A Midgut Digestive Phospholipase A2 in Larval Mosquitoes, Aedes albopictus and Culex quinquefasciatus.

Phospholipase A2 (PLA2) is a secretory digestive enzyme that hydrolyzes ester bond at sn-2 position of dietary phospholipids, creating free fatty acid and lysophospholipid. The free fatty acids (arachidonic acid) are absorbed into midgut cells. Aedes albopictus and Culex quinquefasciatus digestive PLA2 was characterized using a microplate PLA2 assay. The enzyme showed substantial activities at 6 and 8 μg/μl of protein concentration with optimal activity at 20 and 25 μg/μl of substrate concentration in Aedes albopictus and Culex quinquefasciatus, respectively. PLA2 activity from both mosquitoes increased in a linear function up to 1 hour of the reaction time. Both enzymes were sensitive to pH and temperature. PLA2 showed higher enzyme activities in pH 8.0 and pH 9.0 from Aedes albopictus and Culex quinquefasciatus, respectively, at 40°C of incubation. The PLA2 activity decreased in the presence of 5 mM (Aedes albopictus) and 0.5 mM (Culex quinquefasciatus) site specific PLA2 inhibitor, oleyloxyethylphosphorylcholine. Based on the migration pattern of the partially purified PLA2 on SDS-PAGE, the protein mass of PLA2 is approximately 20-25 kDa for both mosquitoes. The information on PLA2 properties derived from this study may facilitate in devising mosquitoes control strategies especially in the development of inhibitors targeting the enzyme active site.

1. Introduction

Phospholipase A2 (PLA2) hydrolyzes the sn-2 ester bond in phospholipids (PLs) [1]. These enzymes make up a large superfamily of proteins that act in a very wide variety of physiological and pathophysiological actions. PLA2 actions include digestion of dietary lipids, remodelling cellular membranes, host immune defenses, signal transduction via production of various lipid mediators, and, in the case of platelet activating factor, inactivation of a lipid mediator. Research into noncatalytic PLA2s and into PLA2 receptors and binding proteins reveals entirely new biological actions in which PLA2 acts as a ligand rather than a catalytic enzyme [2, 3]. Here, we focus attention on PLA2 associated with digestion.

Lipid digestion and absorption take place in the insect midguts. Midgut cells produce and secrete lipases that digest dietary neutral lipids, such as triacylglycerols. PLA2s are responsible for two separate actions in insect physiology. For one, PLA2s hydrolyze a fatty acid from the sn-2 position of dietary PLs. Typically, the fatty acids esterified to the sn-2 positions are C18 and C20 PUFAs. These fatty acids include linoleic acid, 18:2n-6, and linolenic acid, 18:3n-3, one or the other of which is strictly essential nutritional requirements for most insects and nearly all vertebrates. Hence, midgut PLA2s are necessary for insects to meet one of their essential nutritional needs. A few insect and invertebrate species express a Δ−12 desaturase that inserts a double bond into oleic acid (18:1n-9), yielding 18:2n-6 and obviating the nutritional requirement [4-6]. The desaturation and elongation pathways necessary to convert C18 PUFAs to their C20 counterparts have been documented in several insect species [7, 8], from which we infer insects are able to meet all fatty acid requirements via dietary linoleic and linolenic acids, coupled to the desaturase/elongation pathways. The second important PLA2 action contributes to digestion of dietary neutral lipids. Vertebrates, but not insects, produce bile salts that facilitate lipid digestion by solubilizing neutral lipids. In each PLA2 reaction, hydrolysis of the sn-2 fatty acid from PL leads to a free fatty acid and to a lysoPLs and these lipids act as the necessary solubilizers that aid lipase digestion of neutral lipids.

