Journal of Insect Science, (2019) 19(2): 22; 1–9

doi: 10.1093/jisesa/iez029
Research

Control of Callosobruchus maculatus (Coleoptera:

Chrysomelidae) Using Fractionated Extracts from
Cameroonian Hemizygia welwitschii (Lamiaceae) Leaf on
Stored Vigna unguiculata (Fabales: Fabaceae)
Tagne Gabriel Fotso,1,5 Haman Katamssadan Tofel,2 Jean Pierre Abdou,3 Narcis Tchao,1
Calvain Monglo Zourmba,1 Cornel Adler,4 and Elias Nchiwan Nukenine1
1
Department of Biological Sciences, University of Ngaoundere, Ngaoundere, Cameroon, 2Department of Biological Sciences,
University of Bamenda, Bamenda, Cameroon, 3Center for Research on Medicinal Plants and Traditional Medicine (CRPMT), Institute
of Medical Research and Medicinal Plants Studies, Yaoundé, Cameroon, 4Julius Kühn-Institut, Institute for Ecological Chemistry,
Plant Analysis and Stored Products Protection, Königin-Luise Str.19, D-14195 Berlin, Germany, and 5Corresponding author, e-mail:
gabrielfotso2@yahoo.fr

Subject Editor: Christos Athanassiou

Received 19 December 2018; Editorial decision 10 March 2019

Abstract
Bioassays were conducted to assess the individually insecticidal activities of hexane, acetone, and methanol
extracts from Hemizygia welwitschii Rolfe-Ashby leaves powder against Callosobruchus maculatus (F.).The extracts
were applied at 2, 4, 6, and 10 g/kg of cowpea and the untreated seeds served as negative control. Treatments were
arranged in a complete randomized design with four replications. Adult mortality, F1 progeny emergence, as well as
insect population increase, seeds damage, and seeds germination were carried out. The results obtained showed
that the three extracts of H. welwitschii were very effective in protecting stored cowpea against C. maculatus at the
highest dosage (10 g/kg) 7 d after treatment.There was no F1 progeny emergence of C. maculatus in cowpea treated
with hexane extract at the dosage of 10 g/kg, while, at the same dosage, acetone and methanol extracts almost
completely inhibited the F1 progeny emergence. Also, the different concentration levels significantly protected
the seeds with regard to seed damage caused by C. maculatus compared with the untreated control after 3 mo
storage. The viability of seeds was not affected by the extracts. Because of their effectiveness, the three extracts
of H. welwitschii leaves powder could be a good candidate in pest management programs, especially against
C. maculatus in stored cowpea grains, in Cameroon and other developing countries.

Key words: extract, Hemizygia welwitschii, effectiveness, Callosobruchus maculatus, storage

Résumé
Des bio-essais étaient menés afin d’évaluer individuellement les activités insecticides des extraits à l’hexane, à
l’acétone et au méthanol de la poudre des feuilles de Hemizygia welwitschii, contre Callosobruchus maculatus
(F.) (Coleoptera: Chrysomelidae). Les tests ont été réalisés dans des conditions ambiantes de laboratoire. Les
extraits étaient appliqués aux doses de 2, 4, 6 et 10 g/kg et les grains non traités ont servi de témoin négatif.
Les traitements étaient disposés au laboratoire en blocs complétement randomisés avec quatre répétitions. La
mortalité, l’émergence de la progéniture F1, aussi bien que la croissance de la population et les dégâts des graines,
et la germination des graines étaient évalués. Les résultats obtenus ont montré que les trois extraits de la plante
utilisés étaient très actifs dans la protection du niébé stocké contre C. maculatus à la plus grande dose (10 g/kg)
7 jours après traitement. Il n’avait aucune émergence de la progéniture F1 de C. maculatus dans le niébé traité
avec l’extrait à l’hexane à la plus grande dose de 10 g/kg. Tandis qu’à la même dose, les extraits à l’acétone et au
méthanol ont presque complètement inhibé l’émergence de la progéniture F1. De plus, les différentes doses ont
significativement protégé les graines en ce qui concerne les dégâts des graines causés par C. maculatus par rapport

© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
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2 Journal of Insect Science, 2019, Vol. 19, No. 2

au témoin non traité après trois mois de stockage. La viabilité des graines n’était pas affectée par les extraits. En
raison de leur efficacité, les trois extraits de la poudre des feuilles de H. welwitschii pourraient être des bons agents
de protection dans le programme de gestion des ravageurs spécialement contre C. maculatus dans les graines de
niébé stocké au Cameroun et dans d’autres pays en voie de développement.

Mots clés: Extraits, Hemizygia welwitschii, efficacité, Callosobruchus maculatus, stockage.

