Secretagogue Mechanism of Digestive Enzyme Secretion in the Midgut of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) Sahar Sorkhabi-Abdolmaleki & Arash Zibaee
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences ISSN 0369-8211 Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-013-0209-3
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Author's personal copy Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. DOI 10.1007/s40011-013-0209-3 Volum. 82, Issue 2. Pages 373-379
RESEARCH ARTICLE
Secretagogue Mechanism of Digestive Enzyme Secretion in the Midgut of Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) Sahar Sorkhabi-Abdolmaleki • Arash Zibaee
Received: 7 February 2013 / Revised: 10 June 2013 / Accepted: 20 June 2013 Ó The National Academy of Sciences, India 2013
Abstract Time for food processing and enzymatic secretion were evaluated in starved and fed adults of Andralus spinidnes a predatory hemipteran serving as biocontrol agent of rice pests. Adults were kept starved for 8 days then they were allowed to feed on Galleria melonella larvae (one larva per adult). They were dissected at time intervals of 1, 3, 6, 12, 24 and 48 h post-feeding to evaluate digestive enzymatic activities. It was observed that gut of starved adult is small and shrunken but gut of fed adults after 12 h was normal and well shape. No digestive enzymatic activity was observed in starved adults. Activity of general protease was the highest in 24 h post-feeding but the highest activity of a-amylase and lipase were noticed 12 h post-feeding. Also, specific protease activities showed different peaks at different time intervals. The highest trypsin-like, chymotrypsin-like and elastase-like activities were observed 12 and 24 h postfeeding although activity of chymotrypsin-like protease was still high even after 48 h. Cathepsin B showed the highest activity after 24 and 48 h but Cathepsin L and D had the highest activity after 12 h of post-feeding. The highest activities of amino- and carboxypeptidase were observed 24 h post-feeding. These results clearly demonstrate secretagogue mechanism of digestion and digestive enzyme secretions in A. spinidens. Also, secretion of digestive enzymes at least 12 h post-feeding could be attributed to extra-oral digestion made by the insects and liquefaction of the food material.
S. Sorkhabi-Abdolmaleki A. Zibaee (&) Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan-Rasht, 41635-1314 Rasht, Iran e-mail:
[email protected];
[email protected]
Keywords Andrallus spinidens Food processing Digestive enzyme
Introduction Digestion is a multiple and complex mechanism in which ingested macromolecules get digested to mono- and oligomers to be absorbed via epithelial cells of midgut [1]. This process has a close dependency on digestive enzyme secretion and their allocation. Primary digestion refers to dispersion and break-down of ingested polymers changing them to oligomers. In intermediate digestion, molecular size of compounds further reduces and changes to dimers [1]. In the last step, dimers are changed to monomers. Before these phases, secretion of digestive enzymes is mandatory. Digestive enzyme secretion is regulated by food components like proteins, carbohydrates and lipids by acting directly on the midgut epithelium under a mechanism called secretagogue [2]. Mechanisms involved in secretagogue are known to occur in insects with a variety of feeding habits. Different enzymes are controlled independently so that only appropriate one on the ingested food is produced. For example, in blood feeding insects, a blood meal induces enzymatic secretion but sugar meal does not [2]. Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) is a predatory hemipteran in the rice fields of Iran, India and many other places in the world [3]. Both nymphs and adults feed on several caterpillars like Chilo suppressalis Walker (Lepidoptera: Crambidae), Naranga aenescens Moore (Lepidoptera: Noctuidae) and Helicoverpa armigera Hu¨bner (Lepidoptera: Noctuidae) in the rice fields of northern Iran [3]. A. spinidens has five generations per year, each lives for 65 days and hibernates as adult in
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Author's personal copy S. Sorkhabi-Abdolmaleki, A. Zibaee
rice debris and weeds [4]. In consecutive studies, digestive enzymes of A. spinidens were determined and characterized to find their role in prey preference [4–6]. These enzymes are a-amylase, TAG-lipase, trypsin-like protease, chymotrypsin like protease, elastase-like protease, cathepsins B, L, D, amino- and carboxypeptidase. It was found that A. spinidens uses extra-oral digestion to liquefy prey body, the ingested liquids were finally digested in the four sectioned midgut. In laboratory observations, it was found that each adult spends 3–6 h to feed on a Galleria melonella larva. This phenomenon has been studied in grasshoppers but no information is available regarding hemipterans. So, the aim of the current study was to find secretagogue mechanism of digestive enzyme secretion by evaluating several digestive enzymes of A. spinidens.
