Nghiên cứu đặc tính sinh học và biện pháp phòng trừ bọ vòi voi diocalandra frumenti (coleoptera curculionidae) hại dừa tại đồng bằng sông cửu long tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY DOCTORAL DISSERTATION
Major: Plant Protection
Code: 9620112

NGUYEN HONG UNG

STUDY ON BIOLOGICAL CHARACTERISTICS AND
CONTROL METHODS ON LESSER COCONUT
WEEVIL Diocalandra frumenti (COLEOPTERA:
CURCULIONIDAE) IN THE MEKONG DELTA

Can Tho, 2019


THE THESIS HAS BEEN COMPLETED AT THE COLLEGE OF
AGRICULTURE AND APPLIED BIOLOGY, CAN THO UNIVERSITY

Scientific supervisor: Assoc. Prof. Dr. Tran Van Hai

The dissertation will be defended to the University scientific committee
Dissertation seminar at: ………………………….
Time: ……………………………………………..

Reviewer 1: ……………………………………….
Reviewer 2: ……………………………………….

Find more information about dissertation at:
Can Tho University Learning Resource Center

National Library of Vietnam


LIST OF PUBLISHED PAPERS
1. Nguyen Hong Ung, Trieu Phuong Linh, Huynh Ky and Le Van Vang, 2016.
Genetic diversity among Diocalandra frumenti (Coleoptera: Curculionidae) on coconut
by ISSR marker, 2016. Can Tho University Journal of Science (ISSN 1859-2333),
Volume 3/2016, pp. 128-135.
2. Nguyen Hong Ung, Chau Nguyen Quoc Khanh, Le Van Vang and Tran Van
Hai, 2016. Damage situation and morphological characteristics of the coconut weevil,
Diocalandra frumenti Fabricius (Coleoptera: Curculionidae) in Tra Vinh Province,
2016. Journal of Plant protection, No. 5 (268): 36-43.
3. Nguyen Hong Ung, Chau Nguyen Quoc Khanh, Le Van Vang and Tran Van
Hai, 2017. The results of the damage and behaviors of the coconut weevil, Diocalandra
frumenti (Fabricius) (Coleoptera: Curculionidae) in Vinh Long and Ben Tre Province,
2017. Proceedings of the 9th Vietnam National Conference on Entomology, Agriculture
publishing house, pp. 729-736 (ISBN 978- 604-60-2511-5).

1


Chapter 1: INTRODUCTION
1.1 The imperativeness of the dissertation
Coconut (Cocos nucifera L.) is an important tree for oil extraction in the total areas
of 12 millions of hectares in 90 countries of the world. It has been considered a source
of food, material supply for consumers’ products processing, exporting industries and a
tree with economic, social and ecological significance (Vo Van Long, 2007). In addition,
according to Cao Quoc Hung (2015) coconut has high economic potentials, especially
suitable for poor-nutrient coastal, saline intrusion, flood and storm-effected soils. Main
products made from coconuts have been exported to different markets in European,

Middle East, African and North American countries to bring about high profits. Coconut
areas of Vietnam in 2015 were approximately 160 thousands of hectares, mainly
dispersed in the Mekong Delta-82.6% of the total areas. It has created significant
production values for the region. In Ben Tre province, coconut processing is a key
economic industry contributing to creating employment opportunities for numerous
labor and increasing income for farmers. According to Nguyen Thi Thu Cuc (2015),
lesser coconut weevil is scientifically called Diocalandra frumenti Fabricius
(Coleoptera: Curculionidae). This insect, which was firstly recognized in Kien Giang
province in 2012, has quickly caused damages to many provinces in the Mekong Delta,
Southeast and Central areas. Its damages can cause fruit deformity and stunting.
Previously, D. frumenti was recorded to cause damages on areca, nipa palm and other
plants belonging to the Palmae family in many places in the world (EPPO, 2012).
The damages have shown that D. frumenti possibly causes serious effects on
coconut productivity and quality. Since coconut is significantly high; and this insect stays
at places not easy to reach, preventive treatments by chemicals are extremely difficult
which cause environmental pollution and affect human health. At present, official studies
on preventive methods, which are significantly effective to control this pest, have not
been made available. Therefore, the dissertation on “Study on biological characteristics
and control methods on lesser coconut weevil Diocalandra frumenti Fabricius
(Coleoptera: Curculionidae) in the Mekong Delta” was conducted to identify detailed
information on D. frumenti which sets up a foundation for developing an integrated
management strategy with a focus on safe solutions to prevent from this pest.
1.2 The aim and requirements of the study
- Identify the damage capacity of D. frumenti in three provinces: Ben Tre, Vinh
Long and Tra Vinh
- Survey morphological, biological and damage characteristics of D. frumenti
Fabricius on coconut trees
- Identify the genetic diversity of D. frumenti by ISSR molecular markers
2

- Determine natural parasitic fungi of D. frumenti and evaluate efficacy of bio
products against D. frumenti Fabricius
- Establish experimental models to control D. frumenti by environmentally-safety
solutions.
1.3 The scientific meaning of the dissertation
The dissertation provides basic knowledge on lesser coconut weevil D. frumenti in
Ben Tre, Vinh Long and Tra Vinh provinces such as its damage status, morphological
and biological characteristics etc.
It additionally evaluates genetic diversity of D. frumenti in some provinces in the
Mekong Delta and the Southeast region, which makes gene banks of insects more diverse.
The dissertation provides knowledge on managing lesser coconut weevil D.
frumenti under a sustainable direction, an important scientific basis in entomology.
The application of the dissertation results will contribute to ensuring coconut
quality, limiting the use of insecticides, reducing environmental pollution and securing
human health.
1.4 The new contributions of the dissertation
The dissertation results identified damage status, morphological and biological
characteristics, living habits, genetic diversity, genetic relations of lesser coconut weevil
D. frumenti. More importantly, they additionally selected some parasitic fungal strains
with high effectiveness in controlling this pest.
Chapter 3: STUDYING METHODS
3.1 Period and location of study
3.1.1 Period
The dissertation was conducted from November, 2014 to November, 2018.
3.1.2 Locations
The dissertation was conducted in coconut orchards in Ben Tre, Vinh Long, Tra
Vinh and neighboring provinces. Experiments were arranged at laboratories of
Department of Plant Protection, Can Tho University and School of Agriculture and
Aquaculture, Tra Vinh University.

