RESEARCH REPORT


ISJ 11: 66-72, 2014                                                                  ISSN 1824-307X 
 
 

RESEARCH REPORT 
 
Determination of lipase activity in the larval midgut of Bacterocera oleae Gmelin 
(Diptera: Tephritidae) 
 
S Delkash-Roudsari1, A Zibaee1, MR AbbaciMozhdehi2
 
1Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Rasht 41476-1314, Iran 
2Agricultural and Natural Resources Center, Rasht, Iran 
 
 

   Accepted February 7, 2014 

 
Abstract 

In the current study, digestive lipase activity was determined and characterized in the third larval 
instars of olive fly, Bactericera oleae as the first time in dipteran order. By using two sample fractions, 
it was found that the enzyme had higher activity in membrane-bound fraction than that of soluble 
fraction. Optimal pH of soluble lipase was found to be 4 and 6 but membrane-bound lipase showed pH 
4 as optimal value. Optimal temperatures for soluble and membrane-bound lipase were obtained to be 
35 and 50 °C, respectively. Activities of digestive soluble and membrane-bound lipases decreased by 
using various mono- and di-valent ions. Since, fruits of olive are full of various oils, digestive lipases of 
B. oleae larvae have critical role in their digestion. So, these enzymes might be a good target for 
developing inhibitors and resistant varieties. 
 
Key Words: Bacterocera oleae; digestive lipase; characterization 

 

 
Introduction 

 
Bacterocera oleae (Diptera: Tephritidae) is the 

most destructive pest of olive around the world 
(Richard et al., 2003). The insect have been 
introduced to north of Iran as a serious pest since 
2004 (Jafari and Rezaee, 2005; Mirrahimi et al., 
2008). Females lay their eggs in fruits especially 
large green ones, larvae feed on the fruit pulp 
causing severe loss and feasibility of pathogen 
entrances to fruits. Pest control tactics depend on 
chemical treatments against adults, biological 
control and physical control such as sticky and 
pheromone traps (Laskowski and Kato, 1980). 

In insects, lipids are involved in several 
physiological functions like moulting during larval 
and adult development (Kawooya and Law, 1988), 
reproduction (Majumder and Sengupta, 1979), 
energy supplement during starvation (Cheeseman, 
1976; Ziegler, 1991) and immunity. Lipases [EC 
3.1.1.3] are the enzymes that hydrolyze the esteric 
bonds at the interface between insoluble substrate 
and water. The enzymes are synthesized by 
animals, plants, fungi and bacteria (Jaeger et al., 
1999; Gupta et al., 2004; Grillo et al., 2007). Digestive 
___________________________________________________________________________ 

 
Corresponding author: 
Arash Zibaee 
Faculty of Agricultural Sciences 
University of Guilan, Rasht 
Iran, 416351314 
E-mail: arash.zibaee@gmx.com
 

 
 
lipases of insects are divided into triacylglycerol 
lipases (TAG-Lipases), alkaline and acid 
phosphatases as well as phospholipases (Terra and 
Ferreira, 2012). Majority of ingested lipids by insects 
are the storage lipids (Triglycerids) that must be 
digested to di- and monoglycerids in midgut (Zibaee 
et al., 2008). During digestion process, 
triacylglyceride lipases hydrolyze certain positions of 
triacylglycerols while different phospholipases act on 
different ester bonds within phospholipids (Zibae e 
et al., 2008). Digestive lipases has been Studied in 
a few insects such as Rhodnius prolixus L. 
(Lepidoptera: Reduviidae) (Grillo et al., 2007), Chilo 
suppressalis (Lepidoptera: Crambidae) (Zibaee et 
al., 2008), Naranga aenescens (Lepidoptera: 
Noctuidae) (Zibaee and Fazeli-dinan, 2012), Pieris 
brassicae (Lepidoptera: Pieridae) (Zibaee, 2012) 
and Andrallus spinidens (Hemiptera: Pentatomidae) 
(Zibaee et al., 2012). Although lipases have not 
been well studied like other digestive enzymes but 
the enzymes have critical role in digestion process 
of insects. Moreover, characterization of lipases is 
mandatory to find inhibitors against lipases. On the 
other hand, inhibitors do cause severe reduction in 
growth and development and even mortality due to 
the importance of long chain unsaturated fatty 
acids in essential dietary components. This could 
be discovered from host plants that insect 
feeding causes lower activity of digestive 
lipases. Moreover, biochemical behavior of the 
lipases  must be elucidated  before any exposure 