Material and Methods A. spinidens Rearing Colony of A. spinidens was established by adults collected from harvested rice fields in Amol (Mazandaran, North of Iran) during late September of 2012. Insects were reared on the fifth instars of Chilo suppressalis L. (Lepidoptera: Crambidae) as prey and provided with wet cotton plugs fitted into small plastic dishes (2.5 cm diameter) as 80 % moisture sources at 25 ± 1 °C. Starvation and Feeding Experiments Adults (N = 35) were kept starved for 8 days then they were allowed to fed on Galleria melonella larvae (a larva per adult). Fed adults were randomly selected and dissected at time intervals of 1, 3, 6, 12, 24 and 48 h post-feeding to evaluate digestive enzymatic activities. Sample Preparation for Enzymatic Assays Soluble Fractions Insects were randomly selected and dissected under a stereo microscope in ice-cold saline buffer (NaCl, 10 mM). After removing of fat bodies and other undesirable organs, the midgut was separated from body, rinsed in ice-cold distilled water, placed in a pre-cooled homogenizer and grounded before centrifugation. Equal portion of midgut and distilled water was used to have a desirable concentration of enzymes (W/V). Homogenates were separately transferred to 1.5 ml tubes and centrifuged (Kokusan Enshinki Co. Ltd., Tokyo, Japan) at 13,000 rpm for 15 min. The supernatants were pooled and stored at -20 °C for subsequent analyses [4].
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Microvillar-Bound Fraction For solubilization of microvillar-bound enzymes (Exopeptidases) in Triton X-100, membrane preparations (Precipitation from primitive centrifuge, above) were exposed to Triton X-100 for 20 h at 40 °C, in a ratio of 10 mg of Triton X-100 per mg of protein, before being centrifuged at 13,000 rpm for 30 min. No sediment was visible after the centrifugation of this supernatant at 10,000 rpm for 60 min. Activity of enzymes remains unchanged, at -20 °C, for at least a month [7, 8]. A-Amylase Assay The method described by Bernfeld [9] was used to assay a-amylase activity. Ten microlitres of the enzyme was incubated for 30 min at 35 °C with 50 ll of Tris–HCl buffer (20 mM, pH 7) and 20 ll of 1 % soluble starch as substrate. The reaction was stopped by addition of 80 ll dinitrosalicylic acid in boiling water for 10 min prior to the reading of absorbance at 545 nm. One unit of a-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35 °C. The negative control containing all reaction mixtures with pre-boiled enzyme (for 15 min) was used to prove the enzyme presence in the sample. TAG-Lipase Assay The enzyme assay was carried out as described by Tsujita et al. [10]. Gut extract (20 ll) and p-nitrophenyl butyrate (40 ll, 27 mM) as substrate were thoroughly mixed with 100 ll of Tris–HCl buffer (20 mM, pH 11) and incubated at 37 °C. After 1 min, 100 ll of NaoH (1 M) was added to each tube (control and treatment) and absorbance was read at 405 nm. One unit of enzyme releases 1.0 nmol of pnitrophenol per min at pH 11 at 37 °C when p-nitrophenyl butyrate was used as substrate. Standard curve of p-nitrophenol was used to calculate the specific activity of the enzyme. General Proteases Assay Cohen’s method [11] was used to assay the general proteolytic activity in the midgut of P. brassicae by using hemoglobin (20 mg/ml) as substrate. Hemoglobin solution (50 ll) was added to 100 ll of Tris–HCl buffer (20 mM, pH 9) and after addition of 20 ll of enzyme solution, incubation at 30 °C was initiated for 120 min. For termination of proteolysis, 150 ll of 10 % TCA was added to the reaction mixture by cooling down at 4 °C for 20 min. The reaction mixture was centrifuged at 13,000 rpm for 10 min. Blank solution contained all mentioned portions
Author's personal copy Secretagogue Mechanism of Digestive Enzyme Secretion
except the enzyme. The peptides liberated from hemoglobin were estimated using Folin-phenol reagent at 630 nm [12].
negative control was provided for each substrate separately containing all mentioned components except for enzyme pre-boiled at 100 °C for 30 min [13].
Determination of Specific Proteases
Protein Assay
Serine Proteinases
Protein concentrations were assayed according to the method described by Lowry et al. [14]. The method recruits reaction of Cu?, produced by the oxidation of peptide bonds with Folin–Ciocalteu reagent. In the assay, 20 ll of sample was added to 100 ll of reagent, and incubation was made for 30 min prior to read the absorbance at 545 nm (Recommended by Ziest Chem. Co., Tehran-Iran).