3.3 Methods
3.3.1 Content 1: Evaluate damage status of lesser coconut weevil D. frumenti in Ben
Tre, Vinh Long and Tra Vinh provinces
- Interview farmers to obtain information on lesser coconut weevil D. frumenti
Interviewed households, whose areas of at least 1,000m 2, were those involved in
coconut cultivation in Ben Tre, Vinh Long and Tra Vinh provinces. The investigated
contents included information on cultivation, damage status and D. frumenti.
- Investigate damage status of D. frumenti on coconut orchards
3


Ten coconut trees were selected in five locations: four in peripheral and the other
in central coconut orchards to record the damages caused by D. frumenti on main parts
of the trees. The investigation were conducted on 3,300 coconut trees from 330 orchards
in Ben Tre, Vinh Long and Tra Vinh provinces (based on QCVN 0138:2010/BNNPTNT).
- Investigate damage progress of D. frumenti on coconut orchards
Damage progress of D. frumenti was performed at 27 orchards in the three
provinces with areas of more than 3,000m2 each. The investigation was carried out once
a month during 12 months.
3.3.2 Content 2: Investigate the morphological, biological characteristics and
damage caused by lesser coconut weevil D. frumenti
The investigation was carried out with 150 eggs, 29 larvae, 29 female pupae, 20
male pupae, 29 female and 20 male adults in total. The study was proceeded with
collecting pupal individuals from the fields for further additional investigation. Living
habits and damages caused by lesser coconut weevil were investigated at coconut
orchards of cultivation households in Ben Tre, Vinh Long and Tra Vinh provinces,
combined with laboratory investigation.

C

B

A
Adults in the field

laying eggs

raising larvae

E

D

pairing adults after emergence

raising pupae

Figure 3.5: The process of investigating the morphological and biological
characteristics of D. frumenti: Adults in the field (A), laying eggs (B), raising larvae
and pupae (C,D) and pairing adults after emergence (E) (Nguyen Hong Ung, 2016)
3.3.3 Content 3: Evaluate genetic diversity of lesser coconut weevil D. frumenti by
ISSR molecular markers
ISSR molecular markers were used to identify genetic relations of D. frumenti
strains collected respectively in eight and four provinces in the Mekong Delta and
Southeast region. The investigation was carried out on 40 samples of D. frumenti adults
which were grouped with each other based on their morphological characteristics.
4


3.3.4 Content 4: Collect, isolate, select and evaluate effectiveness of parasitic fungi

on lesser coconut weevil D. frumenti
Samples of lesser coconut weevil D. frumenti with parasitic fungi were collected
and transferred to laboratory for isolation, selection and experiment arrangements.
- Evaluating the effectiveness of M. anisopliae on D. frumenti in laboratory condition
The experiment was conducted with six treatments, including five green
muscardine fungus samples collected from local areas and one control with distilled
water. The utilized concentration of the M. anisopliae treatments was 108 spores.mL-1;
all treatments were added with siloxane alkoxylate as an adhesive substance. The
experiment was arranged in a randomized complete block design, four replications with
30 D. frumenti adults for each treatment. The experiment was carried out by dipping the
adults into the fungal spore solution for 30 seconds. Number of alive adults was recorded
at the time of 1, 3, 5, 7, 9, 11, 13 and 15 days after treating.
- Evaluating the efficacy of green muscardine fungus M. anisopliae and some
insecticides on D. frumenti adults in the laboratory condition
This experiment was conducted with seven treatments and four replications
arranged in randomized complete block design. Each replication of one treatment was a
petri dish small pieces of coconut ocrea and 30 adults. The treatments were treated by
spraying solution over the experimented treatments with three M. anisopliae strains (108
spores.mL-1), three insecticides and one control with distilled water. Number of alive
adults was recorded at 1, 3, 5, 7, 9, 11, 13 and 15 days after treating.
- Evaluating the efficacy of green muscardine fungus at different concentrations on
D. frumenti adults in the laboratory condition
The M. anisopliae strain was selected for survey based on the previous results. The
experiment was conducted with five treatments and four replications arranged in
randomized complete design. Each treatment had a petri dish with coconut petiole and
30 D. frumenti adults which were dipped into the fungal spore solution with
concentrations of 106, 107, 108, 109 spores.mL-1 and one control (distilled water). Number
of alive adults was recorded at 1, 3, 5, 7, 9, 11, 13 and 15 days after treating.

Figure 3.8: Arranged experiment to evaluate efficacy of M. anisopliae on D. frumenti

adults in laboratory with coconut petioles as food supply (Nguyen Hong Ung, 2015)
5


- Evaluate preventive efficacy of green muscardine fungus strains and some
insecticides on D. frumenti in the field condition
Two isolates of muscardine fungi were Ma-GT-BT and Ma-LP-ST and two
insecticides Emamectin benzoate and Fipronil were used. The controlled treatments were
treated with distilled water and adhesive substances. The experienment was conducted
at coconut orchards in Ben Tre province with the areas of 3,000 m2. They were in the
stage of blooming and bearing young fruits which were damaged by D .frumenti. The
experiment was arranged in randomized complete block design with five treatments and
four replications. Each treatment was six coconut trees with three times of application
during an interval of two weeks per each time.
3.3.5 Content 5: Establish experimental models to control D. frumenti Fabricius by
environmentally-safety solutions.
A model was implemented on “dua Ta” in Ben Tre province in the dry season and
the other on “dua Xiem” in Tra Vinh province in the rainy season with the areas from
3,000-4,000 m2. Lesser coconut weevil D. frumentri control methods which were mainly
applied were orchard cleaning, periodically spraying Ma-GT-BT entomopathogenic
fungus at 125gx16L-1, 2-3L per tree, once every three weeks. The models in Ben Tre and
Tra Vinh provinces were sprayed three and six times respectively during the
implementation period.
3.4 Statistical analysis
Data were entered and presented by Microsoft Office Excel, collected by Duncan
test and T-tested with MSTATC. The investigation results on D. frumenti genetic
diversity were analyzed under the UPGMA method by SATISTICA 5.5 software.
Chapter 4: RESULTS AND DISCUSSIONS
4.1 Results on the damage capacity of D. frumenti in Ben Tre, Vinh Long and Tra
Vinh provinces.