PLA2 is the key enzyme responsible in the hydrolysis of arachidonic acid which acts as precursors to lipid mediators such as prostaglandins [9]. PLA2s occur abundantly in venoms [10], in pancreatic juices of mammals, and in synovial fluids [11]. While PLA2s are well characterized in terms of protein and gene structures in mammalian physiology, there is relatively little information on the characteristics of insect digestive PLA2. Nonetheless, these enzymes are very important in insect biology and they may become functional targets in some pest management programs. Seen in this light, there is a real need for new knowledge on insect digestive PLA2s. In this paper, we begin to address that need. Here, we report on the presence and the characteristics of a midgut PLA2 in larvae of the mosquitoes, Aedes albopictus and Culex quinquefasciatus.

2. Materials and Methods

2.1. Insect

The larvae of Aedes albopictus and Culex quinquefasciatus were collected from Kampung Semerah Padi, Petra Jaya, Kuching (1°34′59.3″N, 110°19′48.2″E). The larvae were collected with ovitraps filled with a cow-grass infusion solution following methods described by Tang et al. [12].

Ten ovitraps were placed near housing areas at Kampung Semerah Padi, Petra Jaya. The ovitraps were collected and replaced with new ones every week. The larvae collected were pooled together and fed with fish food until the 4th instar. The larvae were identified as Aedes albopictus and Culex quinquefasciatus [13] and midguts were isolated. A larva to be dissected was placed on a glass plate and the water surrounding the larvae was blotted to dry. The midgut was removed by using a pair of forceps. By holding the thorax with forceps, the 8th abdominal segment was gently pulled using the other forceps so that the entire alimentary canal was drown out. The anal papillae, siphon, and Malphigian tubule attaching to the midgut were removed by pinching with the forceps.

2.2. PLA2 Source Preparation

Midgut samples were homogenized in 200 μl buffer (0.1 M Tris[hydroxymethyl]aminoethane, pH 8; Sigma) mixed with 2 mM phenylthiourea (PTU, Sigma) by using a Bio Masher (Optima, Inc., USA). The homogenates were centrifuged at 735g for 3 minutes, then at 11,750g for 10 minutes. The supernatants were collected and used as the enzyme preparation.

2.3. Phospholipase A2 Assay

PLA2 substrate, 4-nitro-3-(octanoyloxy)benzoic acid (NOB; Enzo Life Sciences, Switzerland), was prepared following Nenad et al. [14] and Beghini et al. [15] with several modifications. The substrate was diluted with chloroform to 50 mg/ml. 20 μl (1 mg) aliquots were distributed into Eppendorf tubes and all of the moisture was evaporated to dryness. The dry residue was stored at −20°C. Immediately before the assay, the substrate was resuspended in 1 ml of acetonitrile. The suspension was vortexed until all the substrate dissolved.

A standard PLA2 enzyme assay using 96-well plates was conducted following methods by Beghini et al. [15]. The standard assay mixture contains substrate (NOB), enzyme source, and buffer (0.1 M Tris buffer, pH 8) made up of a 200 μl of mixture in each well. After the addition of the enzyme source, the microplate was incubated (Asys Thermostar) for 40 minutes at 40°C. The NOB, through hydrolysis of an ester bond, will convert into a chromophore (4-nitro-3-hydroxy-benzoic acid). The absorbance of chromophore concentration produced was quantified using a microplate reader at 405 nm. The effects of Ca2+, substrate and protein concentration, incubation time, pH, and temperature were investigated by varying each parameter.

2.4. Localizing the PLA2 in Larvae

The homogenates were prepared from three different sections of the individual larvae. Three groups, A, B, and C, consisted of gut-free larval bodies, guts and contents, and isolated gut contents. Alimentary canals were removed by pulling out the eighth abdominal segment of the larvae with forceps, while holding its thorax with second forceps. The individual gut was quickly removed and placed into an Eppendorf tube containing 0.1 M Tris buffer and 2 mM PTU (Group B). The remaining bodies were collected into different tubes containing the same buffer (Group A). Isolated gut contents were obtained by separating the gut contents (Group C) from forty individual guts. All samples were prepared for enzyme assay as described in Table 1.

Table 1

Mosquito larval preparation for localizing PLA2 enzyme experiment.

