TMOF is a peptide that inhibits trypsin production in mosquitoes. This study tested the acute effects of TMOF formulations (wettable powder and rice husk) and the biopesticide Ecobac-1 on three populations of the oil palm pollinator Elaeidobius kamerunicus. Results showed that TMOF and Ecobac-1 caused no significant mortality of the pollinators after one week of exposure, while the chemical insecticide cypermethrin caused 100% mortality. This suggests that TMOF and Ecobac-1 are safe alternatives to chemical insecticides for controlling pests in oil palms without harming the important pollinator E. kamerunic

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
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
1

2. (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
2

3. 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).
3

4. 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 28oC and relative humidity 50%.
4

5. 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%
5

6. 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).
6

7. 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
TABLE 2. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobius
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
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.
7

8. 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).
Figure 3. Average percentage corrected mortality of Elaeidobius kamerunicus sampled
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
8

9. TABLE 3. AVERAGE PERCENTAGE OF DEAD AND ALIVE Elaeidobius
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
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.
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 a
Figure 4. Average percentage corrected mortality of Elaeidobius kamerunicus sampled
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
9

10. 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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