* Farmer investigation results
- D. frumenti in coconut orchards recognized by farmers
Average percentages of investigated households who recognized damages caused
D. frumenti in 2013-2014 were 32.9% và 41.6%. Farmers in Ben Tre province mainly
realized its damage in 2013 (30.7%) while those in the other two provinces (58.9% for
Tra Vinh and 43.3% for Vinh Long) knew about it in 2014.
- Damage levels of D. frumenti on coconut orchards in 2014 and 2015
According to the farmer investigation results, the damage in 2015 was higher than
that of 2014. Its main damage in 2014 was less than 5% of fruits per orchard (38.4%
households) and that of 2015 was 10 to <30% of fruits per orchard (27.5% households).
- Time and damages caused D. frumenti on coconut orchards
6


Farming households realized damages caused by D. frumenti at many periods of
time in coconut orchards. Of which, percentage of households known the damages all
year round was 43.9%, 29.0% and 14.9% in the dry and rainy seasons, respectively.
- Locations on coconut parts damaged by D. frumenti
The investigation results showed that 100% households acknowledged the damages
caused by D. frumenti at all coconut fruit development stages. Of which, percentages of
households who realized the damages on young, mature fruits were respectively at 57.6%
and 8.24% while 24.7% was possibly on all stages of the fruit.
- D. frumenti control methods by farmers in Ben Tre, Vinh Long and Tra Vinh
provinces
Most farmer who had no effective methods to control D. frumenti accounted for
62.9% of the investigated households and 37.1% of them only used chemicals (Regent
800WG, Basudin 10H …). The chemicals used were available at these households.
* Damage status of D. fumenti in Ben Tre, Vinh Long and Tra Vinh provinces
according to investigations at coconut orchards
- Damage ratios and locations caused by D. fumenti at coconut orchards

The investigation results in Ben Tre, Vinh Long and Tra Vinh in 2015 indicated
that all coconut orchards were damaged by with the ratios of 58.9% on trees, 19.4% on
bunches and 7.77% on fruits. The results investigated in 2017 in Ben Tre province had a
decreasing tendency with the respective ratios of 58.1%, 16.7% and 4.62%.
Table 4.2: Damage ratios of D. fumenti at coconut orchards in Tra Vinh, Vinh Long and
Ben Tre provinces in 2015 and 2017
Average damage ratios (%)
Locations
Tieu Can District
Cang Long District
Cau Ke District
Tra Vinh Province, 2015
Tam Binh District
Vung Liem District
Tra On District
Vinh Long Province, 2015
Binh Dai District
Giong Trom District
Mo Cay Nam District
Ben Tre Province, 2015
Binh Dai District
Giong Trom District
Mo Cay Nam District
Ben Tre Province, 2017

Damaged
orchards
100
100
100

100
100
100
100
100
100
100
100
100
100
100
100
100

Damaged
trees/ orchard
51.3
60.7
56.2
56.1
65.0
61.7
69.0
65.2
50.0
64.8
51.2
55.3
56.7
56.7

61.0
58.1

7

Damaged
bunches/orchard
15.5
18.2
19.0
17.6
19.7
15.8
19.9
18.5
16.0
30.1
20.2
22.1
17.3
14.5
18.4
16.7

Damaged
fruits/orchard
5.90
8.50
9.60
8.00

6.81
6.73
7.20
6.91
6.75
11.2
7.25
8.40
4.35
3.55
5.95
4.62


- Damage progresses on coconut bunches and fruits caused by D. frumenti
Damage progresses on coconut caused by D. frumenti in Ben Tre province
fluctuated from 8.26-29.4% and 3.58-13.6% of damaged bunches and fruits,
respectively. Those in Vinh Long ranged from 13.8-27.3% and 3.78-7.76%. While the
average ratios on bunches damaged by D. frumenti in Tra Vinh province changed from
18.7- 26.0% and those of fruits were 5.31-8.43%. In general, the investigation results on
damage caused by D. frumenti were not much different among the provinces.
4.2 Morphological, biological characteristics and damages of D. frumenti
* Morphological characteristics of D. frumenti
- Eggs: D. frumenti eggs are oval in shape with average sizes of 0.85±0,07 mm
(length) và and 0.29±0.04 mm (width); newly-laid ones are transparent white, later
turning to translucent white which have 2 brown-black spots when they are at prehatching period (Figure 4.19A,B).

B

A

Figure 4.19: D. frumenti eggs: newly-laid (A) and pre-hatching (B)
- Larvae: D. frumenti larvae at the first instar are 1.15 mm long, 0.24 mm wide,
which increase gradually when they get to the 19th instar to the maximum sizes of 6.09
mm long and 1.35 mm wide (Table 4.3, Figure 4.20, 4.21). Newly-hatched larvae have
their bodies in translucent white, brownish yellow heads, later turning to yellow. These
were found in Nguyen Thi Thu Cuc’s (2015) record on morphological characteristics of
D. frumenti. Furthermore, Liao and Chen (1997) also declared that its larvae are white
which get the length of approximately 7.00 mm at full development stage.

1st

2nd

12th

3rd

13th

4th

14th

6th

5th

15th

7th

16th

9th

8th

17th

10th

18th

11th

19th

Figure 4.20: Morphological characteristics of D. frumenti larvae from the 1st instar to
the 19th instar
8


Table 4.3: Sizes of D. frumenti larvae raised in the laboratory conditions of Tra Vinh
University, 2017 (T=28-31oC, RH=68-80%)
Development
stage
Eggs
1st Instar
2nd Instar

3rd Instar
4th Instar
5th Instar
6th Instar
7th Instar
8th Instar
9th Instar
10th Instar
11th Instar
12th Instar
13th Instar
14th Instar
15th Instar
16th Instar
17th Instar
19th Instar
19th Instar
Female pupae
Male pupae
Female adults
Male adults

Sizes (mm)
Sample size

Head capsule
length

Head capsule
width

Body
length

Body
width

150
30
30
30
30
30
30
30
30
26
26
25
25
21
16
11
07
05
04
02
29
20
29

20

0.22±0.05
0.29±0.04
0,.41±0.04
0.51±0.07
0.59±0.88
0.71±0.10
0.82±0.10
0.91±0.11
0.96±0.09
0.98±0.10
1.03±0.11
1.07±0.08
1.08±0.07
1.09±0.06
1.12±0.06
1.14±0.08
1.16±0.09
1.20±0.00
1.20±0.00
-