Groups	Larval sections
A	Gut-free bodies (40 individual larval bodies/pool)
B	Gut and content (40 individual guts/pool)
C	Isolated gut contents (40 individual isolated gut contents/pool)

2.5. The Influence of Ca2+ on PLA2 Activity

We conducted reactions in the presence of three different buffers: (1) Tris buffer with no additions; (2) Tris buffer amended with 5 mM CaCl2; (3) Tris buffer amended with the Ca2+ chelator 5 mM EGTA (ethylene-glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid). Each buffer was used during larvae dissection, homogenization, and enzyme activity assay.

2.6. Characterizing the Mosquito Digestive PLA2

All experiments used midguts plus contents as the enzyme preparations. The influence of substrate and protein concentration, incubation time, pH, temperature, and the effect of site specific inhibitor for PLA2 and OOPC on PLA2 activity were assessed by varying each of the parameters.

2.7. Gel Electrophoresis

Estimation of this digestive PLA2 from Aedes albopictus and Culex quinquefasciatus was performed according to the tricine-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) method by Schägger and von Jagow [16].

Migration of digestive PLA2 was compared to the standard protein markers (Sigma) in a range of 26.6 kDa to 1.06 kDa. The protein marker consisted of triosephosphate isomerase from rabbit muscle (26.6 kDa), myoglobin from horse heart (17.0 kDa), α-lactalbumin from bovine milk (14.2 kDa), aprotinin from bovine lung (6.5 kDa), insulin chain B, oxidized, bovine (3.496 kDa), and bradykinin (1.06 kDa). The protein bands on the electrophoresis gel were directly visualized by using silver staining according to the method by Gromova and Celis [17].

2.8. Statistical Analysis

Data were reported as means ± SEM of n experiments as appropriate. The significance of difference between groups was assessed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test to determine the significant group. The confidence limit for significance was p ≤ 0.05.

3. Results

3.1. Localizing the PLA2 Enzyme in Larvae

To determine the appropriate preparation for characterization of PLA2, three samples were prepared. Substantially high PLA2 enzyme activity from Aedes albopictus was recorded in gut plus content preparation (M = 0.328, SEM = 0.00) and gut content preparation (M = 0.3067, SEM = 0.01). There were no significant differences (F(2,6) = 5.161, p = 0.05) in PLA2 activity between all the preparations. In contrast, significantly higher PLA2 activity was observed in Culex quinquefasciatus' gut content preparation (M = 0.627, SEM = 0.01) (Figure 1). Similarly, lower PLA2 activity was recorded in gut-free bodies of Aedes albopictus (M = 0.2733, SEM = 0.01) and Culex quinquefasciatus (M = 0.220, SD = 0.02). The gut plus content was used as an enzyme source in all subsequent experiments.

Figure 1

The PLA2 activity in different preparations of Aedes albopictus and Culex quinquefasciatus larvae. 10 μg/μl of protein concentration was reacted with 10 μg/μl of substrate concentration. Each histogram bar shows the mean ± SEM of triplicates from a single experiment representative of at least two experiments.

3.2. Calcium Ion (Ca2+) Dependency

Aedes albopictus PLA2 activity was significantly higher in the buffer containing EGTA (M = 0.063, SEM = 0.01) and Tris buffer (M = 0.065, SEM = 0.00), while, for Culex quinquefasciatus, the PLA2 activity was significantly high in Tris buffer (M = 0.089, SEM = 0.01) compared to enzyme activity in Tris buffer with additional calcium (M = 0.0087, SEM = 0.01) (Figure 2). These results suggest that the mosquito larval preparation is independent of Ca2+. Subsequent experiments were conducted in Tris buffer with no added Ca2+.

Figure 2

The PLA2 activity of Aedes albopictus and Culex quinquefasciatus in three different buffers. 10 μg/μl of protein concentration was reacted with 10 μg/μl of substrate concentration. Each histogram bar shows the mean ± SEM of triplicates from a single experiment representative of at least two experiments.