66 
 



 
 
 
Fig. 1 Activity level of soluble and membrane-bound lipases in 3th instar larvae of olive fruit fly. 
 
 
 
 
 
 
to inhibitors. Hence, a biochemical characterization 
of the digestive lipase from B. oleae has been made 
via evaluating pH and temperature, effects of 
different ions and specific inhibitors. This is the first 
report of digestive lipases in a dipteran. 
 
Materials and Methods 
 
Insect rearing 

Larvae of Bacterocera oleae were reared on the 
olive fruits of Arbequina variety in containers of 
20×12 cm under laboratory conditions of 25 ± 1 °C, 
70 % of relative humidity and 16L:8D of 
photoperiod. Rearing containers were daily checked 
and cleaned to remove any contaminations. When 
the larvae reached to 3rd larval instars, they were 
randomly selected and used in biochemical 
experiments. 
 
Sample preparation 

Third larval instars were separated from olive 
fruits and dissected under a stereomicroscope in 
ice cold saline solution (NaCl, 10 mM). Whole 
guts were homogenized in distilled water by a 
glass homogenizer, centrifuged at 25000xg for 20 
min at 4 °C and supernatants were used in lipase 
assay (Zibaee, 2012). Membrane preparations 
were exposed from pellets of centrifuged 
digestive tract (see above) to Triton X-100 for 20 
h at 4 °C, in a ratio of 10 mg Triton X-100 per mg 
of protein. Samples were centrifuged at 25000xg 
for 20 min. Activity of the enzyme remains 
unchanged at -20 °C for at least a month (Zibaee, 
2012). 

Lipase assay 
The enzyme assay was carried out as 

described by Tsujita et al. (1989). Five microliter of 
larval gut extract and 15 μl of p-nitrophenyl-butyrate 
(PNPB, 27 mM) as substrate were added into 40 μl 
of universal buffer (20 mM, pH 7), mixed thoroughly 
and incubated at 30 °C. For negative control, 
samples were placed in a boiling water bath for 15 
min to destroy the enzymatic activity.Finally, 
absorbance was read after 10 min at 492 nm. One 
unit of enzyme released 1.0 nmol of p-nitrophenol 
per min at pH 7.2 and 37 °C when p-nitrophenyl 
butyrate was used as substrate.  
 
Effect of pH and temperature on the activity and the 
stability of the enzyme 

Effects of temperature and pH on lipase activity 
were examined by using p-nitrophenol-butyrate as 
substrate in various pH and temperature values. 
Optimal pH was determined using universal buffer 
(20 mM) with pH set at 3 - 12. The effect of 
temperature on the enzyme activity was determined 
by incubating the reaction mixture at 20, 25, 30, 35, 
40, 45, 50 and 60 °C. The procedures for both pH 
and temperature assays were quite similar to 
mentioned lipase assay (See above).  

 
Effect of mono- and di-valent cations on lipase 
activity 

Different concentrations of cations (1, 3 and 5 
mM) were assayed to find their effects on lipase 
activity in B. oleae larvae. Used cations were Ca2+, 
Cu2+, Fe2+, K+, Mg2+, Na+ and Zn2+. Briefly, 5 µl of a 
solution containing each concentration of ions and 5 

67 
 



 

a) 

 

 

b) 

 
Fig. 2 Effect of pH on the activities of soluble and membrane-bound lipases extracted from the digestive system of 
B.oleae larvae. Statistical deifferences have been shown by various letters (Tukey’s test, p ≤ 0.05). 
 