0.18±0.04
0.27±0.05
0.33±0.05
0.42±0.08
0.53±0.11
0.61±0.11
0.75±0.12

0.81±0.11
0.85±0.11
0.88±0.11
0.92±0.11
0.97±0.09
0.98±0.07
0.99±0.06
1.02±0.06
1.03±0.08
1.08±0.04
1.10±0.00
1.10±0.00
-

0.85±0.07
1.15±0.11
1.61±0.23
2.38±0.63
2.99±0.66
3.66±0.87
4.18±0.97
4.82±0.83
5.28±0.69
5.43±0.70
5.80±0.67
5.96±0.60
6.08±0.53
6.03±0.39
6.03±0.40
5.99±0.40

6.09±0.50
5.94±0.54
5.85±0.57
5.60±0.85
5.22±0.35
5.15±0.32
6.23±0.44
5.14±0.42

0.29±0.04
0.24±0.05
0.30±0.03
0.40±0.07
0.49±0.08
0.55±0.06
0.65±0.11
0.78±0.12
0.88±0.11
0.96±0.12
1.03±0.12
1.09±0.13
1.15±0.10
1.15±0.08
1.18±0.06
1.19±0.05
1.21±0.07
1.26±0.05
1.33±0.05
1.35±0.07
1.16±0.06

1.11±0.06
1.17±0.15
1.08±0.09

- Pupae: D. frumenti pupae are exarate, average length of 5.22±0.35 mm, width of
1.16±0.06 mm (female) and 5.15±0.32 mm and 1.11±0.06 mm (male). They are
translucent white, later turning to yellowish, which have two black markings on elytra
when getting close to emergence (Figure 4.23).

B
A

Figure 4.23: Pupae of D. frumenti after turning to pupae (A) and getting close to emergence
(B)

- Adults: Newly-hatched D. frumenti adults have bright-yellowish brown elytra,
later turning to blackish brown or yellowish brown; they have four big yellowish-brown
9


spots on elytra. Their average sizes are 5.14±0.42 mm x 1.08±0.09 mm (male) 6.23±0.44
mm x 1.17±0.15 mm (female) (Figure 4.24A,B,C).

♀

A

B

C

♂

D

E

Figure 4.24: D. frumenti adults: new emergence (A, B), full development (C), rostrum
of male and female adults (D) and aedeagus of male adults (E)
Male adults have small, short rostrum which is less curved and more setae than the
female’s. They have yellowish brown and slightly curved aedeagus which is 1.00 mm
long (Figure 4.24D, E).
* Biological characteristics and damage capacity of D. frumenti
- Eggs: Egg average development stage takes about 5,62 ±0.62 days in the laboratory
conditions, which is same as that of other study results (Table 4.4). Specifically, Liao
and Chen (1997) indicated that D. frumenti egg development was 6-10 days. Hill (1983)
and Nguyen Thi Thu Cuc (2015) reported that its eggs developed in 4-9 days.
- Larvae: In the laboratory conditions, larvae at the 8th-19th instar develop from 6.21
days (1st instar) to 213 days (the 19th instar) (Table 4.4). This result is different from
Hill’s (1983) in that larvae of this pest developed in 08-10 weeks. Liao and Chen (1997)
identified that larvae developed in 35-40 days. González et al. (2002) also reported that
with differnt nutritional regimes, D. frumenti larvae had of 73.2, 69.2 and 60.9 days in
the temperature conditions of 25±1°C, humidity of 70±10%. As a result, development
stage of D. frumenti larvae has been proven with huge fluctuations and significantly
affected by raising conditions, of which food compositions were also counted.
Changes in larvae instar depends on environmental factors such as temperature,
lighting duration, raising density and humidity (Beckett and Evans, 1994; Esperk et al.,
2007), vulnerability, food compositions and quality (Esperk et al., 2007), nutrition
shortage (Nijhout, 1975), accumulation of necessary lipid to ensure larva existence
(Slansky and Rodriguez 1987). Goettel and Philoge (1978), who conducted a research

on P. isabella, development of B. germanica and A. fasciatus, respectively also indicated
that lighting duration could increase larval instars. Kim et al. (2015) who studied Z.
atratus also pointed out a difference between Z. atratus instars and its larval lengths with
a maximum of 18 instars due to hormone and lack of nutrition. Park et al. (2014) stated
that T. molitor had a great difference on its larval development duration and pupation
proportions in the same raising conditions due to nutritional status of larvae, egg laying
10


period of adults and oxygen concentrations of the raising environment. Ludwig and Fiore
(1960) acknowledged T. molitor instars and larval development duration depended on
egg laying durations of adult parents.
- Pupae: D. frumenti pupae have an average development duration of 9.79±0.86
days (female) and 10.2±1.26 days (male) (Table 4.4). Additional investigations on 150
pupal individuals randomly collected from the fields showed a result of 9.27±1.86 days
on average. The result was same as Hill’s (1983) declaration in that D. frumenti pupae
had a development duration from 09-10 days. Similarly, Liao and Chen (1997) reported
D. frumenti pupae developed in 10-16 days (Table 4.4).
- Adults: In the laboratory conditions, average living duration of male adults is
81.5±34.7 days and that of female adults is 81.8±37.2 days (Table 4.4).
Table 4.4: D. frumenti development cycles in the laboratory conditions of Tra Vinh
University, 2017 (T=28-31oC, RH=68-80%)
Development stage
Eggs
Larvae
1st Instar
2nd Instar
3rd Instar
4th Instar
5th Instar

6th Instar
7th Instar
8th Instar
9th Instar
10th Instar
11th Instar
12th Instar
13th Instar
14th Instar
15th Instar
16th Instar
17th Instar
19th Instar
19th Instar
Female pupae
Male pupae
Female adults
Male adults
Emergence – laying eggs
Egg laying period
Amounts of laid eggs
Average life cycle

Sample size

Average
5.62±0.62
142±34.3
6.21±2.11
14.2±3.69

22.6±5.86
29.9±6.64
39.0±9.04
48.4±11.8
58.3±14.7
70.1±18.3
76.2±14.5
87.1±16.1
96.1±17.3
109±19.9
121±23.6
131±25.2
138±20.9
150±30.2
166±36.2
185±42.8
213±55.9
9.79±0.86
10.2±1.26
81.8±37.2
81.5±34.7
9.79±7.14
67.6±35.2
24.4±13.8
167±34.3

