (1): 61-72.Trypsin Modulating Oostatic Factor (TMOF) (105403) Fact Sheet. United StatesEnvironmental Protection Agency. 11

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