About 870 million people in the world are undernourished because type of botanical material vary from place to place, and appear to
of inadequate intake of proteins, vitamins, and minerals in their diets depend partly on the type and efficacy of suitable plants available in
(FAO 2012). Grain legumes are good source of proteins which offer different locations (Chougourou et al. 2015). Plant extracts are com-
a solution to malnutrition. In fact, they are the second most impor- monly referred to as botanicals and are the secondary plant metabo-
tant group of crops worldwide after cereals. Among legumes, cow- lites synthesized by the plants for protective purposes. Some of these
pea serves as a source of dietary protein for human feeding, mostly compounds are toxic to insects. Since the presence of these different
in developing countries where balanced diet is sometimes a problem bioactive compound groups depend to the polarity of solvent used
(Ojo et al. 2013). The dry seed contains about 25% protein and (Mahmoudi et al. 2013, the choice of solvents determines the nature
67% carbohydrate. Cowpea also contains calcium, iron, vitamins, of the compounds present in each extract fraction. In fact, under
and carotene (Adedire et al. 2011). Unfortunately, the grain is exten- the same extraction time and temperature, solvent and composition
sively infested by a number of insect pests including Callosobruchus of sample are known as the most important parameters (Do et al.
maculatus, which is a primary field to store pest causing consider- 2014). Despite local availability of H. welwitschii, this plant is not
ably great losses to farmers (Ojo et al. 2013) both in the field and yet used by farmers in pest control. In view of the alternatives to
storage (Gupta et al. 2011, Thakur 2012). Their larvae bore into reduce conventional insecticides used in stored cowpea, this research
the seed; now, the seed becomes unsuitable for human consumption was designed to study the efficacy of three fractionated extracts from
and losses its viability during planting. To manage this cowpea pest, H. welwitschii leaves powder for controlling C. maculatus.
different methods have been used by the scientists or local farm-
ers. Synthetic insecticides are effective to control stored insect pests
in commercial storage facilities, but their availability and high cost Materials and Methods
limit their utilization by some local African farmers. Despite their
Substrate: Cowpea
positive effect on the pests, they continued to remain hazardous to
The cowpea seeds (Lori variety) used for this study were purchased
man and the environment. With the perspective of providing quality
from the Institute of Agricultural Research for Development
food for population, the interest of researchers has been focused to
(IRAD), Maroua, Cameroun. Broken seeds and particles were
find alternatives to synthetic chemicals which could be environmen-
removed and then disinfested by keeping them in a freezer at −18°C
tally friendly and do not pose hazards to man (Alice and Srikanth
for 30 d prior to bioassays. The cowpea was then kept under ambi-
2013). As such, more attention is reverted to the usage of natural
ent experimental conditions for acclimation at least 2 wk before
products, including plant extracts, powders, ashes, oils, and cow
use. The moisture content (mc) of the seeds was determined accord-
dung to control pests (Naumann and Isman 1995). Some plant mate-
ing to the method used by AFNOR (1982). For that, 10 g (Mo)
rials have played an important part in those traditional methods in
of cowpea were introduced into the oven at 120°C for 24 h; four
Africa where they have been mixed with stored grains and the use
replications were done. After this period, the grains were removed
of these plant products has assumed significance as an important
and reweighted (M1), and the mc was calculated using the follow-
element of insect pest management because of their economic via-
ing formula and it was 9.88%.
bility and eco-friendly nature. They hold promise as alternatives to
chemical insecticides to reduce pesticide load in the environment. Mo − M1
mc ( % ) = × 100
Phytochemicals possess a wide spectrum of biological properties Mo
against insects. Among them, Hemizygia welwitschii, an herba-
ceous and annual plant, is widely present in tropical environment
such as West, East, Central, and parts of southern Africa (Otieno Insect Rearing
et al. 2006). Currently, a total number of 33 species are recognized The original stock was purchased from infested grains in Ngaoundere
in Hemizygia genus, including H. welwitschii (Rolfe) Ashby (Ashby market, Adamawa Region and maintained in the Laboratory of
1935). The genus Hemizygia sp. has a wide range of use. In fact, in Applied Zoology of the University of Ngaoundere Cameroon.
traditional medicine, aqueous extract of H. bracteosa (Benth) has Cowpea weevils were mass reared on whole clean, undamaged, and
shown promise alternative in the treatment of diabetes mellitus in disinfested cowpea in 5-liter transparent plastic jars. This was done
Benin (Chabi et al. 2015). As insecticidal properties, the volatility under ambient laboratory conditions of [t ≈ 23 ± 2.02°C (18.50–
and repellency effect of H. welwitschii essential oil and its formu- 31.50); relative humidity (r.h.) ≈ 63.69 ± 15.17% (21 – 88.5%)],
lations were evaluated against mosquitoes (Oyedele et al. 2000). registered with a thermo-hygrometer EL-USB-2 (RH/Temp Data
In pest control, Fotso et al. (2018) recently shown the efficacy of Logger) (China). Four kilograms of cowpea seeds were introduced
H. welwitschii leaves powder against C. maculatus and Sitophilus into a clean plastic jar and 200 unsexed adults of C. maculatus were
zeamais in stored cowpea and maize, respectively. Their results then added into that jar containing cowpea and kept in the labora-
showed that, at the content 40 g/kg, H. welwitschii caused signifi- tory for 1 mo for the development of the insects. The subsequent
cant mortality to C. maculatus (82.50%) and S. zeamais (81.25%) progenies were used for all experimentations. All insects used for
at 7 and 14 d after exposure, respectively. But the mode of use and these experiments were not more than 2 d old.
Journal of Insect Science, 2019, Vol. 19, No. 2 3