29
29
29
29

29
29
29
29
29
26
26
25
25
21
16
11
07
05
04
02
29
20
29
20
29
29
29
29

11

Period (days)
Variations
5.00-7.00

99.0-252
2.00-13.0
9.00-25.0
12.0-40.0
20.0-45.0
27.0-67.0
32.0-79.0
39.0-100
47.0-113
54.0-112
63.0-122
70.0-132
75.0-144
95.0-175
108-207
119-191
128-216
138-226
147-234
173-252
8.00-11.0
8.00-13.0
20.0-157
27.0-136
2.00-38.0
5.00-155
2.00-45.0
122-271

Results obtained from additional investigations on 50 male and 50 female adults
randomly collected from the fields provided a living duration of 76.9±32.5 days (male)
and 78.5±32.1 days (female). The results were different from Liao and Chen’s
declaration (1997) in that mature offsprings of D. frumenti had a living duration of 1522 days after emergence. This proved living duration of D. frumenti adults was
remarkably longer than that released by other declarations.
- Life cycle of D. frumenti: In the laboratory conditions (temperature: 28-31oC,
humidity: 68-80%), its cycle is 167±34.3 days (fluctuating from 122 to 271 days) (Table
4.4, Figure 4.26). Study result on D. frumenti life cycle is different from that of previous
reports. Particularly, Hill (1983) stated its cycle was approximately 10-12 weeks and
Liao and Chen (1997) also identified that it lasted about two months. Such difference is
mainly due to development of larval stage.
9.79±7.14 days
(2.00-38.0 days)

Eggs

5.62±0.62 days
(5.00-7.00 days)

Average life cycle
167±34.3 days (122-271 days)
o

(28-31 C, 68-80%)
Adults
Larva
9,79±0.86 days
(8.00-11.0 days)
Pupa

142±34.3 days
(99.0-252 days)

Figure 4.26: Life cycle of D. frumenti in the laboratory conditions
- Egg laying ability of D. frumenti
The investigation revealed female adults started laying eggs at 02-38 days after
emergence (9.79±7.14 days on average) and finished the egg laying process at 15-162
days after emergence (67.6±35.2 days on average). Average amounts of laid eggs were
24.5±13.8 per female adults (fluctuating from 02-45 eggs). Results obtained from
additional investigations on 43 pairs of adults emergence from pupae collected from the
fields showed that each female adult could lay 23.2±20.1 eggs on average (fluctuating
from 02-88 eggs).
- Living habits and damages of D. frumenti
Lesser coconut weevil D. frumenti lays eggs scatterly in tissues, young tree parts
such as fruit stalks, sheaths etc. at the depth of approximately 1.00-2.00 mm, no laid eggs
12


found on the surfaces (Figure 4.27). Such information was also found in studies by
González et al. (2002), Nguyen Thi Thu Cuc (2015).

A

B

Figure 4.27: D. frumenti eggs in female bodies (A) and in tree tissues (B)
D. frumenti larvae and pupae were found in tissues of coconut trunks, sheaths,
flower and fruit stalks, right inside the infested marks (Figure 4.28). Nguyen Thi Thu
Cuc (2015) previously reported that all living processes of D. frumenti larvae occurred
along their boring lines.

A

B

D

C

Figure.28: D. frumenti larvae and pupae inside coconut fruits (A,B,C) and sheaths (D)
D. frumenti adults mainly move by crawling. They usually sneak inside coconut
cracks and bored marks surrounding fruit stalks (Figure 4.29).

B

A

C

Figure 4.29: Living areas of D. frumenti adults on the cracks of coconut trunks (A),
fruits (B) and sheaths (C)
The most noticeable damage of D. frumenti is mainly on fruits and trunks with
popular symptoms such as sap oozing out from the infested areas (Figure 4.30). Studies
by Liao and Chen (1997) and Giblin-Davis (2011) stated that larvae of newly-hatched
lesser coconut weevils bored into tree tissues where eggs were laid causing sap oozing
phenomenon. The investigation results have not yet confirmed deaths of whole coconut
trees caused by D. frumenti.
13

B
C

A

Figure 4.30: Sap oozing phenomenon caused D. frumenti: on trunk (A), sheath (B) and
flower stalk (C)
After being infested by D. frumenti, sap oozing marks got dry and dropped off
leaving small blackish-brown bored holes where larvae could be found (Figure 4.31).

A
D

C

B

Figure 4.31: Infested marks caused by D. frumenti: trunks (A) and sheath (B, C, D)

Lesser coconut weevil D. frumenti damages on both young and mature fruits, but
mainly on young fruits. It can lead to fruit dropping or deformity (Figure 4.23).

A

B

D

C

E

Figure 4.32: Damage symptoms of D. frumenti on coconut with sap oozing
phenomenon (A,C) and sunken lesions at many places on the fruits (B,D,E)
In addition to sap oozing phenomenon (Figure 4.32A), damage symptoms of D.
frumenti also include long, small blackish-brown sunken marks surrounding young areas
of the fruits (Figure 4.32B). These sunken marks in the long run will move to the middle
of the trunks and fruit apexes and possibly form big marks on the exocarps (Figure
4.32C,D). Previously, EPPO (2012) declared a study result on the symptoms recorded
on roots, young leaves, sheaths, fruit stalks and young fruits but no D. frumenti was found
on trunks of host trees. Furthermore, in the Mekong Delta, Nguyen Thi Thu Cuc (2015)
defined that coconut damaged by this pest had its young fruits falling down and big fruits
developing poorly or deforming. However, investigation results showed that besides
young fruits, a significant amount of mature fruits possibly fell down in conjunction with
D. frumenti damage symptoms. Results obtained from field investigations revealed that
14


D. frumenti tended to be found on nipa palms (Nypa fruticans Wurm) as well as attracted
by sap of host trees.
4.3 Genetic diversity of D. frumenti samples collected in provinces of the Mekong
Delta and four provinces in the Southeast region
* Classification based on D. frumenti morphologies
Based on colors of D. frumenti adults, the collected samples were grouped into four
main morphologies (Figure 4.35).