3.3. Characterization of PLA2 Enzyme

The PLA2 activities from Aedes albopictus and Culex quinquefasciatus showed a similar trend where the optimum enzyme activities were recorded at 6–8 μg/μL of protein (Figure 3). Aedes albopictus PLA2 activity was fairly low at 2 and 4 μg/μl and then increased to a high level at 6 μg/μL (M = 0.090, SEM = 0.01) and 8 μg/μL (M = 0.091, SEM = 0.01) before it slightly declined at 10 μg/μL (M = 0.087, SEM = 0.00). Similarly, the Culex quinquefasciatus PLA2 activity increased from 2 μg/μL (M = 0.0243,   SEM = 0.01) of protein until it reached the highest activity at 8 μg/μL (M = 0.0713, SEM = 0.01). For subsequent experiment, 6 μg/μL of protein was used as an enzyme source for PLA2 assay in Aedes albopictus while 8 μg/μL was used as an enzyme source for PLA2 assay in Culex quinquefasciatus.

Figure 3

The influence of protein concentration on Aedes albopictus and Culex quinquefasciatus PLA2 activity. 10 μg/μl of substrate concentration was used to react with each of the protein concentrations, respectively. Each point represents the mean ± SEM of triplicates from a single experiment representative of at least two experiments.

The PLA2 activities from mosquitoes, Aedes albopictus and Culex quinquefasciatus, were increased with increasing concentration of substrate until the enzyme concentration becomes a limiting factor in the reaction. The Aedes albopictus PLA2 activity increased in a linear manner with increasing substrate concentrations, up to 20 μg/μl (M = 0.121, SEM = 0.01) (Figure 4(a)).

Figure 4

PLA2 activity of Culex quinquefasciatus also increased from lower concentration, 5 μg/μL (M = 0.103, SEM = 0.00), until it reached its optimal activity at 25 μg/μL (M = 0.290, SEM = 0.01) (Figure 4(b)). There was no significant increase of enzyme activity at 30 μg/μL for both mosquitoes PLA2. Our standard concentration of substrate was 10 μg/μl for all subsequent experiments.

The PLA2 of Aedes albopictus was fairly low up to 20-minute incubations (M = 0.042, SEM = 0.01) and then increased substantially up to 50-minute incubation time (M = 0.161, SEM = 0.02). There was still another increase at 50 minutes (M = 0.161, SEM = 0.02) (Figure 5(a)). The enzyme activity remained constant at 60 minutes of incubation time.

Figure 5

On the other hand, there is no PLA2 activity of Culex quinquefasciatus recorded in the first 20 minutes of incubation time. Then, the enzyme activity continued to increase steadily even after an hour (M = 0.033, SD = 0.00) (Figure 5(b)). We used 40-minute incubations in all experiments.

The digestive PLA2 was sensitive to temperature. The reaction mixtures were incubated in a range of 24°C (room temperature) to 70°C. The Aedes albopictus and Culex quinquefasciatus PLA2 activity increased in a linear way from room temperature to a peak at 40°C (M = 0.116, SEM = 0.01; M = 0.097, SEM = 0.01) (Figures 6(a) and 6(b)). At higher temperatures (60–70°C) the enzyme activity declined. Our standard incubation temperature was set at 40°C.

Figure 6

pH of the reaction mixtures influenced PLA2 (Figures 7(a) and 7(b)). The PLA2 activity from Aedes albopictus and Culex quinquefasciatus increased from acidic condition to a mild alkaline condition. Aedes albopictus PLA2 increased gradually from pH 5.0 (M = 0.067, SEM = 0.01) until it reached a maximum PLA2 activity at pH 8.0 (M = 0.130, SEM = 0.01). At pH 9.0, the enzyme activity slightly decreased, but it is not statistically significant. Similarly, Culex quinquefasciatus reached its highest PLA2 activity at pH 9.0 (M = 0.1093, SEM = 0.01) before it dropped drastically at pH 10.0 (M = 0.035, SEM = 0.02). Post hoc multiple comparison (Tukey) analysis showed there was no significant difference in the enzyme activity from pH 6.0 to 9.0. Therefore, pH 8 was selected as a reaction pH for all subsequent reactions.