Collection and Preparation of Plant Material F1 Progeny Bioassay

Green leaves of H. welwitschii were collected between August After 7 d mortality recorded, all insects were discarded, and jars
and October 2016 at Ngaoundere, Cameroon (latitude 7°22′′ containing treated and untreated cowpeas were maintained in
North and longitude 13°34′′ East, altitude of 1,100 m a.s.l.), and the same laboratory conditions until progeny emergence. The
shade-dried at the room temperature until they became crisp. The counting of F1 progeny was carried out once a week, for four
identification of the plant was done at the Cameroon National consecutive weeks commencing fourth week after infestation.
Herbarium in Yaounde. The sample was identified in compari- After each counting session, the insects were removed from the
son with voucher specimen of H. welwitschii (Rolfe) M. Ashby jars and recorded. The inhibition rate of F1 progeny (%IR) was
under the serial number 6910/SRFK. The dried leaves were calculated as
hand crushed into powder using locally made pestle and mortar.
Cn − Tn
Powder was stored in a deep-freezer at the temperature of −4°C %IR = × 100
Cn
until needed for extraction.
where Cn is the number of newly emerged insects in the untreated
jar and Tn is the number of insects in the treated jar (Rajashekar
Extraction of the Plant Powder et al. 2010).
The dried leaves powder of H. welwitschii (2.3 kg) was mixed
with 10 liters of hexane (apolar solvent) in the perforated con-
Insect Population Increase and Seed Damage
tainer and agitated for 30 min and allowed to stand for 24 h in
Assessment
the Phytochemistry Laboratory of Institute of Medical Research
Similar doses of each extract for toxicity bioassay were considered
and Medicinal Plants Studies, Yaoundé, Cameroon, and then
for 150 g cowpea grains. A group of 30 unsexed adult insects was
restirred again. The solvent was removed after 48 h by filtration.
introduced into each glass jar containing treated or untreated seeds.
The residue obtained after filtration was put through the above
Control consisted of seeds with respective pure solvent. All treat-
process again and the filtrate was admixed with the one obtained
ments were maintained under ambient laboratory conditions in a
initially. After the hexane extraction was done, the paste left was
complete randomized design with four replications. After 3 mo of
dried for 10 h at room temperature in the laboratory and then
storage, the numbers of live and dead insects were registered for each
used for acetone (intermediate solvent) extraction and followed
jar. Seed damage assessment was performed by counting the dam-
by methanol (polar solvent) extraction. The filtrates obtained
aged and undamaged cowpea seeds. The seed damage percentage
with hexane, acetone, and methanol were then separately con-
was calculated using the following formula:
centrated in a Rotavapor at 70, 60, and 65°C, respectively, at
120 rpm (Kosini et al. 2015). The extract yield of 3.42, 2.42, and Number of seeds damged
6.44% was obtained by using hexane, acetone, and methanol, Seed damage = × 100
Total number of seeds
respectively. Extracts were stored in a refrigerator at 4°C until
needed for bioassays.
Seed Viability Evaluation
To assess the viability of seeds, germination test was conducted
Phytochemical Screening of H. welwitschii Extracts
using 20 undamaged seeds from each jar after the evaluation of
The plant extracts were phytochemically screened using standard
seeds damage. The grains were placed in moistened sand in perfo-
procedures for the detection of phenolic compounds, sterols, sap-
rated plastic plates. Germination was counted and recorded after
onins, glycosides, tannins, flavonoids, terpenoids, and alkaloids as
10 d (Rao et al. 2006, Demissie et al. 2008) and percentage of seed
described in Adeniyi et al. (2010).
germination was thereafter determined according to the following
formula:
Toxicity Bioassay
Four dosages 0.1, 0.2, 0.3, and 0.5 g were introduced in 500 ml Number of germinated seeds
% seed germination = × 100
glass jar containing 50 g of cowpea corresponding to 2, 4, 6, and Total number of seeds
10 g/kg. Untreated controls for each set of treatments consisted of
grains treated with 2 ml of pure respective solvents. Each jar was
then hand-shaken for 2 min to ensure uniform distribution of each Statistical Analysis
extract or pure solvent to the entire seed mass. After this, the glass Data on % cumulative mortality, % reduction of F1 progeny, %
jars containing treated cowpeas or negative control were kept open damaged grains, and % of germination were subjected to the analy-
for 2 h for complete solvent evaporation (Nukenine et al. 2010). sis of variance (ANOVA) procedure using the Statistical Package for
After the solvent evaporation, 20 unsexed adults of C. maculatus the Social Science (SPSS 16.0). Turkey’s test (P = 0.05) was applied
were introduced in each glass jars. Each jar was then covered with for mean separation. A logarithmic transformation (log10(x+1),{10,
cotton clothes to prevent insects from escaping and closed with a subscript, x italics} where x = content in %) was performed before
perforated metal lid for sufficient ventilation. All treatments were regression analysis. Abbott’s formula (Abbot 1925) was used to cor-
maintained under ambient laboratory conditions (t ≈ 24.62 ± rect control mortality when mortality in the control is comprised
1.04°C; r.h. ≈ 76.36 ± 3.23%) in a complete randomized design between 3 and 10% before submission to the ANOVA. Probit anal-
with four replications. Mortality was recorded after 1, 3, 5, and 7 ysis (Finney 1971; SPSS 16.0) was applied to determine lethal con-
d of treatment. The percentage of control mortality was corrected centration causing 50% (LC50) mortality of C. maculatus after 1, 3,
according to Abbot (1925). 5, and 7 d of exposure.
4 Journal of Insect Science, 2019, Vol. 19, No. 2