 
 
 
 
µl of enzyme were pre-incubated for 10 min at pH 7 
and room temperature. The pre-incubated mixture 
was added to a solution including 30 µl of universal 
buffer and 10 µl of substrate (PNPB, 27 mM). Other 
steps were carried out as mentioned earlier. 
 
Effect of specific chelating agent on lipase activity 

The effects of enzyme inhibitors on lipase 
activity were studied using different concentrations 
(2, 4, 6, 8 and 10 mM) of ethylene glycolbis (β-
aminoethylether) N,N, N′, N′-tetraacetic acid 
(EGTA), triethylenetetramine hexa acetic acid 

(TTHA), Phenylmethylsulfonyl fluoride (PMSF), 
diethyldithiocarbamate (DTC), and 
Ethylenediaminetetraacetic acid (EDTA). The 
enzyme (5 µl) was pre-incubated with inhibitors for 
10 min at pH 7 and room temperature. The pre-
incubated mixture was added to a solution including 
10 µl of substrate. Other steps were carried out as 
mentioned earlier. 

 
Protein determination 

Protein concentrations were assayed according 
to the method described by Lowry et al. (1951). 

68 
 



 

a) 

 
 

 

b) 

 
Fig. 3 Effect of temperature on the activities of soluble and membrane-bound lipases extracted from the digestive 
system of B.oleae larvae. Statistical deifferences have been shown by various letters (Tukey’s test, p ≤ 0.05). 
 
 
 
 
 
 
Statistical analysis 

The data were compared by one-way analysis 
of variance (ANOVA) followed by Tukey’s test when 
significant differences were found at p ≤ 0.05 using 
SAS program (SAS, 1997). 
 
Results and Discussion 

 
Lipases have important roles in digestion of 

lipids by insects and intermediary metabolism 

including lipid storage and mobilization (Horne et al., 
2009). Our results clearly demonstrated presence of 
a digestive lipase in the gut of B. oleae. On the other 
hands, it could be inferred that alimentary canal of 
larval olive fruit fly has ability to digest lipid by the 
action of soluble and membrane-bound lipases.In 
the current study, lipase activity was observed in 
both soluble and membrane-bound fractions by 
higher activity of membrane-bound fraction (Fig. 1) 
althopugh, majority of studies have been determined 

69 
 



in soluble content of the midgut. Since digestive 
enzyme secretion is a secretagogue process, 
digestive enzymes are presence as enzymatic 
vesicle inside of epithelial cells during starvation 
(Klowden, 2007). During ingestion, these vesicles 
are released into midgut lumen. Hence, higher 
activity of membrane-bound lipase could be 
attributed to the higher amounts of enzyme as 
vesicles inside the epithelial cells. 

Temperature and pH are the two factors that 
affect biochemical reactions via various approaches 

 
 
 
 
 

Table 1 Effect of mono- and divalent cations on 
soluble lipase activity of B.oleae 
 

compound concentration Specific activity 

 C 1.857±0481 a 

Ca2+

1 
3 
5 
 

C 

0.312±0.064 b 
0.122±0.093 b 
0.047±0.037 b 

 
1.857±0481 a 

Cu2+

1 
3 
5 
 

C 

0.442±0.034 b 
0.289±0.75 b 

0.537±0.074 b 
 

1.857±0481 a 

Fe2+

1 
3 
5 
 

C 

1.088±0.194 b 
1.598±0.119 ab 
1.714±0.167 ab 

 
1.857±0481 a 

K+

1 
3 
5 
 

C 

0.741±0.134 ab 
0.224±0.081 b 
0.047±0.013 b 

 
1.857±0481 a 

Mg2+
1 
3 
5 

8.913±0.649 b 
2.415±0.350 b 
1.592±1.354 b 

 C 1.857±0481 a 

Na+

1 
3 
5 
 

C 

0.340±0.018 b 
0.115±0.013 b 

1.769±0.426 ab 
 

1.857±0481 a 

Zn2+
1 
3 
5 

1.585±0.204 a 
0.122±0.031 b 
0.394±0.013 b 

 
*Statistical differences have been shown by various 
letters (Tukey’s test, p ≤ 0.05). 

