insecticidal activity with the renown bioinsecticides
being nicotine, pyrethrum, rotenone, neem and plant
essential oils (Philogene, Regnault-Roger, & Vincent,
2005). Essential oils have been shown to control
stored product pests by fumigant and contact toxicity or as repellents and this maybe by affecting
growth rate and oviposition (Chen, Akinkurolere, &
Zhang, 2011).Thus, essential oils from aromatic plants
such as Pinus caribaea could provide environmentally
friendly alternatives to the currently used synthetic
pesticides (Seixas et al., 2018).
Pinus caribaea Morelet (Pinaceae) is a resinous
woody tree with evergreen needle-like leaves and
grows up to 30 m tall, usually free from branches to
a considerable height (Lee & Lee, 1991). It has many
varieties native to Central America and the Caribbean
(Chowdhury, Bhuiyan, & Nandi, 2008). The plant
bark is grey to reddish-brown. Seeds are usually mottled grey or light brown, narrowly ovoid and about
6 mm long, with a well-developed usually persistent
wing (Stanley & Ross, 1989). On the essential oils
of P. caribaea needles, there are a few reports on
its phytochemical composition (Barnola & Cedeño,
2000; Barnola, Cedeño, & Hasegawa, 1997; Chowdhury et al., 2008; Moronkola et al., 2009; Sonibare &
Olakunle, 2008). Thus, the current study investigated
the chemical constituents of essential oils in fresh needles P. caribaea growing in Buikwe district of Uganda,
EastAfrica and evaluated their potential as insecticides
for control of maize and bean weevils.
2 MATERIALS AND METHODS
2.1 Collection, extraction and phytochemical
analysis of Pinus caribaea needles
Fresh P. caribaea needles (3 kg) were collected from
Ferdsult Pine Forest, Luwombo village, Buikwe district of Uganda from identified cultivated plants.
Needles were collected from various parts of the
crowns to overcome plant plasticity phenomena (Bradshaw, 1965; Roussis, Petrakis, Ortiz, & Mazomenos,
1995). They were identified as P. caribaea Morelet
var. hondurensis by a botanist at Makerere University Herbarium, Kampala (Uganda) where a voucher
sample (No. JM-001) was deposited.
Aliquots (500 g) of crushed needles were hydrodistilled using a modified Clevenger-type apparatus for
3 hrs. The oil was dried using anhydrous sodium sulphate and stored in amber bottles away from UV light
at 4
◦ . Gas chromatography/mass spectrometry analysis was carried out with an Agilent 5975 GC/MSD
system. One mL of the oil was withdrawn by an autosampling system. Innowax FSC column (60 m × 0.25
mm, 0.25 mm) was used with helium as the carrier
gas (0.8 mL/min). GC oven temperature was kept at
60
◦ for 10 minutes, programmed to 220
◦ at a rate
of 4
◦ /min, kept constant at 220
◦ for 10 minutes, and
then programmed to 240
◦ at a rate of 1
◦ /min. Split
ratio was adjusted at 40:1. The injector temperature
was set to 250
◦ . Mass spectra were recorded at 70 eV.
GC analysis was carried out using an Agilent 6890N
GC system. The flame ionization detector temperature was set at 300
◦ . To obtain the same elution order
with GC/MS, simultaneous autoinjection was done on
a duplicate of the same column applying the same conditions described before. Relative percentage amounts
of the separated compounds were calculated from FID
chromatograms. Constituents were identified by comparison of their retention indices with those reported
in literature (Akın, Saraço˘ glu, Demirci, Ba¸ ser, &
Küçüködük, 2012; Baser, Demirci, Kurkcuoglu, Satin,
& Tumen, 2009; Baser, Demirei, Özek, Akalin, &
Özhatay, 2002a; Baser et al., 2002b; Demirci et al.,
2017; Kaya, Benirci, & Baser, 2007; Moronkola et
al., 2009; Saglam, Gozler, Kivcak, Demirci, & Baser,
2001; Tabanca, Demirci, Ozek, Tumen, & Baser,
2001). Matching against libraries such as Adams
Library and those of reference compounds from NIST
web book were done.
2.2 Insecticidal assay
Maize weevils (S. zeamais) and bean weevils (C. maculatus) were obtained from Banda stores, Kyambogo,
Kampala (Uganda) and the colonies were mass reared
on whole maize grains and beans, respectively in glass
containers at optimal growth conditions (ambient temperature of 25–28
◦ C, 60–70% relative humidity) in the
dark at Kyambogo University Chemistry laboratory
for three days prior to the bioassays.
Fumigant toxicity assay
Fumigant toxicity was tested using the method
described by Liu and Ho (1999). Range-finding studies were run to determine the appropriate testing
concentrations. A serial dilution of the essential oil
(four concentrations) was prepared in acetone. Different concentrations of 2, 4, 6, 8 and 10 µL and were
prepared by diluting each quantity in 1 ml acetone.
They were then applied on filter papers separately and
air dried for 10 minutes. Each dried filter paper was
placed at the bottom of a glass jar. Counted 10 adult
maize or bean weevils were placed in muslin clothes
each with 12 g of whole maize grain or bean seeds.
The clothes were tightly closed with rubber bands and
hung at the center of the jars. The control was performed using a filter paper treated with acetone alone.
The experiments stood for five days. Every day, the
clothes were removed from the jars and the number
of dead insects were counted. Each experiment was
replicated three times and averaged.
The percentage mortality (PM) was calculated
using Equation 1 (Asawalam, Emosairue, & Hassanali,
2006; Omara et al., 2018).
PM =
N D
N T
× 100
(1)
Where N D and N T are the number of dead and total
number of test insects per jar, respectively.