Trypsin-, chymotrypsin- and elastase-like activities (as three subclasses of serine proteinases) were assayed using a concentration 1 mM of BApNA (Nabenzoyl-L-arginine-p-nitroanilide), 1 mM SAAPPpNA (N-succinyl-alanine-alanineproline-phenylalanine-p-nitroanilide) and 1 mM SAAApNA (N-succinyl-alanine-alanine-alanine-p-nitroanilide) as substrates, respectively. The reaction mixture consisted of 35 ll of Tris–HCl buffer (20 mM, pH 8), 5 ll of each substrate and 5 ll of enzyme solution. The reaction mixture was incubated at 30 °C for a period from 0 to 10 min before adding 30 % TCA to terminate the reaction. The absorbance of the resulting mixture was then measured spectrophotometrically at 405 nm by p-nitroaniline release. To prove the specific proteolytic activity, a negative control was provided for each substrate separately containing all mentioned components except for pre-boiled enzyme at 100 °C for 30 min [13]. Cysteine Proteinases Cathepsin B, L and D activities were assayed using a concentration 1 mM of Z-Ala-Arg–Arg 4-metjoxy-bnaphtylamide acetate, N-benzoyl-Phe-Val-Arg-p-nitroanilide hydrochloride and cathepsin D (Sigma-Aldricht Co. Switzerland, LM0342) as substrates, respectively. The reaction mixture consisted of 35 ll of Tris–HCl buffer (pH 5), 5 ll of each mentioned substrate and 5 ll of enzyme solution. The reaction mixture was incubated at 30 °C for a period from 0 to 10 min before adding 30 % TCA to terminate the reaction. It was read at 405 nm. To prove the specific proteolytic activity, a negative control was provided for each substrate separately containing all mentioned components except for enzyme pre-boiled at 100 °C for 30 min [13]. Exopeptidases Activities of the two exopeptidases in the midgut of A. spinidens were obtained by using hippuryl-L-arginine and hippuryl-L-phenilalanine for carboxy- and aminopeptidases, respectively. The reaction mixture consisted 35 ll of Tris–HCl buffer (pH 7), 5 ll of each mentioned substrate and 5 ll of enzyme solution. The reaction mixture was incubated at 30 °C for a period from 0 to 10 min before adding 30 % TCA to terminate the reaction. It was read at 340 nm. To prove the specific proteolytic activity, a
Statistical Analysis All data obtained from a complete randomized design were compared by one-way analysis of variance followed by Tukey’s test. Significant differences were found at p B 0.05.
Results and Discussion Results of the current study clearly prove secretagogue mechanism of digestive enzyme secretions in A. spinidens. It was observed that gut of starved adults is small and shrunken but gut of fed adults after 12 h was normal and well shaped (Fig. 1). This finding could be attributed to perimicrovillae membrane in starved and fed hemipterans. Mehrabadi and Bandani [15] studied structural changes of midgut epithelial cells in the starved and fed conditions Eurygaster integriceps Puton (Hemiptera: Scutelleridae) by TEM and SEM observations. TEM showed that PMM was a little developed in starved insects so midgut cells had no PMM covering. SEM results revealed that midgut cells were not covered by PMM and margin of microvilli could be clearly seen. Similar results have been reported on Dysdercus peruvianus Gue´rin-Me´neville (Hemiptera: Pyrrhocoridae) and Rhodnius prolixus L. (Hemiptera: Reduviidae) [16, 17]. Meanwhile, Khan and Ford [18] reported cytologic changes in midgut epithelium of hemipterans. So, different shape of gut in starved and fed A. spinidens might be attributed to perimicrovillar membrane and cytology of epithelial cells of midgut. It was observed that there is little enzymatic activity in the adults kept under starvation for 8 days (Figs. 2, 3, 4, 5). Activity of general protease was stable in hungry adults up to 6 h post-feeding. It sharply increased and showed the highest activity at 24 h post-feeding (Fig. 2). Activity of a-amylase increased 6 h post-feeding and showed the highest activity at 12 h although the slight statistical
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Author's personal copy S. Sorkhabi-Abdolmaleki, A. Zibaee Fig. 1 Gut morphology in starved and fed adults of A. spinidens 12 h post-feeding. a Intact gut of starved adult, b determined gut sections in starved adult, c intact gut of fed adult, d determined gut sections in fed adult
difference was observed at 12 and 24 h post-feeding (Fig. 2). Digestive lipase activity increased 1 h post feeding and showed the highest activity after 12 h (Fig. 2). In case of specific proteases, the highest activities of trypsin-like, chymotrypsin-like and elastase-like proteases as three subclasses of serine proteases demonstrated the highest activity 12 and 24 h post-feeding (Fig. 3). It must be indicated that chymotrypsin-like protease activity was the highest at 12–48 h post feeding (Fig. 3). Activities of cathepsin B, L and D as three subclasses of cysteine proteases were the highest after 12 and 24 h post-feeding (Fig. 4). In case of two exopeptidases, the highest enzymatic activities were observed at 24 h post-feeding (Fig. 5). It has been reported that a minimal enzyme release is typical for many insect
