MOUSTICIDE™ - TMOF™ Published in The Journal of Tropical Medicine & Parasitology


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MOUSTICIDE™ - TMOF™ Published in The Journal of Tropical Medicine & Parasitology

  1. 1. J Trop Med Parasitol 2010;33:69-76. ORIGINAL ARTICLE Available online at Larvicidal Effect of Trypsin Modulating Oostatic Factor (TMOF) Formulations on Aedes aegypti Larvae in the Laboratory Norashiqin Misni, Hidayatulfathi Othman, Sallehudin Sulaiman Department of Biomedical Science, Faculty of Allied Health Sciences,Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz 50300 Kuala Lumpur, Malaysia T Abstract rypsin Modulating Oostatic factor (TMOF), a peptide hormone originally isolated from the ovaries of adult Aedes aegypti, is currently under commercial development as a new insecticide. This study was carried out to evaluate the various formulations of TMOF viz recombinant TMOF yeast cell paste form, TMOF yeast cell dried powder form, a combination of TMOF and Bti rice husk, TMOF and Bti wettable powder and TMOF and Bti mosquito fudge cubes formulated against Aedes aegypti larvae in the laboratory. The TMOF yeast cell paste and dried powder were found to cuase mortality in larvae from 24 hours exposure to 96 hours exposure. These products had a prolonged residual effect for four weeks of observation. The TMOF and dried yeast powder (Pichia 11 and 12) caused 100% larval mortality after 96 hours of treatment. The TMOF and Bti in rice husk, wettable powder and mosquito fudge cubes were effective in causing mortality within 1 hour of treatment and gave a prolonged residual effect (no larva survived) against all larval stages for four weeks after treatment. The mosquito fudge cubes showed high larvicidal activity for three months after treatment. The TMOF and yeast cell and TMOF and Bti formulations have the potential to be utilized for dengue vector control.Keywords: Trypsin Modulating Oostatic Factor (TMOF) formulations, Bacillus thuringiensis (Bti), vector control, Aedes aegyptiIntroduction chemical insecticides; there are being phased out Despite advances in medical science and in many countries due to insecticide resistance innew drugs for treating mosquito-borne diseases, mosquito populations. Thus, there is an urgentthey remain important diseases in humans, need for new agents and strategies to controlwith an estimated two billion people worldwide these diseases. Today, the advent of recombinantliving in areas where these diseases are endemic DNA technology is having an enormous impact[1]. Since World War II, disease control methods on agriculture and medicine. The ability tohave relied heavily on broad-spectrum synthetic manipulate and recombine genes using this technology may be applied to improving larvicidesCorrespondence: for vector control [2]. Norashiqin Misni, Control of vector mosquitoes using microbial E-mail: <> agents such as Bacillus thuringiensis H-14 (Bti), is Vol 33 (No. 2) December 2010 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 69
  2. 2. Larvicidal Effect of TMOF on Ae. aegypti Larvaea relatively recent development in Malaysia [3,4]. insectarium of the Department of BiomedicalThe effectiveness of Bti in control of mosquitoes Science, Universiti Kebangsaan Malaysia (Nationalhas been demonstrated [5]. The most widely used University of Malaysia). First instar larvae of thisare Vectobac® and Teknar®, which are based on species were used for the test.B. thuringiensis subsp israelensis. VectoLex ® aproduct based on B. sphaericus (Bs), has come to Larvicidal testingmarket recently for control of mosquito vectors For the bioassay of the toxicity of TMOFof viral diseases. These products have achieved with yeast (Pichia 7, 11 and 12) against Ae. aegyptimoderate commercial success in developed