Table 1. Phytochemical analyses of extracts from Hemizygia wel- extracts at all the exposure periods, except at 5 and 7 d exposure for
witschii leaf powder hexane extract where the R2 values were 0.57 and 0.48, respectively.
Acetone extract within 1 d acted more rapidly (slope = 4.55 ± 0.74)
Compounds Extracts
than methanol extract (slope = 1.18 ± 0.21) and hexane extract
Hexane Acetone Methanol (slope = 1.08 ± 0.42) and 5 d of exposure (slope = 1.25 ± 0.13).
While within 3 d of exposure, the hexane extract acted more rapidly
Phenolic compounds ─ + + (slope = 1.19 ± 0.13) than acetone and methanol extract. In 7 d of
Alkaloids ─ ─ +
exposure, methanol extract acted more rapidly (slope = 1.76 ± 0.14).
Saponins + + +
All the χ2 values were not significant for the three extracts at all the
Tannins ─ + +
exposure periods, except for the hexane extract which was signifi-
Flavonoids ─ + +
Sterols + + ─ cant at 1 and 7 d of exposure.
Terpenoids + ─ ─
Glycosides ─ ─ ─ F1 Progeny Inhibition
The emergence of F1 progeny was significantly affected by extract
─ absent, + present.
treatments compared with untreated control. The number of C. mac-
ulatus progeny emerged in the untreated control was significantly
Results higher (number) than in the treated cowpea grains (236 adults for
hexane, 292.75 adults for acetone, and 109.75 adults for metha-
Phytochemical Constituents of H. welwitschii nol) (Table 3). Classification in terms of efficacy for each extract in
Extracts progeny reduction on the treated cowpea seeds from the content of
Important phytochemicals, such as phenolic compounds, alkaloids, 4 g/kg was as follows: hexane extract > methanol extract >> ace-
terpenoids, sterols, tannins, saponins, and flavonoids, were screened tone extract. At 4 g/kg, the number of F1 progeny was 8.00, 11.00,
for their presence in H. welwitschii leaves powder using hexane and 28.25, respectively, for hexane, methanol, and acetone extracts.
(apolar), acetone (intermediate), and methanol (polar) as presented At the lower concentration (2 g/kg), the classification of extracts
in Table 1. The qualitative phytochemical studies indicate that sap- in terms of progeny inhibition was as follows: methanol extract >
onins, sterols, and terpenoids groups are found in hexane extract, hexane extract > acetone extract with, respectively, 19.50, 19.75,
whereas phenolic compounds, saponins, tannins, flavonoids, and and 36.75 C. maculatus emerged. Complete percentage in terms
sterols groups were present in the acetone extract. Concerning meth- of inhibition rates of C. maculatus adult was obtained by hexane
anol extract, the chemical groups which were present are phenolic extract from the dosage of 6 g/kg, while with methanol extract the
compounds, alkaloids, saponins, tannins, and flavonoids. It is noted percentages of reduction were 95.68 and 98.40% at 6 and 10 g/kg,
that alkaloids were exclusively present in the methanol extract. respectively. At the highest dosage (10 g/kg), acetone extract almost
achieved complete inhibition of F1 progeny (99.06%) (Table 3).
Adult Mortality
Adult mortality of C. maculatus caused on treated cowpeas by each Population Increase and Grain Damage
leaf extract of H. welwitschii is presented in Fig. 1. The results show The results of Table 4 indicate significant decrease of number of live
that extracts were toxic to C. maculatus adults, and this varies with insects among the three extracts tested compared with the untreated
ascending concentrations and exposure times. The highest concen- control. At the lowest dosage (2 g/kg), acetone extract recorded less
tration (10 g/kg) of each extract of H. welwitschii within 1 d caused live insects (13.25) compared with hexane and methanol extracts
less than 15% mortality of C. maculatus. At this same time point, with 19.00 and 52.00 live insects, respectively. The hexane extract
mortality recorded in cowpea seeds treated with acetone extract was of H. welwitschii possesses the strongest effect against C. macula-
lower and had no significant effect on adult mortality compared with tus population growth with no live insects recorded at the highest
untreated control. Within 3 d of exposure, only the hexane extract dosage of 10 g/kg, followed by acetone extract (2 living insects) and
recorded more than 50% of mortality at the highest dosage of 10 g/ methanol extract (14.25 living insects). The dead insects recorded in
kg. At this same dosage and same time point, acetone and meth- cowpea treated with hexane and acetone significantly decreased with
anol extracts recorded 38.75 and 32.50% mortality, respectively. the dosages compared with control. At the highest dosage (10 g/kg),
After 7 d of exposure, the mortality of C. maculatus increases sig- hexane extract killed all the 30 insects introduced at the beginning of
nificantly with concentrations (hexane extract: F = 1766.55; df = 4, the experiment; acetone extract recorded 66.50 dead insects, while
15; P < 0.001; acetone extract: F = 397.37; df = 4, 15; P < 0.001; 167.27 dead insects were observed in cowpea treated with methanol
methanol extract: F = 1197.40; df = 4, 15; P < 0.001). Treatments extract.
with hexane extract at the rate of 10 g/kg caused complete mortal-
ity within 7 d after treatment application. However, the mortality Seeds Damage
of C. maculatus recorded with methanol and acetone extracts, at Figure 2 shows that all treatments were effective to protect cowpea
this same exposure period and same dosage was 94.03 and 81.62%, against the attack of C. maculatus and it is dose-dependent (dam-
respectively. aged seeds decrease significantly with an increase of dosages (hexane
Table 2 shows the mortality parameters of C. maculatus. For 3, extract: F = 591.98; df = 4, 15; P < 0.001; acetone extract: F = 18.87;
5, and 7 d after exposure, hexane extract was more toxic to the df = 4, 15; P < 0.001; methanol extract: F = 7.12; df = 4, 15;
bruchids. The lowest LC50 was obtained with the three extracts on P < 0.001). Higher seeds damage was recorded in untreated control
cowpea within 7 d of exposure. The hexane extract (LC50 = 0.31 g/kg) (up to 39.38% seeds damage). Among the three extracts tested for
was the most toxic to the bruchids than acetone (LC50 = 1.53 g/kg) their efficacy against C. maculatus in stored cowpea grains, hexane
and methanol (LC50 = 1.87 g/kg) extracts, respectively. Concerning extract of H. welwitschii leaves powder was found to be the most
the R2 values, they ranged between 0.65 and 0.89 for the three protective by recording no seeds damaged at highest tested contents
Journal of Insect Science, 2019, Vol. 19, No. 2 5

2 g/Kg 4 g/kg
Untreated control
100 Hexane extract 100 D
D
Acetone extract
Metahnol extract
80 80
C
C DD

Mortality (%)
Mortality (%)

60 DD 60 C
C
C
C
40 40
B
BB
BB B
20 20

A A A A
A A
0 0
0 1 3 5 7 0 1 3 5 7
Exposure periods (Days) Exposure periods (Days)

6 g/kg 10 g/kg
D 100 D
100 D
C D
C D
80 C 80 C
C C
Mortality (%)
Mortality (%)

C
60 60 B

B B
40 40
B B
B

20 20 A
A
A A A
A
0 0
0 1 3 5 7 0 1 3 5 7
Exposure periods (Days) Exposure periods (Days)

Fig. 1. Corrected cumulative mortality of Callosobruchus maculatus exposure to hexane, acetone and methanol leaf extracts of Hemizygia welwitschii.

of 10 g/kg. While at the same dosage, acetone and methanol extracts with hexane extract (47.50%) at the dosage 4 g/kg and the cowpea
recorded 6.12 and 6.94% damaged seeds, respectively. seeds treated with methanol extract (41.25%) at the lowest tested
content of 2 g/kg.