A

B

C

D

Figure 4.35: Four morphologies of D. frumenti adults collected in provinces of the
Mekong Delta and Southeast region: morphology 1 (A), morphology 2 (B),
morphology 3 (C) and morphology 4 (D)
D. frumenti adults have four basic morphologies: black elytra, four yellow spots on
elytra, black head (morphology 1); blackish-brown elytra, four yellow spots on elytra,
brown or yellowish-brown head (morphology 2); brown or blackish-brown elytra, four
invisible spots on elytra, like a bright yellow stripe, yellow or brown head (morphology
3) and brown or blackish-brown elytra, four visible yellow spots, numerous big bright
yellow spots on head (morphology 4) Figure 4.35.
* Investigation on genetic diversity among four morphologies of D. frumenti
samples based on COI gene sequences
The electrophoresis results presented at Figure 4.37 illustrated that the collected
amplification primers of COI gene sequences showed only one line with a size of 648 bp
containing typical sequences for insect nomenclature.

Figure 4.37: PCR Electrophoresis results of D. frumenti samples on gel agarose 1.5%
15


Additionally, the high similarity of the gene sequence proved the morphologies of
lesser coconut weevil collected in the 12 provinces belong to D. frumenti species and
have the same inheritance (Figure 4.38).

Figure 4.38: Similar positions of D. frumenti nucleotide sequences with morphology 1
(KH1), morphology 2 (KH2), morphology 3 (KH3) and morphology 4 (KH4)
16

* Investigation results on D. frumenti genetic diversity by ISSR molecular markers
The process to analyze groups of and construct a genetic linkage map of 40 lesser
coconut weevil samples based on the PCR data indicated that the 40 samples were
classified into four main groups.
Thereby, Group I included two lesser coconut weevil samples of the morphologies
three and four collected in Can Tho (CT4, CT3), group II included four lesser coconut
weevil samples from Hau Giang province with 1,2,3 (HG1, HG2, HG3) morphologies
and morphology 2 collected in Can Tho (CT2). In addition, compositions of lesser
coconut weevil of group III included 20 samples collected in provinces of the Southeast
region (DN4, DN3, DN2, DN1, VT3, VT1, VT4, VT2, TP2, TP3, TP1, BD3, BD2, BD1)
and in 02 provinces of the Mekong Delta, namely Vinh Long and Soc Trang (VL2, VL3,
VL1, ST2, ST3, ST1). The results also revealed that group III was divided into two subgroups with numerous additional ones with closer genetic distances. Of which, ST3 and
ST1 were two samples with the closet genetic distances (3,16). Table 4.7 illustrated
group IV consisting of 14 samples with four sub-groups and CT1 with genetic distances
bigger than those of the remaining 13 samples. The genetic distances among D. frumenti
samples fluctuated from 3.16-8.54. The branching map illustrated the genetic relations
among 40 samples was shown in Figure 4.40.

I

II

III

IV

Figure 4.40: Genetic linkage map to show the genetic relation among 40 D. frumenti
samples in provinces of the Mekong Delta and Southeast region

In general, most of D. frumenti samples collected from a same place were grouped
together. In some cases, there were also presence of other individuals from different
geographical locations possibly due to migration, exchange and transportation of
coconuts carrying individuals which created conditions for genetic exchanges among
17


lesser coconut weevil populations. According to Kerdelhué et al., (2002), a great number
of factors contributed to genetic differences in populations such as disperse capacity,
geographical isolation, influence from living environment or food sources. The study
results indicated that D. frumenti samples were related between genetic and geographical
distances. This was also found in study results of Gadelhak and Enan (2005) on genetic
diversity in Rhynchophorus ferrugineus Olivier populations by RAPD technology in
some regions in Dubai. Moreover, in a study under the ISSR technology, Conotrachelus
humeropictus Fielder species in Amazon grouped together appropriate to their
geographical structure (Souza et al., 2015). Furthermore, genetic changes of Chrysomya
megacephala populations recorded from different areas in Malaysia after being analyzed
by ISSR technology also had a relation to genetic and geographical distances (Chong et
al., 2014). Similarly, study results on genetic diversity based on ISSR technology on
white-back planthopper Sogatella furcifera in China showed such genetic relation (Xie
et al., 2014). Thereby, genetic diversity of D. frumenti is influenced by geographical
distances and ecological conditions.
4.4 Results on collection, isolation, selection and effectiveness evaluation of parasitic
fungi on lesser coconut weevil D. frumenti
* Collection, isolation, selection of parasitic fungi on D. frumenti
Results obtained from collecting fungi parasited on D. frumenti in some provinces
in the Mekong Detal revealed that all M. anisopliae parasitic fungal strains collected in
Soc Trang, Ben Tre and Tra Vinh provinces shared the same characteristics such as green
myceliums, oval spores with 14 strains in Soc Trang, four in Ben Tre and one strain in
Tra Vinh (Figure 4.41).

D
A

C

B

Figure 4.41: M. anisopliae parasitizing D. frumenti (A,B), morphological structure of
conidiophore branching (B) and spores after isolated (C,D.)
* Efficacy of M. anisopliae collected on D. frumenti in the laboratory conditions
Recorded results presented in Table 4.10 showed that at three days after being
treated, death causing effectiveness of green muscardine fungus on lesser coconut weevil
18


adults was not high, no statistical differences among treatments. Of which, treatment
with the highest effectiveness was Ma-BD2-BT (14.2%), while Ma-MCN-BT got the
least effectiveness (6.70%).
Results recorded in Table 4.10 indicated that after three days of treatment, the
efficacy of M. anisopliae on D. frumenti adults is not good enough, only 14.2% for MaBĐ2-BT and 6.70% for Ma-MCN-BT as highest.
Table 4.10: Efficacy of M. anisopliae at the concentration of 108 spores.mL-1 on D.
frumenti adults in the laboratory condition, Can Tho University, 2015
Treatments
Ma-GT-BT
Ma-BD1-BT
Ma-BD2-BT
Ma-MCN-BT
Ma-LP-ST
Control

CV (%)
Significant levels

3
12.5
8.3
14.2
6.7
9.2
0.0
14.5
ns

Corrected effectiveness (%) at the days after treated
5
7
9
11
13
71. 7ab
80.8ab
85.0ab
89.2ab
95.8ab
69.2b
83.3ab
89.2ab
91.7ab
94.2ab
c

c
c
c
35.8
44.2
49.2
57.5
60.8c
bc
b
bc
b
50.0
67.3
71.7
75.8
85.0 b
92.5a
94.2a
96.7a
98.3a
100a
0.0d
0.0d
0.0d
0.0d
0.0d
19.3
17.8
16.2