163
being nicotine, pyrethrum, rotenone, neem and plant
essential oils (Philogene, Regnault-Roger, & Vincent,
2005). Essential oils have been shown to control
stored product pests by fumigant and contact toxicity or as repellents and this maybe by affecting
growth rate and oviposition (Chen, Akinkurolere, &
Zhang, 2011).Thus, essential oils from aromatic plants
such as Pinus caribaea could provide environmentally
friendly alternatives to the currently used synthetic
pesticides (Seixas et al., 2018).
Pinus caribaea Morelet (Pinaceae) is a resinous
woody tree with evergreen needle-like leaves and
grows up to 30 m tall, usually free from branches to
a considerable height (Lee & Lee, 1991). It has many
varieties native to Central America and the Caribbean
(Chowdhury, Bhuiyan, & Nandi, 2008). The plant
bark is grey to reddish-brown. Seeds are usually mottled grey or light brown, narrowly ovoid and about
6 mm long, with a well-developed usually persistent
wing (Stanley & Ross, 1989). On the essential oils
of P. caribaea needles, there are a few reports on
its phytochemical composition (Barnola & Cedeño,
2000; Barnola, Cedeño, & Hasegawa, 1997; Chowdhury et al., 2008; Moronkola et al., 2009; Sonibare &
Olakunle, 2008). Thus, the current study investigated
the chemical constituents of essential oils in fresh needles P. caribaea growing in Buikwe district of Uganda,
EastAfrica and evaluated their potential as insecticides
for control of maize and bean weevils.
2 MATERIALS AND METHODS
2.1 Collection, extraction and phytochemical
analysis of Pinus caribaea needles
Fresh P. caribaea needles (3 kg) were collected from
Ferdsult Pine Forest, Luwombo village, Buikwe district of Uganda from identified cultivated plants.
Needles were collected from various parts of the
crowns to overcome plant plasticity phenomena (Bradshaw, 1965; Roussis, Petrakis, Ortiz, & Mazomenos,
1995). They were identified as P. caribaea Morelet
var. hondurensis by a botanist at Makerere University Herbarium, Kampala (Uganda) where a voucher
sample (No. JM-001) was deposited.
Aliquots (500 g) of crushed needles were hydrodistilled using a modified Clevenger-type apparatus for
3 hrs. The oil was dried using anhydrous sodium sulphate and stored in amber bottles away from UV light
at 4
◦ . Gas chromatography/mass spectrometry analysis was carried out with an Agilent 5975 GC/MSD
system. One mL of the oil was withdrawn by an autosampling system. Innowax FSC column (60 m × 0.25
mm, 0.25 mm) was used with helium as the carrier
gas (0.8 mL/min). GC oven temperature was kept at
60
◦ for 10 minutes, programmed to 220
◦ at a rate
of 4
◦ /min, kept constant at 220
◦ for 10 minutes, and
then programmed to 240
◦ at a rate of 1
◦ /min. Split
ratio was adjusted at 40:1. The injector temperature
was set to 250
◦ . Mass spectra were recorded at 70 eV.
GC analysis was carried out using an Agilent 6890N
GC system. The flame ionization detector temperature was set at 300
◦ . To obtain the same elution order
with GC/MS, simultaneous autoinjection was done on
a duplicate of the same column applying the same conditions described before. Relative percentage amounts
of the separated compounds were calculated from FID
chromatograms. Constituents were identified by comparison of their retention indices with those reported
in literature (Akın, Saraço˘ glu, Demirci, Ba¸ ser, &
Küçüködük, 2012; Baser, Demirci, Kurkcuoglu, Satin,
& Tumen, 2009; Baser, Demirei, Özek, Akalin, &
Özhatay, 2002a; Baser et al., 2002b; Demirci et al.,
2017; Kaya, Benirci, & Baser, 2007; Moronkola et
al., 2009; Saglam, Gozler, Kivcak, Demirci, & Baser,
2001; Tabanca, Demirci, Ozek, Tumen, & Baser,
2001). Matching against libraries such as Adams
Library and those of reference compounds from NIST
web book were done.
2.2 Insecticidal assay
Maize weevils (S. zeamais) and bean weevils (C. maculatus) were obtained from Banda stores, Kyambogo,
Kampala (Uganda) and the colonies were mass reared
on whole maize grains and beans, respectively in glass
containers at optimal growth conditions (ambient temperature of 25–28
◦ C, 60–70% relative humidity) in the
dark at Kyambogo University Chemistry laboratory
for three days prior to the bioassays.
Fumigant toxicity assay
Fumigant toxicity was tested using the method
described by Liu and Ho (1999). Range-finding studies were run to determine the appropriate testing
concentrations. A serial dilution of the essential oil
(four concentrations) was prepared in acetone. Different concentrations of 2, 4, 6, 8 and 10 µL and were
prepared by diluting each quantity in 1 ml acetone.
They were then applied on filter papers separately and
air dried for 10 minutes. Each dried filter paper was
placed at the bottom of a glass jar. Counted 10 adult
maize or bean weevils were placed in muslin clothes
each with 12 g of whole maize grain or bean seeds.
The clothes were tightly closed with rubber bands and
hung at the center of the jars. The control was performed using a filter paper treated with acetone alone.
The experiments stood for five days. Every day, the
clothes were removed from the jars and the number
of dead insects were counted. Each experiment was
replicated three times and averaged.
The percentage mortality (PM) was calculated
using Equation 1 (Asawalam, Emosairue, & Hassanali,
2006; Omara et al., 2018).
PM =
N D
N T
× 100
(1)
Where N D and N T are the number of dead and total
number of test insects per jar, respectively.
163