Acute effect of tmof against op polinators not transformed

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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

Abstracts

Trypsin Modulating Oostatic Factor (TMOF) is a decapeptide hormone originally

purified from ovaries of females Aedes aegypti inhibits the biosynthesis of trypsin and

chymotrypsin like enzymes in midgut epithelial cell of female and larval mosquitoes by a

translational control mechanism. The lack of free amino acids liberated from the blood

meal in adult females or larval gut causes inhibition of egg development (sterility) to

adult, 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 by

spraying oil palm spikelets from anthesizing male flower containing adult pollinating

weevils 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 the

spikelets were directly exposed to the different treatments prior to feeding by the weevil

for one week. The bioassay was conducted for three different populations of Elaeidobius

kamerunicus. Results indicated that like MPOB Bt1 Ecobac-1(EC), TMOF (WP) and

TMOF (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.

Introduction

Mosquitoes are vectors for major diseases such as malaria, dengue, encephalitis and are

nuisance in the temperate zones (Borovsky et al., 2010). An active ingredient known as

Trypsin Modulating Oostatic Factor (TMOF) is used against mosquito larvae in aquatic

environments. TMOF is a small protein containing 10 amino acids that interferes with

digestion in mosquito larvae. TMOF is a decapeptide hormone originally purified from

the ovaries of female Aedes aegypti (Borovsky et al, 1990). It inhibits the biosynthesis of

trypsin and chymotrypsin like enzymes in the midgut epithelial cells of female and larval

mosquitoes 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 or

from digested proteins liberated in the larval gut causes inhibition of egg development

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(sterility) to adults and anorexia and death to larval mosquitoes, respectively (Borovsky

et al., 2003; Borovsky et al., 2006). TMOF also inhibits de novo biosynthesis of trypsin in

the midgut cells and this will lead to the insect starvation (Danuta et al., 1998). Oogenesis

in insects is a well-studied and complex process. Because the target tissue of the hormone

is the mosquito midgut and not the ovary or the brain, the hormone was named “Trypsin

Modulating Oostatic Factor” (TMOF).

Two oostatic factors called Aea-TMOF was isolated from yellow fever mosquito Aedes

aegypti (Borovsky et al., 1991) and Neb-TMOF found in fleshfly, Neobellieria bullata

(Bylemans et al., 1994) have been identified. Both are Dipteran and are anautogenous

species, 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 vitellogenin

production by fat body. The vitellogenins are then selectively taken up by the oocyctes in

a process called vitellogenesis. At the end of vitellogenesis the ovaries release oostatic

factor (TMOFs) which inhibits denovo biosynthesis of trypsin in the midguts (Danuta et

al., 1998)

TMOF has been used against larvae of mosquitoe larvae of Anopheles Mosquito

(transmitter malaria), Aedes aegypti (aedes) and Aedes albopictus (Chikungunya). It can

be applied in mosquito larvae habitat such as ponds, streams, ditches, puddles, and other

sources of standing water. TMOF can be applied directly to water or any mosquito larva

breeding site. TMOF protein is broken down quickly in the human gut and doesn't have

the 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 characterization

of trypsin modulating oostatic factor (TMOF) from the ovaries of the grey fleshfly,

Neobellieria (Sarcophaga) bullata instead of Aedes aegypti. There are also study on

trypsin 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 on

the synergistic of TMOF and Bacillus thuringiensis -endotoxin was reported by

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Borovsky et al. (2010). This study is conducted to investigate the acute effect of TMOF

as compared to MPOB BT1 Ecobac-1(EC) and chemical on three populations of oil palm

pollinators.

MATERIALS AND METHOD

Source of E. kamerunicus

Three populations of pollinating weevil, E. kamerunicus were obtained from a week old

anthesizing male inflorescences of oil palms. The three populations of oil palm

pollinators used in the study were collected from the MPOB Research Station at Bangi,

Selangor, Teluk Intan Perak and Kluang Johore.

Experimental design

The acute tests were conducted for the three populations of pollinators using five

treatments. Each treatment was conducted in replicates of three. For each replicate four

spikelets containing both living weevils and grubs of the pollinators were subjected to the

treatments. Total of twelve spikelets were exposed to the different treatments.