13.1
9.90
**
**
**
**
**

15
95.8ab
95.0ab
64.2c
89.2b
100a
0.0d
8.90
**

Means within the same column followed by the same letters are not significantly different according to
Duncan’s multiple range test; ns: not significant; **: significant at 1%; Ma: Metarhizium anisopliae

At 5th day after treatment, the efficacy of all treatments increased and was
statistically different compared to the control. From 13 to 15 days after treating, the
treatments had high effective ratios (over 60%). Three treatments gave high controlling
efficacy on D. frumenti adults were Ma-LP-ST (100% at 13 days), Ma-GT-BT (95.8%
at 13 and 15 days) and Ma-BĐ1-BT (95.0% at 15th day after treatment), but not
statistically different. Evidently, green fungus (Ma) was recorded to be able to kill many
insects belonging to Coleoptera order (Zelazny, 1989; Nussenbaum and Lecuona, 2012),
and M. anisopliae was also identified with abilities to parasitize on insects such as
Isoptera, Orthoptera etc. (Pham Thi Thuy, 2004).

* Efficacy of green muscardine fungus and some insecticides on D. frumenti adults
in the laboratory condition
At day one after being treated, the treatments with Ma-GT-BT, Ma-BĐ1-BT and
Ma-LP-ST all did not indicate any efficacy while those with Emamectin benzoate and
Fipronil got high ratios and were significantly different in comparison with the remaining
treatments, with 62.5% and 63.3%, respectively (Table 4.11).

19


Table 4.11: Corrected effectiveness of M. anisopliae and some insecticides on D.
frumenti adults at the time of treatment in the laboratory condition, Can Tho University,
2015
Treatments

Concentrations

Ma-GT-BT
Ma-BD1-BT
Ma-LP-ST
Abamectin
Emamectin benzoate
Fipronil
Control
CV (%)
Significant levels

108 spores/mL
108 spores/mL
108 spores/mL

1.25g/16L
1.25g/16L
1.5g/16L

Effectiveness (%) at days after being treated
1
3
5
7
11
15
0.0c
2.5c
36.8c
44.6bc 55.1bc 66.3b
0.8c
7.5c
11.9d
22.8c 39.8c 60.7b
c
c
cd
0.0
10.0
30.8
53.4bc 58.7bc 63.3b
b
b
b
18.3

52.5
66.6
67.1b 74.5b 77.7b
62.5a
100a
100a
100a
100a 100a
63.3a
100a
100a
100a
100a 100a
c
d
e
d
0.0
0.0
0.0
0.0
0.0d
0.0c
20.8
26.1
20.3
22.1
18.5 24.8
**
**

*
**
**
*

Means within the same column followed by the same letters are not significantly different according to
Duncan’s multiple range test; ns: not significant; **: significant at 1%; *: significant at 5%; Ma: Metarhizium
anisopliae

From three days after being treated, Emamectin benzoate and Fipronil gave highest
effective ratio on D. frumenti adults at 100%, while that of Abamectinonly reached
52.5%. All treatments of muscardine fungus concentrations gave slow efficacy and
reached highest at 15th day after treatment with 60.7 to 66.3% without significant
difference between 3 isolates. Similarly, Tran Van Hai et al. (2009b) also showed that
efficacy of M. anisopliae on Lepidiota cochinchinae causing damages on peanut and
corn roots was of 70.8-79.2% and lasted until 28 days.
* Influence of different concentrations of muscardine fungus on D. frumenti adults
Results in Table 4.12 showed 4 concentrations of the investigated green muscardine
fugus with controlling efficacy on D. frumenti adults. Starting from 5 to 15 days after
being treated, the effective ratios of all concentrations increased.
Table 4.12: Effective ratios of M. anisopliae at different concentrations on D. frumenti
adults at days after being treated in the laboratory condition, Can Tho University, 2015
Treatments
Ma-GT-BT
Ma-GT-BT
Ma-GT-BT
Ma-GT-BT
Control
CV (%)
Significant level

Concentrations
106 spores/mL
107 spores/mL
108 spores/mL
109 spores/mL

Corrected effectiveness (%) at days after being treated
3
5
7
9
11
13
15
3.3b 34.2b 60.0b 70.0b 75.0b 79.2a 80.8a
3.3b 44.7b 64.8b 72.0b 76.7b 79.2a 83.2a
5.8b 46.2b 68.6b 75.5b 77.8b 79.2a 84.3a
10,0a 80.0a 90.0a 93.3a
95.0a
95.0a 96.7a
0.0c
0.0c
0.0c
0.0c
0.0c
0.0b
0.0b
15.2
18.1

16.3
15.8
18.8
16.3
15.9
**
**
**
**
**
**
**

Means within the same column followed by the same letters are not significantly different according to
Duncan’s multiple range test; **: significant at 1%; Ma: Metarhizium anisopliae

20


In the investigated treatments, green muscardine fungal treatment with the
concentration of 109 spores.mL-1 gave the highest efficacy started from five days until
15 days after being treated (96.7%). Three treatments with the concentrations of 10 6, 107
and 108 spores.mL-1 had effective ratios at 80.8%, 83.2% and 84.3%, respectively at the
time of 5 days after being treated. However, the ratios were lower compared to those of
109 spores.mL-1 but had no significant difference since 13 to 15 days after being treated.
Another study (Tran Van Hai et al. (2009c) on efficacy of green muscardine fungal
concentrations on peanut leaf roller Archips micacerana Walker showed the highest
effective ratio from 92.8-94.7% which could last until 17 days after being treated (in the
laboratory condition). The efficacy accordingly tended to increase strongly at 7 th day
after spraying and reached at 80% after 12 days. In addition, Pham Kim Son et al. (2016)

also reported their results on efficacy of M. anisopliae on sweet potato weevil Cylas
formicarius at the concentrations from 106 to 109 spores.mL-1 with the effective ratios
from 88.2% to 100%. This demonstrates that muscardine fungus have potentials to
reduce damages caused by pests.
* Results on the efficacy of green muscardine fungus M. anisopliae and some
insecticides on lesser coconut weevil D. frumenti adults in the field conditions
The results indicated that at 7th day after being treated, damage percentage (%) of
coconut fruits among treatments was similar, with no statistically significant differences.
Until 21 days after being treated, such percentage increased significantly, especially at
controlled treatments, percentage of infected fruits reached 17.4%. Those used Fipronil
revealed remarkably high efficacy and total difference from the remaning treatments (the
lowest percentage of infected fruits was at 2.3%) (Table 4.12).
Table 4.13: Preventive efficacy of M. anisopliae and some insecticides on D. frumenti
adults at days after being treated in the coconut orchards in Giong Trom district, Ben Tre
province
Treatments