Germination Rate
The results obtained in this study for the viability test showed that
the germination of cowpea seeds was influenced differently by con- Discussion
centrations and extract types of H. welwitschii (Fig. 3). There is Phytochemical investigation of the extracts from the plant used in
significant difference of germination rate between the control and the present study showed that it is rich in many active compounds
treated seeds (hexane extract: F = 4.86; df = 4, 15; P < 0.05; ace- including phenolic compounds, alkaloids, saponins, tannins, triter-
tone extract: F = 142.30; df = 4, 15; P < 0.001; methanol extract: penes, flavonoids, and sterols groups. The presence of these different
F = 18.69; df = 4, 15; P < 0.001). Germination rate of cowpea seeds in compound groups depend on the polarity of the solvent used. In fact,
treatments ranged from 28.75% in the untreated control to 47.50% the solvent polarity used and the solubility determined the nature
with hexane extract, 13.75% in the untreated control to 60% with of the compounds present in each plant extract (Mahmoudi et al.
acetone extract, and 15.00% in the untreated control to 41.25% 2013). Currently, many farmers in Africa and Asia are using botan-
with methanol extract. The highest percentage of germination was icals to protect their legumes from attack by bruchids, with varying
obtained in cowpea seeds treated with acetone extract (60%) at its success degrees (Chellapa and Chelliah 1976), due to the type of
highest tested content (10 g/kg), followed by cowpea seeds treated formulations and the commodities used among others. Results from
6 Journal of Insect Science, 2019, Vol. 19, No. 2

Table 2. Toxicity parameters of adult Callosobruchus maculatus in grains treated with different extracts from H. welwitschii leaf powder

Products N Slope ± SE R2 LC50 (95% FL) (g/kg) χ2

1d

Hexane extract 20 1.08 ± 0.42 0.81 123.22 (52.12–806.22) 15.07**

Acetone extract 20 4.55 ± 0.74 0.84 19.04 (15.66–27.53) 17.31ns
Methanol extract 20 1.18 ± 0.21 0.84 91.07 (44.08–395.70) 15.51ns
3d
Hexane extract 20 1.19 ± 0.13 0.89 8.93 (7.64–11.05) 5.10ns
Acetone extract 20 0.67 ± 0.13 0.76 25.86 (15.69–75.77) 3.18ns
Methanol extract 20 0.49 ± 0.13 0.69 83.74 (30.12 ± 2234.77) 4.17ns
5d
Hexane extract 20 0.71 ± 0.13 0.57 0.51 (0.14–0.94) 6.73ns
Acetone extract 20 1.25 ± 0.13 0.78 2.88 (2.43–3.28) 8.34ns
Methanol extract 20 0.71 ± 0.12 0.65 2.36 (1.54–3.03) 7.28ns
7d
Hexane extract 20 1.56 ± 0.25 0.48 0.31 (0.03–0.69) 27.12*
Acetone extract 20 1.08 ± 0.13 0.69 1.53 (1.06–1.94) 9.76ns
Methanol extract 20 1.76 ± 0.14 0.78 1.87 (1.43–2.24) 23.27ns

ns
P > 0.05, nsP < 0.05, *P < 0.05, **P < 0.01.

Table 3. Progeny production of Callosobruchus maculatus in C. maculatus is in accordance with the previous works done by
grains treated with three extracts of Hemizygyia welwitschii leaf Kosini and Nukenine (2017), which reported similar outcome on
powder under ambient laboratory conditions C. maculatus with Gnidia kaussiana hexane extract at 5 g/kg within
Products Mean number of F1 % reduction in adult emergence 2 d of exposure. The results obtained in our study also agree with the
Contents adult progeny relative to control findings of Mahama et al. (2018) and those of Danga et al. (2015).
(g/kg) In the findings of these authors, hexane extract was also the most
effective product against C. maculatus among the plant extracts they
Hexane used. In addition to the presence of saponins and sterols, H. wel-
extract witschii hexane extract contains other secondary metabolites like
0 236.00 ± 10.73c 0.00 ± 0.00a
triterpenoids, which exhibit antifeedant and/or lethal contact actions
2 19.75 ± 2.22b 91.62 ± 0.94b
on insect. This can explain the high activity of hexane extract from
4 8.00 ± 2.5a 96.60 ± 0.62c
H. welwitschii leaves powder. In fact, Kostyukovsky et al. (2002)
6 2.50 ± 3.00a 98.92 ± 1.28d
10 0.00 ± 0.00a 100.00 ± 0.00d reported that terpenoids cause symptoms that suggest a neurotoxic
F(4, 15) 1,599.18*** 12,967.22*** mode of action of these secondary metabolites which was observed
Acetone to C. maculatus exposed to orange peel essential oil-treated seeds.
extract However, before the current study, Fotso et al. (2018) recently shown
0 292.75 ± 0.96e 0.00 ± 0.00a the efficacy of H. welwitschii leaves powder against C. maculatus
2 36.75 ± 1.50d 87.44 ± 0.54b and Sitophilus zeamais in stored cowpea and maize, respectively.
4 28.25 ± 1.50c 90.35 ± 0.49c These authors recorded significant mortality (82.50%) of C. macu-
6 11.00 ± 1.41b 96.24 ± 0.49d
latus at the highest dosage (40 g/kg), in 7 d after exposure caused by
10 2.75 ± 2.21a 99.06 ± 0.75e
H. welwitschii leaves powder. The present study demonstrated the
F(4, 15) 24,477.28*** 26,068.39***
potential of using H. welwitschii leaves powder extracts to control
Methanol
extract C. maculatus in stored cowpea but with the high effect pronounced
0 109.75 ± 0.96d 0.00 ± 0.00a when compared with H. welwitschii leaves powder. In fact, in this
2 19.50 ± 3.00c 82.22 ± 2.76b finding, the hexane and methanol extracts recorded more than 90%
4 11.00 ± 1.41b 89.98 ± 1.25c of C. maculatus which are more effective than H. welwitschii leaves
6 4.75 ± 0.96a 95.68 ± 0.85d powder plant extract. The effectiveness of the plant extracts indi-
10 1.75 ± 0.50a 98.40 ± 0.46d cates a possible contact action of the active constituents of H. wel-
F(4, 15) 3,158.53*** 3,388.60*** witschii with the bruchids. In fact, volatile compounds of plant
extracts contain many bioactive molecules, which may have contact
Means within the column followed by the same small letter do not differ
and fumigant properties. The efficacy of these plant extracts could be
significantly at the 5% level according to Tukey test. *** P < 0.001.
attributed to the presence of phytochemical secondary metabolites
present in each extract. Indeed, the plant secondary metabolites are
this study showed that H. welwitschii leaves powder extracts also responsible for diverse activities including their insecticidal proper-
have significant insecticidal effects against C. maculatus which vary ties (Rubabura et al. 2014). This shows that H. welwitschii leaves
with the contents, exposure periods, and extracts. Among the tested powder extracts are highly effective in controlling bruchids infes-
extracts in the present investigation, it was found that the H. wel- tations, which could be due to the presence of some bioactive com-
witschii hexane extract appeared to be more toxic to C. maculatus at pounds, including phenolic compounds, alkaloids, saponins, tannins,
its highest treatment (10 g/kg) than methanol and acetone extracts, flavonoids, triterpenoids, and sterols. Aniszewski (2007) found that
respectively in 7 d of exposure. This result on adult mortality of alkaloids are the toxic secondary metabolites which can block ion
Journal of Insect Science, 2019, Vol. 19, No. 2 7