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species at starvation [2]. In discontinuous feeders, such as seed-sucking insects, a regulated release of digestive enzymes occurs because of releasing stored enzymes in the epithelial cell’s cytoplasm [19]. Woodring et al. [20] suggested that some digestive enzymes such as amylase, trypsin, and aminopeptidase are secreted continuously at a basal rate in unfed Gryllus bimaculatus (Orthoptera: Gryllidae). The time for activity of digestive enzymes in A. spinidens varied from 6 to 24 h. This variation could be explained by the studies on grasshoppers [21]. A meal usually leaves foregut after 90 min. It is then pushed back by newly eaten food and it will reach hindgut to form feces. In the absence of more food, foregut is completely emptied in 5 h, and midgut is emptied after 8 h [21]. In Periplaneta
Author's personal copy Secretagogue Mechanism of Digestive Enzyme Secretion Fig. 2 Changes in general protease, a-amylase and lipase in the midgut of A. spinidens in starvation and different intervals post-feeding. Different letters show statistical differences among values by Tukey’s test (Pr [ F: 25.31, p B 0.0001, df = 3)
Fig. 3 Changes in serine protease activities in the midgut of A. spinidens in starvation and at different intervals of postfeeding. Different letters show statistical differences among values by Tukey’s test (Pr [ F: 14.71, p B 0.0001, df = 3)
Fig. 4 Changes in cysteine protease activities in the midgut of A. spinidens in starvation and at different intervals of postfeeding. Different letters show statistical differences among values by Tukey’s test (Pr [ F: 34.29, p B 0.0001, df = 3). Activity of Cathepsin D as OD/ min
americana L. (Blattodea: Blattidae), solid food passes through the gut in 20 h, but some of it can still be found in the crop after days of starvation [2]. Since A. spinidens does not eat another prey for about 24 h, long period of
digestive enzyme secretion is similar to the findings on grasshoppers and P. americana. The present findings indicated secretagogue mechanism of digestive enzyme secretions in A. spinidens. The mechanism
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Author's personal copy S. Sorkhabi-Abdolmaleki, A. Zibaee Fig. 5 Changes in exopeptidase activities in the midgut of A. spinidens in starvation and at different intervals of postfeeding. Different letters show statistical differences among values by Tukey’s test (Pr [ F: 18.97, p B 0.0001, df = 3)
has been clarified at molecular level in carnivorous and bloodfeeding insects. Felix et al. [22] believed that production of proteases especially trypsin-like in Aedes aegypti L. (Diptera: Culicidae) occurs in two phases after a blood meal. First secretion occurs 3 h after feeding. In fact, trypsin synthesis is a translation process of mRNA being stimulated by a protein in the ingested blood [22]. Then, the broken peptides from the initial phase of digestion stimulate production of further mRNA (transcription) in which additional trypsin is produced 8–10 h after feeding. In some insects like Spodoptera exigua Hubner (Lepidoptera: Noctuidae) and Heliothis zea Boddie (Lepidoptera: Noctuidae), trypsin activity as the amount of protein increases in the food [23]. Since increasing of digestive enzymes of A. spinidens occurs somehow in a linear way, so these findings could be attributed to the suggestions of Felix et al. [22], and Broadway and Duffey [23]. The current findings clarify that perimicrovilar membrane and physiological status of midgut epithelial cells in A. spinidens are different in starved and fed insects. Also, secretion and activity of digestive enzymes is regulated by feeding and digestion process. So, secretagogue mechanism of digestion and utilizing of extra-oral digestion has been proved in A. spinidnes and it may explain long period feeding on a prey. To find efficiency of a biological control agent (Both predator or parasitoid), a given number of preys are exposed to an agent within 24 or 48 h. In case of hemipterans, it takes about 20 h to eat a prey so this kind of experiment cannot be considered. Hence, the current study is useful to confirm long period feeding of the bug and second to find efficiency of a bug by biochemical and molecular approaches.
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