larvaeWHO standard procedures [11] were used.countries, but their high cost deters use in many Twenty first instar larvae were introduced intodeveloping countries. Moreover, concerns have each 500 ml beaker containing 200 ml distilledbeen raised about their long-term utility due to water. A minimum of three replicates was made forresistance, which has already been reported to B. each concentration of TMOF with yeast tested. Allsphaericus in field populations of Culex mosquitoes TMOF with yeast and Bti samples were fermentedin several different countries [2]. and formulated at the Chemical Engineering The use of the peptide hormone, Trypsin Power Plant (CEPP), Universiti Teknologi Malaysia,modulating oostatic factor (TMOF), as a pesticide, Johor. They came inboth wet paste and dryrepresents a novel biorational approach to insect powder.control using a new mode of action different Prior to testing, 160 ml distilled water wasfrom that of Bti [6]. TMOF is an insect hormone added to each beaker. Then, a stock solutionoriginally isolated from the ovaries of Aedes of 20,000 ppm TMOF with yeast was preparedAegypti (Diptera: Culicidae) that regulates trypsin to make the desiredconcentration. Using thebiosynthesis in the mosquito digestive system [7]. formula, C1V1 = C2V2, the correct amount of stockTMOF has been shown to inhibit the growth and solution was added to each of the three replicatedevelopment of mosquito larvae feeding on this beakers and the mixture was stirred with a glasspeptide, resulting in death by starvation [8]. TMOF rod. For the negative and positive control beakers,have been shown to inhibit trypsin biosynthesis in a 20,000 ppm stock solution containing normalother medically important insects, including the yeast (Nona Instant Yeast©) and another solutionhousefly, Musca domestica L (Diptera: Muscidae), containing 10,000 ppm Bti were prepared to yieldstable fly, Stomoxys calcitrans L (Diptera: Muscidae) 400 ppm normal yeast and 1 ppm Bti. After eachand the cat flea, Ctenocephalides felis Bouche of the beakers was prepared with TMOF with(Siphonaptera: Publicidae) [9]. TMOF can be easily yeast, Bti or normal yeast, 20 first instar Ae. aegyptiengineered for high expression in recombinant larvae were transferred from a breeding tray intobacteria [10]. This study presents the effects of each beaker. Finally, distilled water was added toTMOF fermented with yeast cells, TMOF and Bti bring the volume of the beaker to 200 ml each.wettable powder, the combination of TMOF and Observations were conducted after 1 hour and 24Bti in rice husk form and TMOF with Bti in the hours. The mortality of the larvae was recordedform of mosquito fudge cubes as larvicides against until all test larvae died. The larval survivalthe dengue vector, Ae. aegypti. was recorded at 1 hour, 24 hours, 48 hours, 72 hours and 96 hours post-treatment. Dead larvaeMaterials and methods were replaced with live larvae and the test wasColonization of mosquitoes conducted for four weeks to determine the residual An established colony of insecticide effect of each formulation.susceptible Ae. aegypti susceptible mosquitoes After a number of TMOF with yeast and Btiwhich originated from the Institute for Medical fermentations in the form of wet paste and dryResearch (IMR) of Malaysia was reared at the powder were tested, Entogene-X Sdn Bhd with70 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 33 (No. 2) December 2010