Table 4. Population increase of Callosobruchus maculatus in cowpea treated with of Hemizygia welwitschii leaf extracts and stored for
3 mo

Insects

Products Number of insects (means ± standard error)

Contents (g/kg) Dead insects Live insects Total insects

Hexane extract Callosobruchus maculatus
0 244.00 ± 4.76c 207.50 ± 5.74d 451.50 ± 1.91d
2 205.00 ± 49.89c 19.00 ± 5.22c 224.50 ± 44.78c
4 114.00 ± 8.98b 9.75 ± 0.96b 123.75 ± 8.26b
6 74.25 ± 5.38ab 6.75 ± 1.26ab 81.00 ± 4.24b
10 30.00 ± 0.00a 0.00 ± 0.00a 30.00 ± 0.00a
F(4,15) 61.03*** 2,526.37*** 265.16***
Acetone extract
0 290.25 ± 65.27c 140.50 ± 12.50b 430.75 ± 64.39c
2 213.25 ± 76.42bc 13.25 ± 3.30a 223.50 ± 80.04b
4 121.75 ± 57.95ab 4.25 ± 3.77a 126.00 ± 56.12ab
6 109.25 ± 38.87ab 3.75 ± 2.63a 113.00 ± 41.00ab
10 66.50 ± 26.45a 2.00 ± 2.16a 68.50 ± 27.96a
F(4,15) 10.40*** 377. 79*** 25.93***
Methanol extract
0 197.50 ± 79.94a 98.50 ± 6.76c 296.00 ± 83.29a
2 175.75 ± 78.64a 52.00 ± 15.56b 227.75 ± 78.53a
4 131.00 ± 43.41a 22.00 ± 4.08a 153.00 ± 44.43a
6 83.00 ± 17.26a 17.75 ± 3.03a 100.75 ± 19.72a
10 167.25 ± 49.31a 14.25 ± 2.87a 181.50 ± 50.84a
F(4,15) 0.52ns 77.97*** 1.42ns

Means within the column and line followed respectively by the same small letter do not differ significantly at the 5% level according to Tukey’s test.
ns
P < 0.05; ***P < 0.001.

60 doses of applied substances, which lead to complete or almost complete

b
Hexane extract efficacy, with no variation in the insect responses (mortality). Therefore,
Acetone extract the botanical extracts were greatly responsible for the responses of
50 C. maculatus on the treated cowpea. The values of χ2 were generally not
Methanol extract
significant for all the three extracts, implying that the obtained regres-
Seeds damaged (%)

40 sion models approximate the theoretical model, concerning the toxicity

c of the used substances to C. maculatus (Finney 1971).
The H. welwitschii leaves powder extracts also reduced sig-
30 e ab nificantly the production and inhibited F1 progeny emergence of
ab
d b C. maculatus. The same tendencies were recorded by other authors
and plants extracted with the same solvents used in this investiga-
20
a a tion. Mahama et al. (2018) reported similar result with a significant
c a
reduction in the progeny F1 production of C. maculatus on seeds
b a a
10 treated with Eucalyptus camaldulensis leaf extracts on Bambara
groundnut grains. Reduction in the F1 progeny emergence of C. mac-
a ulatus in the Bambara groundnut treated with Ocimum canum Sims
0
leaf extract fractions was also obtained by Kosini et al. (2015).
0 2 4 6 10
Chudasama et al. (2015) stated that the reduction in adult emer-
Contents (g/kg) gence could either be due to egg mortality, larval mortality, or even
reduction in hatching of the eggs. This shows that H. welwitschii
Fig. 2. Percentage of seeds damaged by Callosobruchus maculatus on
leaves extracts probably have oviposition deterrent, ovicidal, and
cowpea treated with Hemizygia welwitschii leaf extracts and stored for 3 mo.
lavicidal properties. The highest percentage of inhibition of C. mac-
ulatus F1 progeny could be attributed to the highest mortality of the
channels, inhibit enzymes, or interfere with neurotransmission, loss adult C. maculatus in the mortality test observed at the last day of
of coordination, and death. Alkaloid compounds were found only exposure at these concentrations. According to Hertlein et al. 2011,
in methanol extract of H. welwitschii, which could be responsible effective control of protectants is attributed to the mortality of adult
for higher mortality caused by methanol extract than that caused by and/or immature stages, confirmed by lack of progeny generation.
acetone extract in this study. Grain damage indicated the quantitative loss in stored grains due
Faraway (2002) reported that in the Biological Sciences when the to insect feeding showing a direct relationship between insect pop-
coefficient of determination R2 ≥ 0.6, then the favorable results are attrib- ulation and grains damage. In our findings, all the extracts provide
utable to the products used. In the present study, most of the R2 ≥ 0.6. a significant reduction in seed damage activity compared with the
The few smaller values of coefficient of determination are linked to high untreated seeds. These effects resulted in reduced weight; especially,
8 Journal of Insect Science, 2019, Vol. 19, No. 2

70 product protection by the farmers who store cowpea for consumption

e and planting, and industrial level.
60
b
Seeds germination (%)

d
Acknowledgments
50
c Some of the equipments used to carry out this research were purchased with
ab c
the financial support of the Alexander-von-Humboldt Stiftung, Bonn, Germa-
40 ab ny, under grant no. 3.4-B151/11016 (Equipment Grant) to E.N.N., which we
b a
a gratefully acknowledged. Also, we are thankful to the Deutsche Akademischer
b
30 b b Austauschdienst (DAAD) for providing small equipment grants to K.H.T. We
would like to thank Drs G. Agbor and Tchinda of the Medical Research In-
stitute and Studies of Medicinal Plants (IMPM), Yaounde, Cameroon for their
20 assistance in extracting botanical insecticides.
a a