Table 2 Effect of mono- and divalent cations on 
membrane-bound lipase activity of B. oleae 
 

 
*Statistical differences have been shown by various 
letters (Tukey’s test, p ≤ 0.05). 
 
 
 
 
 
like substrate and enzyme stability, their 
combination, tertiary structure of the enzyme and 
etc. Both of these factors provide optimal conditions 
to more adjustment of enzyme leading to better 
affinity and velocity during enzymatic reactions 
(Zibaee et al., 2012). It was found that optimal pH of 
the digestive soluble- and membrane-bound lipases 
in the gut of B. oleae larvae were obtained 4 and 6 
for soluble lipase and 6 for membrane-bound one 
(Fig. 2). Meanwhile, optimal temperatures for 
soluble and membrane lipases were observed at 50 

compound concentration Specific  activity 

 C 6.800±0.322 a 

Ca2+

1 
3 
5 
 

C 

0.628±0.081 b 
0.052±0.028 b 
0.231±0.087 b 

 
6.800±0.322 a 

Cu2+

1 
3 
5 
 

C 

0.304±0.066 b 
1175±62.915 b 
0.555±0.277 b 

 
6.800±0.322 a 

Fe2+

1 
3 
5 
 

C 

0.463±0.176 b 
1.283±0.162 b 
0.972±0.251 b 

 
6.800±0.322 a 

K+

1 
3 
5 
 

C 

0.807±0.184 b 
142.719±719 b 

140.778±12.165 b 
 

6.800±0.322 a 

Mg2+
1 
3 
5 

3.380±0.587 b 
1.574±0.767 b 
1.316±0.324 b 

 C 6.800±0.322 a 

Na+

1 
3 
5 
 

C 

0.469±0.017 b 
0.205±0.102 b 
0.608±0.283 b 

 
6.800±0.322 a 

Zn2+
1 
3 
5 

1.223±0.160 b 
0.205±0.102 b 
0.641±0.509 b 

70 
 



°C and 35 °C, respectively (Fig. 3). Grillo et al. 
(2007) reported that digestive lipase from the midgut 
of Rhodnius prolixus (Hemiptera: Reduviidae) had 
maximal activity in pH 7-7.5. Zibaee et al. (2008) 
found the optimal pH and temperature of 10 and 37 
- 40 °C in larvae of Chilo suppressalis. Zibaee 
(2012) found optimal pH and temperature of 
digestive lipasein P. brassicae as pH 11 and 
temperature of 30 °C. The optimal pH of 10 and 
temperature of 35 - 40 ◦C observed for a lipase from 
larvae of N. aenescens (Zibaee and Fazeli-Dinan, 
2012). The optimal pH for TAG-lipase activity was 
obtained at pH 9 and temperature 40 °C for A. 
spinidens (Zibaee et al., 2012). The optimal 
temperature for activity of an enzyme reflects 
temperature in which organism is living in it. Also, 
activity of enzymes increase along with elevation of 
temperature up to optimal value then it decrease 
because the hydrogen bonds in enzyme structure 
break in extreme temperature and disrupt three-
dimensional structure leading to enzyme 
denaturation (Zeng and Cohen, 2000). 