  3. 3. Larvicidal Effect of TMOF on Ae. aegypti Larvaethe help of CEPP had formulated the possible end pattern to Pichia-P7. The dried form of TMOFproducts in the form of rice husks and fudge to with yeast had a better effect than the paste forminvestigate the best medium in which the TMOF throughout the study. By the end of 4 weeks, thewith Bti would be most effective. The rice husks dried TMOF with yeast resulted in a 5.8% larvalwere tested at 50 g, 100 g and 200 g in 200 ml survival rate.distilled water by adding the rice husks to 160 ml The combination of TMOF with Bti indistilled water and topping up the water to 200 wettable powder (WP) gave 12.5% larval survivalml just after transferring the 20 first instar Ae. 1 hour post-treatment. The TMOF with Bti riceaegypti larvae to the beakers. For the mosquito husk (4% Bti plus 4% TMOF) formulation causedfudge cubes, each cube was added to a beaker rapid mortality among 1st instar Ae. aegypti larvaeand observed for larval survival at the same time 1 hour after exposure. At concentrations of 50 mg,intervals from 1 hour to 3 months. 100 mg and 200 mg The TMOF with Bti rice husk (4% Bti plus 4% TMOF) resulted in larval survivalResults rates of 24.1%, 22.5% and 16.7%, respectively. The efficacy of TMOF paste (Pitchia-P7), dried From 48 hours until 4 weeks no larvae survivedTMOF with yeast, TMOF with Bti wettable powder, in the groups treated with both formulationsTMOF with Bti rice husk (4% TMOF plus 4% Bti) TMOF with Bti WP and rice husk. One ppm Btiand Bti only were evaluated against Ae. aegypti gave a 15% larval survival at 1 hour; thereafter,larvae (Fig 1). TMOF paste (Pichia-P7) caused no larvae survived until 2nd weeks after treatment.mortality in 1st instar larvae at 24 hours, with the However, at the 3rd and 4th weeks after treatmentpercent of larval survival of 55.7% at 24 hours onset, the larvae survival rates were 0.8% andand 37.5% at 96 hours. Pichia-P7 caused mortality 18.3%, respectively.(10.8% larval survival) until four weeks even Fig 2 describs the larvicidal activity of Pichiawithout adding any more active ingredient. The 11 and 12 from one hour to 120 hours afterdried TMOF with yeast gave a similar mortality treatment. Pichia 11 and 12 had no mortality 120 Control 100 1 ppm Bti only% Larval survival 80 400 ppm pitchia-P7 400 ppm dried 60 TMOF_yeast 400 ppm TMOF_Bti WP 40 50 mg husk- TMOF+Bti 20 100 mg husk- TMOF+Bti 200 mg husk- 0 TMOF+Bti 0 5 10 15 20 25 -20 Days of observationFig 1 Larvicidal activity of TMOF with yeast cell paste and dried form compared with TMOF and Bti rice husk and wettable powder from the 1st to the 4th week of treatment. Indicates when the 1st instar larvae were addedVol 33 (No. 2) December 2010 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 71
  4. 4. Larvicidal Effect of TMOF on Ae. aegypti LarvaeFig 2 Larvicidal activity of TMOF with yeast cell dried form (Pichia 11 and Pichia 12) against 1st instar larvae from the 1st hour until the 144 th hour of treatment. 120 100% Larval survival 80 Reference no 02072007 A 60 02072007 B 02072007 C 40 02072007 D 02072007 E 20 Control (yeast) 0 Week Week Week Week Week Week Week Week Week Week Week Week 1 2 3 4 5 6 7 8 9 10 11 12 -20 Days of observation (larvae added)Fig 3 Larvicidal activity of mosquito fudge cubes for three months of treatment against Ae. aegypti larvae. Indicates when 1st instar larvae one hour of treatment, and at 24 hours there three months is shown in Fig 3. The five differentwas 70% and 97.5% larval survival, respectively. types were labeled A, B, C, D and E.However, Pichia 11 and 12 caused complete A was the control, without TMOF or Bti added,mortality at 96 and 120 hours, respectively. The while B, C, D and E consisted of TMOF and Bti.larvicidal activity of the mosquito fudge cubes for Cube A caused a low mortality against the larvae72 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 33 (No. 2) December 2010