10
References Cited
0 Abbot, W. 1925. A method of computing the effectiveness of an insecticide. J.
Econo. Entomol. 18: 265–267.
0 2 4 6 10
Adedire, C. O., O. O. Obembe, R. O. Akinkurolele, and O. Oduleye. 2011.
Contents (g/kg)
Response of Callosobruchus maculatus (Coleoptera: Chysomelidae:
Hexane extract Bruchidae) to extracts of cashew kernels. J. Plant Dis. Prot. 118: 75–79.
Acetone extract Adeniyi, S. A., C. L. Orjiekwe, J. E. Ehiagbonare, and B. D. Arimah. 2010.
Methanol extract Preliminary phytochemical analysis and insecticidal activity of etha-
nolic extracts of four tropical plants (Vernonia amygdalina, Sida acuta,
Fig. 3. Seed viability of cowpea treated with three extracts from Hemizygia Ocimum gratissimum and Telfaria occidentalis) against beans weevil
welwitschii leaves powder after 3 mo storage. (Acanthscelides obtectus). Int. J. Phys. Sci. 5: 753–762.
Association Française de Normalisation (AFNOR). 1982. Recueil des normes
françaises des produits dérivés des fruits et légumes. Jus de fruits. p. 27.
in the control seeds and the feeding activities of C. maculatus; in 1st edn. Paris, France.
particular, resulted in holes in the seeds. Alice, J., and N. Srikanth. 2013. Non destructive method to manage the
The percentage seed germination recorded in the present study most dreaded pest Callosobruchus maculatus (Fab.) in Black gram Vigna
showed that there was a significant difference (P > 0.05) compared mungo L. J. Stored Prod. Post. Res. 4: 30–34.
with the control. This may be that the three extracts have no effect on Aniszewski, T. 2007. Alkaloids–secrets of life: alkaloid chemistry, biological signifi-
the seeds viability. This result is similar with the work of Umar (2008) cance, applications and ecological role, pp. 185–186. Elsevier, Amsterdam.
who reported that the cowpea seeds remained viable which were treated Ashby, M., 1935. The genus Hemizygia Briquet. J. Botanic (London). 81: 653–686.
Chabi, N. W., C. T. R. Konfo, M. Adjagba, L. Moussedikou, E. Ahoussi-
with Jatropha curcas powder. The same tendency was also reported by
Dahouenon, A. Laleye, C. Gbaguidi, and M. M. Soumanou. 2015.
Ojianwuna et al. (2014), which reported highest germination rate of
Evaluation of the toxicity of Hemizygia bracteosa (Benth) plant used
cowpea viability when treated with crude extracts of Ocimum suave leaf
in traditional medicine for the treatment of diabetes mellitus in Benin.
oil. Our studies show that H. welwitschii leaf extracts cannot also have American J. Biomed. Res. 3: 40–44.
a negative effect on seed viability. But the lowest percentage of germina- Chellapa, K., and S. Chelliah. 1976. Preliminary trials with oil emulsions for
tion recorded in this study could be attributed to some factors such as the control of insect pests. Madras Agri. J. 63: 10–11.
environmental conditions and immature stages of bruchids. According Chougourou, C. D. Y. A. Zoclanclounon, A. Agbaka, and A. Togola 2015.
to Couturon (1980), when environmental conditions are not well con- Toxicity of two plant powders as biopesticides in the management of
trolled, germination rate decreases quickly. The lowest percentage of Callosobruchus maculatus F. (Coleoptera: Chrysomelidae, Bruchinae) on
germination recorded in this study could be related to the development two stored grain legumes. J. Applied Biosci. 86: 7900–7908.
Chudasama, J. A., N. B. Sagarka, and S. Sharma, 2015. Deterrent effect of
of bruchids, which emerged at these level contents by feeding on seed
plant extracts against Callosobruchus maculatus on stored cowpea in
germ, which prevents germination and reduces germination rate. Also,
Saurashtra (Gujarat, India). J. Applied and Natural Sci. 7: 187–191.
the seeds used for germination were nonperforated but they would con-
Couturon, E. 1980. Le maintien de la viabilité des graines de caféiers Par
tain the immature stages of bruchids that could destroy the parts of seed le contrôle de leur teneur en eau et de la température de stockage. Café
embryo. Cacao Thé. 24: 27–32.
The finding obtained in this study revealed that, all the three Danga, S. P. Y., E. N. Nukenine, L. Younoussa, C. Adler, and C. O. Esimone.
solvent extracts were toxic against C. maculatus adults in the pro- 2015. Efficacy of Plectranthus glandulosus (Lamiaceae) and Callistemon
tection of cowpea (Vigna unguiculata) and having significant inhi- rigidus (Myrtaceae) leaf extract fractions to Callosobruchus maculatus
bition of F1 progeny emergence in treated grain. Among the extracts (Coleoptera: Bruchidae). J. Insect Sci. 15: 1–6.
tested, hexane extract showed best result as insecticidal product on Demissie, G., T. Tefera, and A. Tadesse. 2008. Efficacy of SilicoSec, filter cake
and wood ash against the maize weevil, Sitophilus zeamais Motschulsky
reduction in stored cowpea damage caused by C. maculatus. With
(Coleoptera: Curculionidae) on three maize genotypes. J. Stored Prod. Res.
the three extracts, the seeds did not lose their viability. Therefore,
44: 227–231.
the use of H. welwitschii leaves powder extracts as insecticide of
Do, Q. D., A. E. Angkawijaya, P. L. Tran-Nguyen, L. H. Huynh, F. E. Soetaredjo,
plant origin in postharvest protection should be encouraged in S. Ismadji, Y.-H. Ju 2014. Effect of extraction solvent on total phenol con-
stored cowpea management because of its great toxicant poten- tent, total flavonoid content, and antioxidant activity of Limnophila aro-
tial against cowpea weevils in storage. However, further research matic. J. Food and Grud Analysis. 22: 296–302.
is required to study the active ingredients of this plant, mode of Food and Agriculture Organization (FAO). 2012. The State of Food Insecurity
action, and consumer safety, before promoting their use in stored in the World. Executive summary report. Rome, Italy. p. 2.
Journal of Insect Science, 2019, Vol. 19, No. 2 9