Figure 7

3.4. The Influence of OOPC on PLA2 Activity

Reactions in the presence of 50 μM to 500 μM of OOPC led to a dose-related decline in PLA2 activity (Figures 8(a) and 8(b)). The PLA2 of Aedes albopictus was significantly inhibited in the presence of 5000 μM of OOPC [F(3,8) = 5.886, p = 0.020]. However, the PLA2 of Culex quinquefasciatus was not statistically inhibited in the presence of PLA2 inhibitor, OOPC [F(2,6) = 3.651, p = 0.092].

Figure 8

3.5. Protein Mass Determination of Partially Purified Digestive PLA2

The protein electrophoretic profile of partially purified PLA2 from Aedes albopictus showed bands in different sizes which range from 14.6 to 20.3 kDa (Figure 9(a)) while for partially purified PLA2 from Culex quinquefasciatus it showed only one band which was at 25 kDa (Figure 9(b)). The presence of band at a similar size (25 kDa) suggests the presence of similar protein, which is the PLA2.

Figure 9

4. Discussion

In this paper, we report on the characterization of a digestive PLA2 in Aedes albopictus and Culex quinquefasciatus larvae. During our initial experiment, we compared the PLA2 activities in selected fractions of the alimentary canal. Higher PLA2 enzyme activity was recorded in the midgut plus content and isolated gut content preparations. Similar findings were reported for a related mosquito species, Aedes aegypti, where higher PLA2 enzyme activity was recorded in midgut plus content preparation. These findings suggested that midgut cells secrete more PLA2 than they store [18].

Secretory PLA2 is characterized as a low molecular weight molecule (13–55 kDa) [10, 19] that catalyzes substrate in full activity in the presence of calcium. sPLA2 differs from other PLA2 as it acts extracellularly [20]. The enzyme was secreted from the cells before catalyzing the substrate which usually occurs in the lumen of the insects' midgut [21].

The characterization of PLA2 in insects was assayed with radioactive substrate in the past. Here, we used a microplate assay using the chromogenic substrate, NOB. NOB is widely used in characterizing PLA2 from snake venom [22-24] and human serum [14]. Our PLA2 assays were performed on mosquito samples that have been partially enriched using Heparin column chromatography (1 ml HiTrap Heparin, Sigma, USA). This method successfully enriched the target PLA2 in primary screwworm preparations [25]. For this work, our simple microplate assay used only a small amount of protein and substrate to obtain an optimal PLA2 activity. This is an advantage for an investigation with limited amounts of protein sample. Since we collected our larval mosquito from the field, the number of individual larvae in each collection varied depending on their local environmental condition. The microplate assay is a practical but effective method to conduct our experiments with limited enzyme source.

Ca2+ is essential for both catalysis and binding of some enzymes to the substrate [26]. A study of Ca2+ requirement in primary screwworm PLA2 preparations showed that the enzyme activity was almost abolished in the presence of calcium chelator, EGTA. The PLA2 dependency on Ca2+ varied across species [21]. Previous studies on PLA2 from primary screwworm, C. hominivorax [25], robber flies, Asilis sp. [27], and adult tiger beetles, Cicindela circumpicta [28], showed strict Ca2+ requirement for catalysis. Several studies on PLA2 from venom sources such as rattlesnake, Crotalus durissus cascavella, venom [15] and sea anemone, Aiptasia pallid, nematocyst venom [29] also showed strict Ca2+ requirement [30].

PLA2 activity in the Aedes albopictus and Culex quinquefasciatus preparations revealed slightly higher enzyme activity in Tris buffer without additional calcium. This is in agreement with PLA2 from the midgut of tobacco hornworm [31] and Aedes aegypti larvae midgut [18].

Generally, enzyme activities are influenced by biophysical parameters, protein and substrate concentrations, pH, temperature, and reaction time. Increasing the amount of either enzyme or substrate generally will increase reaction rates because more active sites are available for reaction and more substrate molecules can bind with the active sites.