  5. 5. Larvicidal Effect of TMOF on Ae. aegypti Larvaefrom the 1st week to the 12th week of treatment. Bti, 1% TMOF plus 1% Bti, 3% TMOF plus 1% BtiCubes B through E caused high mortality until and 1% TMOF plus 3% Bti). These formulationsthe 12th week; cube E had the best larvicidal also caused high mortality (100%) among allactivity. Pichia 11 and 12 were the dried TMOF larval stages until 96 hours post-treatment. Nowith yeast. Table 1 shows the efficacy of TMOF significant differences in percent mortality werewith Bti rice husk at different ratios of TMOF plus seen among the different concentrations testedBti (viz: 4% TMOF plus 4% Bti, 2% TMOF plus 2% (p > 0.05).Table 1 Mortality caused by TMOF with Bti rice husk at different concentrations from one hour to 96 hours treatment against Ae. aegypti larvae. Concentrations % Mortality (mg) 1h 24 h 48 h 72 h 96 h 120 h 4% TMOF; 4% Bti 50 85.9 100 100 100 100 100 100 87.5 100 100 100 100 100 200 94.3 100 100 100 100 100 3% TMOF; 1% Bti 50 100 100 100 100 100 100 100 95.0 100 100 100 100 100 200 97.5 100 100 100 100 100 2% TMOF; 2% Bti 50 100 100 100 100 100 100 100 100 100 100 100 100 100 200 100 100 100 100 100 100 1% TMOF; 3% Bti 50 95 100 100 100 100 100 100 100 100 100 100 100 100 200 97.5 100 100 100 100 100 1% TMOF; 1% Bti 50 97.5 100 100 100 100 100 100 97.5 100 100 100 100 100 200 100 100 100 100 100 100Discussion the ovary 12-35 hours after a blood meal. TMOF Insects, including blood-sucking pests, is then released into the hemolymph and bindsmust consume and digest a proteinaceous meal to a specific receptor on midgut epithelial cellsto acquire sufficient essential amino acids for signaling the termination of trypsin biosynthesis.growth, development and the production of Mosquito larvae also synthesize trypsin as theirmature eggs. Mosquitoes and houseflies produce major protease and use the enzyme to digestoostatic hormones that inhibit egg development decaying organic material or small organisms likeby inhibiting digestion of the protein meal, algae that are found in ponds and marshes [12].thereby limiting the availability of the essential Thus, controlling insects by interfering with theiramino acids necessary for egg development. They digestive enzymes, using trypsin inhibitors hasproduce TMOF in the follicular epithelium of been used to try to control insects [13-15]. Our Vol 33 (No. 2) December 2010 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 73
  6. 6. Larvicidal Effect of TMOF on Ae. aegypti Larvaestudy showed TMOF fermented in yeast cells has to 8-20%. Previous studies reported Bti causeda larvicidal effect against Ae. aegypti. mortality quickly but had a shorter duration Recombination TMOF protein and its analogs of activity, especially under natural conditionsusing yeast cells to express the protein and release [19,20]. Bti toxin is composed of two proteinsit into the larval medium will cause larval mortality that co-crystallize into a single parasporal bodyby preventing normal food digestion essential for [21]. After ingestion by a mosquito larva, thegrowth and development [16]. One study reported proteins are cleaved by proteases, yielding peptides38-83% of larvae died after being fed with TMOF that form active toxin [21,22]. These proteinscloned in yeast cells [17]. They also demonstrated bind to a receptor, a glucosidase in the midgutthe peptide’s larvicidal properties in a field test. microvilli [23] and cause lysis of midgut cells afterTMOF with yeast paste (Pichia-P7) and TMOF with internalization [24].dried yeast caused no mortality within one hour This study showed all concentrations ofof exposure. This indicates TMOF does not cause TMOF with Bti rice husk tested (4% TMOF plus 4%rapid mortality among Ae. aegypti larvae. However, Bti, 3% TMOF plus 1% Bti, 1% TMOF plus 3% Bti,by 24 hours, Pichia-P7 led to only 55.7% larval 2% TMOF plus 2% Bti and 1% TMOF plus 1% Bti)survival and dried TMOF with yeast led to only caused high mortality (95-97%) within one hour of40% larval survival. The