Faraway, J. J. 2002. Practical Regression and Anova using R. http://www- Nukenine, E. N., C. Adler, and C. Reichmuth. 2010. Bioactivity of fen-
cranr-projectorg/doc/contrib/ Faraway-PRApdf chone and Plectranthus glandulosus oil against Prostephanus trunca-
Finney, D. J. 1971. Probit analysis. Cambridge University Press, London. tus and two strains of Sitophilus zeamais. J. Applied Entomol. 134:
Fotso, T. G., E. N. Nukenine, R. Tchameni, J. W. Goudoungou, D. Kosini, 132–141.
V. Tigamba and C. Adler. 2018. Use of Cameroonian Hemizygia wel- Ojianwuna, C. C., P. A. Umoru, and J. Ugbebor. 2014. Toxicity of crude
witschii Rolfe Ashby (Lamiaceae) leaf powder against Callosobruchus extracts of Ocimum suave leaf oil on cowpea weevil (Callosobruchus
maculatus and Sitophilus zeamais. J. Entomol. Zool. Stud. 6: 1261–1269. maculatus) (f) (Coleoptera; Bruchidae) and test of cowpea seeds’ viability.
Gupta, K., B. Shashi, L. Beche, L. K. Manju, S. Charan, B. Ruquaeya, Biores. Bull. 3: 1–5.
S. Subadas, T. Manju, and R. K. Khetarpal. 2011. Interception of insect Ojo, J. A., A. A. Olunloyo, and E. O. Akanni. 2013. Efficacy of Moringa oleifera leaf
pests in exotic seed germplasm. Ind. J. Agri. Sci. 81: 100–103. powder against Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae)
Hertlein, M. B., G. D. Thompson, Bh. Subramanyam, and C. G. Athanassiou. on stored cowpea (Vigna unguiculata L. Walp). Res. 5: 240–244.
2011. Spinosad: a new natural product for stored grain protection. J. Otieno, D. F., K. Balkwill, and A. J. Paton. 2006. A multivariate analysis of
Stored Prod. Res. 47: 131–146. morphological variation in the Hemizygia bracteosa complex (Lamiaceae,
Kosini, D., and E. N. Nukenine. 2017. Bioactivity of novel botanical insec- Ocimeae). Plant Systematics Evol. 261: 19–38.
ticide from Gnidia kaussiana (Thymeleaceae) against Callosobruchus Oyedele, A. O., L. O. Orafidiya, A. Lamikanra, and J. I. Olaifa, 2000. Volatility
maculatus (Coleoptera: Chrysomelidae) in stored Vigna subterranea and mosquito repellency of Hemizygia welwitschii oil and its formula-
(Fabaceae) grains. J. Insect Sci. 17: 31; 1–7. tions. Insect Sci. Applied. 20: 123–128.
Kosini D., E. N. Nukenine, and K. H. Tofel. 2015. Efficacy of Cameroonian Rajashekar, Y., N. Gunasekaran, and T. Shivanandappa. 2010. Insecticidal
Ocimum canum Sims (Lamiaceae) leaf extract fractions against activity of the root extract of Decalepis hamiltonii against stored-product
Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae), infesting insect pests and its application in grain protection. J. Food Sci. Technolo.
Bambara groundnut. J. Entomol. Zool. Stud. 3: 487–494. 43: 310–314.
Kostyukovsky, M., A. Rafaeli, C. Gileadi, N. Demchenko, and E. Shaaya, Rao, N. K., J. Hanson, M. E. Dulloo, K. Ghosh, D. Nowell, and M. Larinde.
2002. Activation of octopaminergic receptors by essential oil constituents 2006. Manuel de manipulation des semences dans les banques de gènes.
isolated from aromatic plants: possible mode of action against insect pests. Manuels pour les banques de gènes No. 8. Biodiversité Internationale,
Pest Manag. Sci. 58: 1101–1106. Rome, Italie. p. 165.
Mahama, A., C. Saidou, H. K. Tofel, A. Ali, M. B. Adji, and E. N. Nukenine. Rubabura, K., M. Nsambu, B. Muhigwa, M. Bagalwa, and S. Bashwira. 2014.
2018. Efficacy of Eucalyptus camaldulensis leaf extracts against the pea Evaluation in vitro activity of insect alkaloid, saponins, terpenoids or ster-
beetle Callosobruchus maculatus and their impact on biochemical and oids extracts capscicum frutescens L. (Solanaceae) against Antestiopsis
microbiological properties of the treated Bambara groundnut grains. J. orbitalis ghesquierei, pests of coffee trees. Intern. J. Inno. Applied Stud.
Entomol. Zool. Stu. 6: 869–877. 8: 1231–1243.
Mahmoudi, S., M. Khali, and N. Mahmoudi. 2013. Etude de l’extraction des Thakur, D. R. 2012. Taxonomy, distribution and pest status of Indian biotypes
composés phénoliques de différentes parties de la fleur d’artichaut (Cynara of Acanthoscelides obtectus (Coleoptera: Chrysomelidae: Bruchinae). A
scolymus L.). Nat. Technolo. 9: 35–40. New Recorded. Pak. J. Zool. 44: 189–195.
Naumann, K., and M. B. Isman. 1995. Evaluation of neem (Azadirachta indica Umar, Y. F. 2008. Comparative potentials of leaf, Bark and wood pow-
A. Juss.) seed extracts and oils as oviposition deterrents to noctuid moths. ders of Joctropha cuncas (L) as protectants of stored cowpea against
Entomol Exp Applied. 76: 115–120. Callosobruchus maculatus (f). Savannah J. Agri. 3: 86–92.