The Aedes albopictus and Culex quinquefasciatus preparations responded to the usual biophysical parameters in a way fairly similar to the Aedes aegypti preparations [18] except for its sensitivity to OOPC. PLA2 activity in Aedes aegypti [18] was less sensitive to OOPC than the Aedes albopictus and Culex quinquefasciatus preparations. Aedes albopictus PLA2 was inhibited at 5000 μM of OOPC as compared to Culex quinquefasciatus PLA2, which was inhibited at a lower concentration of OOPC (500 μM). In contrast, the PLA2 activity from Aedes aegypti [18] did not show any significant decrease in the presence of OOPC (5–5000 μM).

Similar finding was also recorded for tobacco hornworm, Manduca sexta [31], digestive PLA2 where there was no inhibition in its enzyme activity when exposed to different concentrations of OOPC in a range of 5–500 μM. On the other hand, the primary screwworm PLA2 preparation was more sensitive to OOPC, 50 μM [25].

The Aedes albopictus and Culex quinquefasciatus PLA2 activity increased with time in reactions up to 1 hour, in agreement with PLA2 enzyme from other insects, Aedes aegypti [18] and C. hominivorax [25], and PLA2 from venoms, Bothrops jararacussu [22].

PLA2 from Aedes albopictus and Culex quinquefasciatus shares some similarity and differences with a related species, Aedes aegypti, where the optimal enzyme activity was at 40–50°C. However, the optimum pH condition differs. Aedes aegypti PLA2 activity was optimal at pH 9.0, which is similar with Culex quinquefasciatus PLA2, while for Aedes albopictus the enzyme activity declined at pH 9. Although the maximum enzyme activity differs, all mosquitoes PLA2 reaction studied slowed down in acidic conditions. This is in broad agreement with the pH conditions of insect midguts, which can be very high in lepidoptera and more acidic in mosquitoes [25].

This study has estimated the size of PLA2 from Aedes albopictus and Culex quinquefasciatus and provided information on partially purified PLA2 from crude homogenate of mosquito larval midgut by using Heparin column. In this study, both Aedes albopictus and Culex quinquefasciatus larval midgut PLA2 estimated molecular weights were found in a range of secretory insect PLA2. The molecular weights were estimated at 14.6–25 kDa and 25 kDa for Aedes albopictus and Culex quinquefasciatus, respectively. Distinct band at 25 kDa was shown in both PLA2 preparations of Aedes albopictus and Culex quinquefasciatus which are more likely the protein of interest in this study.

The estimated sizes agreed with the classic characteristic of sPLA2, which consists of small MW enzyme ranging from 13 to 15 kDa which were obtained from various organisms such as snake venoms, porcine pancreas, fungus, and bacteria [1] and for tiger beetle and human PLA2 which were reported to be 22 kDa and 55 kDa, respectively [19].

However, other investigations such as amino acid sequence and X-ray crystal structures of Aedes albopictus and Culex quinquefasciatus need to be conducted in order to classify the type of the digestive PLA2.

5. Conclusions

Aedes albopictus PLA2 from different sample preparations showed no significant difference in their activity, while for Culex quinquefasciatus significantly higher PLA2 in gut contents was shown if compared to other preparations. PLA2 from both enzymes did not require calcium (Ca2+) for full enzyme activity and both showed increasing of enzyme activity with increasing concentration of substrate. The PLA2 enzymatic assay from both mosquitoes showed accumulation of chromogenic substance up to 60 minutes of incubation time at 40°C. Both enzymes reacted in full catalytic activity in alkaline condition. Aedes albopictus PLA2 was significantly inhibited by site specific PLA2 inhibitor, OOPC. However, Culex quinquefasciatus PLA2 was not significantly inhibited by the same inhibitor. Based on the electrophoretic pattern of the enzyme samples, protein band at 20–25 kDa was observed in both mosquitoes. To conclude, there were no differences between the characteristic of PLA2 from Aedes albopictus and Culex quinquefasciatus except for its inhibition toward site specific inhibitor PLA2, OOPC, where the inhibitor does not affect the Culex quinquefasciatus PLA2 activity.