mechanism of action treatment and caused complete mortality (100%)of TMOF against larvae may need more than until 96 hours after treatment. From this data, weone hour to cause mortality. The process begins can conclude even at lower ratios of TMOF and Btiwhen mosquito larvae consume heat-killed yeast, (1%:1%), the effectiveness was as good as higherthen the gene inside the yeast is expressed and concentrations. When compared to the previousa sufficient amount of the hormone crosses the sample of TMOF with dried yeast powder in Pichiadigestive system into the hemolymph stopping 11 and 12, it gave better results causing completeprotein digestion and development. The larvae mortality by 96 hours of treatment.eventually die from starvation or other causes The mosquito fudge cubes labeled B, C, D and Eresulting from delayed development [10]. TMOF caused high mortality against Ae. aegypti larvae fromwith yeast may cause mortality amongst all larval the 1st to the 12th weekafter treatment. Cube E causedstages from the 1st instar to the 4th instar and still the highestmortality, resulting in 0% larval survivalcause mortality even four weeks after exposure during the 1st through the 12th week after treatment.without any added active ingredient. TMOF Cube A (control without active ingredient) alsocaused an LC50 value of 0.45 mM against Cx. pipiens caused mortality (70% to 98% larval survival).five days after exposure [18]. This suggests the fudge cubes themself have killing TMOF with Bti was formulated from rice husk, properties. These cubes were formulated to attractTMOF with Bti wettable powder and mosquito larvae to ingest the TMOF causing starvation andfudge are possible end products for commercial death. The fudge cubes caused rapid killing withinused. TMOF with Bti rice husk and wettable one hour of treatment.powder caused high mortality even within one TMOF with dried yeast in powdered formhour after exposure and resulted in complete (Pichia 11 and 12) were toxic against larvae ofmortality (0% larval survival) from 24 hours up Ae. aegypti and may be developed into commercialuntil 4 weeks after exposure at each concentration product. The combination of TMOF and Btitested. The mortality effect of TMOF with Bti improved the larvicidal activity of TMOF resultingmay be influenced by the rapid killing effect of in rapid mortality and a prolonged residual effect.Bti and prolonged killing effect of TMOF due to Further studies of the larvicidal activity of TMOFthe residual effect. Our results showed 1 ppm with yeast, TMOF with Bti rice husk and mosquitoBti gave complete mortality for the first 25 days, fudge cubes under field conditions needs to beand after day 25 the larval survival increased carried out to determine the effect of TMOF with74 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 33 (No. 2) December 2010
  7. 7. Larvicidal Effect of TMOF on Ae. aegypti Larvaeyeast and TMOF with Bti against other mosquito midgut. FASEB J. 1990;4:3015-20.species in the field. 10. Borovsky D, Carlson DA, Griffin PR, Shabanowitz J, Hunt DE. Mass spectrometryAcknowledgements and characterization of Aedes aegypti trypsin This work was supported by a grant from the modulating oostatic factor (TMOF) and itsEntogenex Sdn Bhd of Malaysia, research grant analogs. Insect Biochem Mol Biol.1993;23:NN-001-2008 and the Universiti Kebangsaan 703-12.Malaysia (National University of Malaysia) for 11. WHO. Guidelines for laboratory and fieldproviding the research facilities. testing of mosquito larvicides. WHO/CDC/ WHOPES/GCDPP/2005.13.References 12. Borovsky D, Schlesinger Y, Nauwelaers SMI. 1. WHO. World Health Report. Geneva: World Transformed cells useful for the control of Health Organization; 1999. pests. 2000. US Patent number 6,566,129. 