Treatments

Five different treatments namely, Trypsin Modulating Oostatic Factor (TMOF) wettable

powder, TMOF rice husk, Bt product, Ecobac-1 (EC), chemical control, Cypermethrin

and untreated were prepared. TMOF is a small mosquito-derived peptide expressed

within Pichia pastoris yeast cells. TMOF has synergistic effect with strain of B.

thuringiensis israelensis, therefore, can be formulated together with this protein. The

Ecobac-1 (EC) is an emulsified concentrate of MPOB Bt1 indigenous isolate propagated

and formulated at MPOB Microbial Technology and Engineering Center (MICROTEC).

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Preparation of test pollinating weevils and feed

Adult weevils and grubs in the four spikelets were transferred into clean sterilized

cylinder measured 25 cm in height with diameter of 13.5 cm. The cylinder containing

test oil palm pollinators were sealed using cheese cloth with rubber bands. The cylinders

and 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 on

as feed for the adult weevils.

Bioassay

TMOF wettable powder solution was prepared by weighing 10g of TMOF wettable

powder mixed with 1 L of distilled water. As for the TMOF rice husk, the same

procedure 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 wettable

powder, TMOF rice husk and Ecobac-1 (EC). Each of the four spikelets containing grubs

for each replicate were thoroughly sprayed with the products in the fumehood using 2

ml/spikelets of the product using a hand held sprayer. For chemical control, the spikelets

were sprayed with 5% Cypermethrin at 2 ml/spikelet. For the untreated control, the

weevils were sprayed with only distilled water at 2ml/spikelet. Spraying of weevils was

done in the fume cupboard using a 1L hand held sprayer.

Each group of the four treated spikelets containing the grubs and the adult weevils were

transferred into a 500 ml sterilized cylinder (measured 25 cm in height with diameter of

13.5 cm.) labelled according to the treatments and replicates. The cylinders were sealed

using cheese cloths and dried under the bright sunlight for 30 minutes prior to incubation

in the indoor insectory at temperature 24oC to 28

oC and relative humidity 50%.

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Data recording

The mortality of E. kamerunicus population from Bangi was recorded at 7 days after

treatment (DAT). At the end of 7 days the spikelets were dissected and the numbers of

live and dead weevils and grubs were recorded. The dissected spikelets containing live

and dead grubs were photographed using dissecting microsope with a camera attached.

Data Analysis

Data on survival and mortality was calculated for each replicate and treatments. The

mortality was then converted to corrected mortality using the Abbot formula, then

analyzed in one-way ANOVA using SPSS software version 11.5. The means were

analyzed by the Least Significant Difference (LSD) test using the same software.

RESULTS AND DISCUSSION

Table 1 shows that the exposure of E.kamerunicus population from Bangi, 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 13.8%, 16.1%, 12.4% and 10.8 % for TMOF WP, TMOF

RH, MPOB Ecobac-1(EC) and untreated control, respectively. This implies that both

forms of TMOF and Ecobac-1(EC) have no acute effect on the oil palm pollinating

weevils. The contrasting effect was noted for chemical insecticide, in this case

cypermethrin which resulted in 100% mortality of E.kamerunicus (Table 1)

The corrected mortality of E. kamerunicus population from Bangi subjected to the

various 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%

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TABLE 1. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobius

kamerunicus SAMPLED FROM MPOB STATION IN BANGI SELANGOR AT 7

DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS

______________________________________________________________________

Treatments dead weevil alive (weevils & grubs)

_____________________________________________________________________

Control 10.8±2.4 a 89.2±2.4 a

TMOF WP 13.8±2.9 a 86.2±2.9 a

TMOF RH 16.1±5.3 a 83.9±5.3 a

Ecobac-1(EC) 12.4±1.5 a 87.6±1.5 a

Cypermethrin 100+0.0 b 0+0.0 b

The 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% for

cypermethrin. Unlike chemical, TMOF WP, TMOF RH, MPOB Ecobac-1(EC) are safe

for the oil palm pollinating weevils and grubs found in the florets (Figure 2).