Concentrations

Ma-GT-BT
Ma-LP-ST
Emamectin benzoate
Fipronil
Control
CV (%)
Significance level

108 spores/mL
108 spores/mL
1.25 gr/16L

1.5 gr/16L
Water

Infected ratio (%) of coconut fruits at days after
being treated
7
21
35
50
65
1.20
4.20bc
5.95b
7.30b
4.00b
1.80
6.90b
6.80b
9.30ab
5.60b
1.10
9.10b
7.50b
16.5a
14.7a
1.20
2.30c
5.40b
8.5b
11.4a

a
a
a
0.00
17.4
14.6
15.8
11.2a
24.9
23.4
16.4
37.7
34.6
ns
*
**
*
**

Means within the same column followed by the same letters are not significantly different according to
Duncan’s multiple range test; **: significant at 1%; *: significant at 5%; Ma: Metarhizium anisopliae

21


At 35th day after being treated, the percentage of infected coconut fruits at
treatments applying with Ma-GT-BT, Ma-LP-ST, Emamectin benzoate and Fipronil all
reduced and differentiated from the control ones. At 50th day after being treated, the
percentage of fruits damaged by D. frumenti using Ma-GT-BT was the lowest (7.30%)
and differentiated from the controlled ones. Percentages of damaged fruits at two

treatments using Ma-GT-BT and Ma-LP-ST were at 4.00% and 5.60% respectively while
those at treatments using insecticides with Emamectin benzoate and Fipronil got back to
increase. Therefore, the results showed that both insecticides and green muscardine
fungus could reduce the percentage of infected by D. frumenti on fruits in laboratory and
field conditions.
4.5 Results on establishing experimental models to control D. frumenti Fabricius by
environmentally-safety solutions.
*Results on establishing experimental models to control lesser coconut weevil D.
frumenti in Ben Tre province
The model implementation results to control lesser coconut weevil D. frumenti by
environmentally-safe and friendly solutions included cultivation methods and usage of
green muscardine fungus M. anisopliae in Mo Cay Nam district, Ben Tre province were
presented in Table 4.15.
Table 4.15: Ratios of coconut bunches damaged by D. frumenti at the model and
controlled orchards in Mo Cay Nam district, Ben Tre province, 2016
Treatments

Ratios (%) of coconut bunches damaged at investigation periods

Model

BBT
5.30

Control

6.10

18.3

18.6

31.5

37.1

ns

ns

ns

*

*

F test

Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Batch 7 Batch 8
19.4
18.2
20.2
24.2
26.6
24.6
25.5
18.3
50.4
*

58.5

60.9

*

*

50.0
*

ns: no significant difference,*: significant difference at 5% via T-test; Ma: Metarhizium anisopliae, BBT:
before being treated.

The period at which the ratios of damaged coconuts having the significant
difference level at 5% started from the third investigation during the model
implementation. Accordingly, the average ratios of damaged cononut bunches by D.
frumenti fluctuated from 18.3% to 26.6% while those of controlled orchards were 31.5%
to 60.9%.
Results at Table Bảng 4.16 incidated that average ratios of damaged fruits at model
orchards at the 3rd to the 8th record investigation fluctuated from 9.10% to 17.1%,
difference with the significant level at 5% in comparison with controlled orchards (7.6%
to 39.2%). Study ressults of Kunimi (2005), Pham Kim Son et al. (2016) and Tran Thi
22


Thanh (2000) stated that green muscardine fungus also had death causing ability on
numberous pests.
Table 4.16: Ratios of damaged fruits caused by D. frumenti at model and controlled
orchards in Mo Cay Nam district, Ben Tre province, 2016

Treatments

Ratios (%) of coconut fruits damaged at investigation periods

Model

BBT Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Batch 7
3.90 4.60
8.50
10,4
13.3 17.1
15.1
14.9

Batch 8
9.10

Control

5.30

20.9

F test

ns

5.30

5.40

17.6

ns

ns

*

39.2
*

23.6

33.5

*

*

34.8
*

*

ns: no significant difference,*: significant difference at 5% via T-test; Ma: Metarhizium anisopliae, BBT:

before being treated.

- Results on establishing experimental models to control lesser coconut weevil D.

frumenti in Tra Vinh province
Ratios of coconut bunches damaged by D. frumenti at model orchards tended to
decrease gradually and fluctuated from 10.2% to 18.0% at the 6th investigation after
fungus was sprayed. These ratios at controlled orchards were 18.6% to 26.1% with no
differences via statistical analysis. Nevertheless, until the 7th investigation, the ratios of
damaged bunches started showing differences with 4.42% (7th batch), 6.23% (8th batch)
at the model orchards; and these results were 24.5% and 25.2% (Table 4.17), respectively
at the controlled orchards.
Table 4.17: Ratios of coconut bunches damaged by D. frumenti at model and controlled
orchards in Cang Long district, Tra Vinh province, 2017.
Treatments

Ratios (%) of coconut bunches damaged at investigation periods
BBT Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Batch 7 Batch 8

Model

18.0 19.7

18.5

20.2

19.7

13.8

10.2

4.42

6.23

Control

18.6 19.7
ns ns

21.1
ns

22.2
ns

24.0
ns

19.5
ns

26.1
ns

24.5
*

25.2
*

F test

ns: no significant difference,*: significant difference at 5% via T-test; Ma: Metarhizium anisopliae, BBT:

before being treated.

Similarly, investigation results on the ratios of damaged fruits at the model orchards
in Tra Vinh province also had no statistical differences between the model and controlled
orchards at the periods before being treated until the 5 th investigation after being treated.
Ratios of damaged fruits differed from 3.98% to 9.30% (model orchards) and 6.31% to
12.8% (controlled orchards), started showing differences at the 6th, 7th, 8th investigations
with the respective results of 21.5%, 23.9%, 21.3% (controlled orchards) and 3.55%,
2.67%, 2.07% (model orchards) (Table 4.18).
23