Ions are one of the significant components in 
active sites of enzymes. Ions can take and release 
electrons; affect electrophiles and nucleophiles, 
increase efficiency of enzyme-substrate complex 
and stability of enzymes (Zibaee, 2012). In the 
present study, different concentrations of ions and 
chelating agents caused various effects on lipase 
activity in B oleae larvae (Tables 1 - 4). Activities of 
the digestive soluble and membrane-bound lipases 
in the gut of B. oleae larvae were decreased by 
using all ions (Tables 1, 2). The effects of several 
chelating agent and PMSF were examined to find 
their possible effects on digestive soluble- and 
membrane-bound lipase activities (Tables 3, 4). All 
the chelating agents except for TTHA in soluble 
lipase had no inhibitory effects on the enzymatic 
activity but PMSF sharply decreased enzymatic 
activity in both fractions (Tables 3, 4). Applebaum 
1985) reported that Ca2+ ion increased lipase activity 

 
 
 
 
 

Table 3 Effect of specific inhibitors on soluble lipase 
activity of B. oleae 
 

compound concentration Specific activity 

 C 0.217±0.013 a 

DTC 10 0.442±0.114 a 

PMSF C 10 
1.857±0.481 a 
0.421±0.118 b 

EGTA C 10 
925±0.156 b 

1.809±0.119 a 

EDTA C 10 
0.217±0.013 a 
0.442±0.114 a 

TTHA C 10 
1.687±0.203 a 
0.591±0.142 b 

 
*Statistical differences have been shown by various 
letters (Tukey’s test, p ≤ 0.05). 

Table 4 Effect of specific inhibitors on membrane-
bound lipase activity of B. oleae 
 

compound concentration Specific activity 

 C 0.555±0.052 b 

DTC 10 1.223±0.152 a 

PMSF C 10 
6.800±0.322 a 
1.706±0.558 b 

EGTA C 10 
1.574±0.220 a 
1.488±0.045 a 

EDTA C 10 
0.840±0.262 a 
1.303±0.304 a 

TTHA C 10 
1.580±0.077 a 

0.077±0.0.056 a 
 
*Statistical differences have been shown by various 
letters (Tukey’s test, p ≤ 0.05). 
 
 
 
 
 
of Callosobruchus chinensis. Zibaee (2012) found 
that Mg2+, Na+, EDTA and TTHA significantly affect 
digestive lipase activity in P. brassicae. Grillo et al. 
(2007) found that Ca2+ increase activity of lipases in 
R. prolixus. Similar results were found in case of C. 
suppressalis and N. aenescens (Grillo et al., 2007; 
Zibaee et al., 2008; Zibaee and Fazeli-Dinan, 2012). 
Ca2+, Mg2+, K+, Na+, Mn+, PMSF and EGTA showed 
significant effects on lipase activity in A. spinidens 
(Zibaee et al., 2012). 

Although digestive lipases of insects have been 
less studied but they have a great potential to be 
inhibited like amylases and proteases. This property 
could be used to develop resistant varieties in 
sustainable agricultural system. There are many 
reports on inhibition of vertebrate pancreatic lipases 
by secondary metabolites of plants but relevant 
studies on insect digestive lipases are few 
(Markwick et al., 2011). Senthil-Nathan et al. (2006) 
and Zibaee and Bandani (2010) reported that 
feeding of Eurygaster integriceps (Hemiptera: 
Scutelleridae) and Cnaphalocrocis medinalis on the 
diets containing botanical extracts lead to significant 
inhibition of digestive lipases. Also, Markwick et al. 
(2011) demonstrated effects of tetrahydrolipstatin 
(THL), on neonate Epiphyas postvittana 
(Lepidoptera, Tortricidae) larvae by feeding on 
control artificial diets and diets containing one of 
three concentrations of THL (0.011 %, 0.037 % and 
0.11 %). The authors reported significant decrease 
in growth, pupation and time to pupation in 
comparison with control. 

Although reports on digestive lipases of insects 
revealed promising results but it is necessary to 
carried out more studies to confirm that digestive 
lipases of insects could be a new control for insect 
pest or not. Lipase inhibitors do cause severe 
reduction in growth and development and even 
mortality due to the importance of long chain 
unsaturated fatty acids being essential dietary 
components. Hence, a plant breeding program might 

71 
 



be adopted to increase levels of naturally occurring 
lipase inhibitors. The current study was the first one 
to characterize a digestive lipase of dipteran larvae. 
This is a basic study that will continue to develop 
inhibitors leading to an efficient control of B. oelae. 

 
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