2. Federici BA, Park HW, Bideshi DK, Wirth Available from: MC, Johnson JJ. Recombinant bacteria for US-Patent-6566129/transformed-cells-useful- mosquito control. J Exp Biol. 2003;206: for-the-control-of-pests 3877-85. 13. Hilder V, Gatehouse A, Sheerman S, Barker 3. Foo AES, Yap HH. Comparative bioassays R, Boulter D. A novel mechanism of insect of Bacillus thuringiensis H-14 formulations resistance engineered into tobacco. Nature. against four species of mosquitoes in Malaysia. 1987;330:160-3. Southeast Asian J Trop Med Public Health. 14. Johnson R, Narvaez J, An G, Ryan C. 1982;13:1-5. Expression of proteinase inhibitors I and II in 4. Foo AES, Yap HH. Fiels trials on the use Bacillus transgenic tobacco plants: effects on natural thuringiensis H-14 formulations against defense against Manduca sexta larvae. Proc Mansonia mosquitoes in Malaysia. Mosq News. Natl Acad Sci USA. 1989;86:9871-5. 1983;43:306-10. 15. Ryan CA. Protease inhibitors in plants: 5. Fry-O’brien LL, Mulla MS. Effect of tadpole genes for improving defenses against insects shrimp, Triops longicaudatus, (Notostraca: and pathogens. Annu Rev Phytopathol. Triopsidae), Bacillus thuringiensis var. israelensis 1990;28:425-49. in experimental microcosms. J Am Mosq 16. Borovsky D, Nauen R. Biological and Control Assoc. 1996;12:33-8. biochemical effects of organo-synthetic 6. Thompson DM, Young HP, Edens FW, analogues of Trypsin Modulating Oostatic Olmstead AW, LeBlane GA, Hodgson E, et Factor (TMOF) on Aedes aegypti, Heliothis al. Non-target toxicology of new mosquito virescens. Pestycydy. 2007;3:17-26. larvicide, trypsin modulating oostatic factor. 17. Philip K. Mosquitocides take out the Pest Biochem Physiol. 2004;80:31-142. middleman. J Exp Biol. 2003;206:3723-6. 7. Borovsky D. DNA encoding peptide hormone 18. Vanderherchen MB, Isherwood M, Thompson that inhibits digestion in insects. 1997. US DB, Linderman RJ, Roc RM. Toxicity of novel Patent number 5,629,196. Available from: aromatic and aliphatic organic acid and ester analogs of trypsin modulating oostatic factor 5629196/dna-encoding-peptide-hormone- to larvae of the northern house mosquito, that-inhibits-digestion-in-insects Culex pipiens complex and the tobacco 8. Borovsky D, Carlson DA, Griffin PR, hornworm, Manduca sexta. Pest Biochem Shabanowitz J, Hunt DE. Mosquito oostatic Physiol. 2005;81:71-84. factor: a novel decapeptide modulating 19. Lee YW, Zairi J. Field evaluation of Bacillus trypsin-like enzyme biosynthesis in the thuringiensis H-14 against Aedes mosquitoes. Vol 33 (No. 2) December 2010 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY 75
  8. 8. Larvicidal Effect of TMOF on Ae. aegypti Larvae Trop Biomed. 2006;23:37-44. Bacillus spharicus toxins: molecular biology20. Lee YW, Zairi J, Yap HH, Adnan CR. Integration and mode of action. Annu Rev Entomol. of microbial agent (Bacillus thuringiensis 1996;41:451-72. H-14, water dispersible granule and liquid 23. Darboux I, Neilsen-LeRoux C, Charles JF, formulations) and analog Insect Growth Pauchet Y, Puron D. The receptor of Bacillus Regulator (pyriproxyfen) for control of spharicus binary toxin in C. pipiens (Diptera: larvae of dengue vectors (Aedes aegypti and Cullicidae) midgut: molecular cloning Aedes albopictus). J Am Mosq Control Assoc. and expression. Insect Biochem Mol Biol. 2005;21:84-9. 2001;31:981-90.21. Baumann P, Clark MA, Baumann L, Broadwell 24. Davidson EW. Binding of the Bacillus spharicus AH. Bacillus spharicus as a mosquito pathogen: (Eubacteriales: Bacillaceae) toxin to midgut properties of the organism and its toxins. cells of mosquito (Diptera: Cullicidae) larvae: Microbiol Res. 1991;55:425-36. relationship to host range. J Med Entomol.22. Charles JF, Neilsen-LeRoux C, Delecluse A. 1988;25:151-7.76 THE JOURNAL OF TROPICAL MEDICINE AND PARASITOLOGY Vol 33 (No. 2) December 2010