Figure 1. Average percentage corrected mortality of Elaeidobius kamerunicus sampled

from MPOB Station in Bangi Selangor at 7 days after exposure to different treatments

Spraying of cypermethrin was detrimental to both the weevil and grubs because it

resulted in 100% mortality of both stages of the pollinator. The rest of the treatments

including untreated resulted in almost 100% survival of the grubs and majority of the

weevils (Figure 2).

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a b

c d

Figure 2. Microscopy of E.kamerunicus with magnification 40x. Showing live adults

weevil (a) and grubs (b) of E.kamerunicus subjected to TMOF and dead weevil treated

with cypermethrin outside (c) and within the spikelet (d).

Table 2 shows that the exposure of E.kamerunicus originated from MPOB Station in

Teluk Intan Perak, to TMOF WP, TMOF RH, for 1week resulted in no significant


kamerunicus SAMPLED FROM MPOB STATION IN TELUK INTAN PERAK AT

7 DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS

_____________________________________________________________________

Treatments dead weevils alive (weevils & grubs)

Control 32.4±7.5 a 67.6±7.5 a

TMOF WP 45.6±5.3 a 54.4±5.3 a

TMOF RH 32.4±3.5 a 67.6±3.5 a

Ecobac-1(EC) 22.9±11.1 ab 77.1±11.1 ab

Cypermethrin 100+0.0 c 0+0.0 c



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difference in mortality at P< 0.05 as compared to untreated control. However, MPOB

Ecobac-1 (EC) resulted in much lower mortality, 22.9% than TMOF WP, TMOF RH

and 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 acute

effect on the oil palm pollinating weevils. The contrast effect was noted for chemical

insecticide, cypermethrin which resulted in 100% mortality of E.kamerunicus (Table 2).

The corrected mortality of E.kamerunicus sampled from MPOB Station in Teluk Intan

Perak, 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).


from MPOB Station in Teluk Intan Perak at 7 days after exposure to different treatments

Dissection of the spikelets at 7 DAT revealed the same result as in the case for pollinators

from MPOB Station in Bangi. Unlike cypermethrin which resulted in 100% mortality of

both 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 from

MPOB Stations in Perak and Kluang (Table 3 and Figure 4). Table 3 shows that the

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kamerunicus SAMPLED FROM MPOB STATION IN KLUANG, JOHORE AT 7

DAYS AFTER EXPOSURE TO DIFFERENT TREATMENTS

____________________________________________________________________

Treatments dead weevils alive (weevils & grubs)

__________________________________________________________________

Control 34.0±3.4 a 66.0±3.4 a

TMOF WP 34.4±0.8 a 65.6±0.8 a

TMOF RH 44.9±3.5 a 55.1±3.5 a

Ecobac-1(EC) 33.9±7.4 a 66.1±7.4 a

Cypermethrin 100+0.0 b 0+0.0 b



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


from MPOB Station in Kluang Johore at 7 days after exposure to different treatments

Ecobac-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

a

a

a

b

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Station in Kluang, Johore subjected to the various treatments showed minimal corrected

mortality of E. kamerunicus of 0%, 16% and 0% for TMOF WP, TMOF RH, MPOB

Ecobac-1(EC) ) as compared to 100% for cypermethrin (Figure 4).

Conclusions

Like other biological insecticide such as MPOB Ecobac-1 (EC), TMOF WP and TMOF

RH are safe for beneficial insects such as the oil palm pollinating E.kamerunicus, as

proven by the acute toxicity trials. Unlike chemical, the use of these biological

insecticides, MPOB Ecobac-1 (EC), TMOF WP and TMOF(RH) did not detriment the

pollinators particularly the grubs.

Acknowledgements

The authors would like to thanks Y Bhg Datuk Choo Yuen May, Director General of

MPOB, Y Bhg Dato Seri Utama Shahrir Abdul Samad, the Chairman of MPOB, MPOB

Board Members for their permission to undertake this joint project between MPOB and

EntoGenex Sdn Bhd. The authors are very grateful for the continuous support given by

the subordinates or supporting staff of Microbial Technology Research Group of MPOB.

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