Journal of Insect Physiology 60 (2014) 58–67

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Journal of Insect Physiology

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Role of adipokinetic hormone in stimulation of salivary gland activities:The fire bug Pyrrhocoris apterus L. (Heteroptera) as a model species

0022-1910/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jinsphys.2013.11.005

⇑ Corresponding author at: Institute of Entomology, Biology Centre, Academy ofSciences, Branišovská 31, 370 05 Ceské Budejovice, Czech Republic. Tel.: +420 387775 271; fax: +420 385 310 354.

E-mail address: kodrik@entu.cas.cz (D. Kodrík).1 These authors contributed equally to this work.

Konstantin Vinokurov a,1, Andrea Bednárová a,b,d,1, Aleš Tomcala c, Tereza Stašková a, Natraj Krishnan d,Dalibor Kodrík a,b,⇑a Institute of Entomology, Biology Centre, Academy of Sciences, Branišovská 31, 370 05 Ceské Budejovice, Czech Republicb Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 Ceské Budejovice, Czech Republicc Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Flemingovo Sq. 2, 166 10 Praha 6, Czech Republicd Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 September 2013Received in revised form 8 November 2013Accepted 11 November 2013Available online 21 November 2013

Keywords:AKHSalivary glandsDigestive enzymescAMPPolygalacturonase

The effect of adipokinetic hormone (Pyrap-AKH) in stimulating the function of insect salivary glands(SGs) in extra-oral digestive processes was studied in the firebug, Pyrrhocoris apterus L. (Heteroptera).The analyses were performed on samples of SGs and extracts of linden seeds, a natural source of the bug’sfood. The SGs from 3-day old P. apterus females (when the food ingestion culminates), primarily con-tained polygalacturonase (PG) enzyme activity, whereas the level of lipase, peptidase, amylase and a-glu-cosidase was negligible. The transcription of PG mRNA and enzymatic activity were significantlyincreased in SGs after Pyrap-AKH treatment. The piercing and sucking of linden seeds by the bugs stim-ulated the intrinsic enzymatic cocktail of seeds (lipase, peptidase, amylase, glucosidase), and moreoverthe activity of these enzymes was significantly enhanced when the seeds were fed on by the Pyrap-AKH treated bugs. Similarly, a significant increase in PG activity was recorded in linden seeds fed onby hormonally-treated bugs or when injected by SG extract from hormonally treated ones as comparedto untreated controls. The mechanism of AKH action in SGs is unknown, but likely involves cAMP (andexcludes cGMP) as a second messenger, since the content of this compound doubled in SGs afterPyrap-AKH treatment. This new and as yet undescribed function of AKH in SGs is compared with theeffect of this hormone on digestive processes in the midgut elucidated earlier.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The insect alimentary tract provides a complex physical andchemical environment for processing of ingested food, and its mor-phology and physiology reflects the character of intaken food. Thesalivary glands (SGs) play an important role in digestive processesof many insect species. The saliva secreted by the SGs moistens thefood, adjusts its pH and ionic content, and often initiates the oral orextra-oral digestion. The enzymatic cocktail frequently containscarbohydrases and peptidases, as described in the saliva of somephytophagous and predaceous bugs: e.g. a-amylase has beenreported in Lygus hesperus and Lygus lineolaris (Zeng and Cohen,2000; Cooper et al., 2013) and serine peptidase in Oncopeltus fasci-atus (Francischetii et al., 2007); interestingly Woodring et al.(2007) also reported the presence of a cysteine peptidase in

salivary glands of the latter species. In a number of insects, SGsproduce specific enzymes which facilitate disruption of cell protec-tive barriers to reach the food source: the saliva of phytophagousbugs Pyrrhocoris apterus, L. hesperus or representatives of the Mir-idae family contains pectinase polygalacturonase (PG; the enzymedegrading polygalacturonan present in the cell walls of plants byhydrolysis of the glycosidic bonds) (Courtois et al., 1968; Fratiet al., 2006; Allen and Mertens, 2008; Walker and Allen, 2010);the saliva of predaceous and mycophagous species often containschitinases (Chapman, 1998). The salivary secretion of plant-sucking Hemiptera is ample in oxidases (peroxidase, catechol oxi-dase) which upon being injected into the plant tissue, participatein detoxification of phytochemicals (terpenes, alkaloids) and to-gether with other components of saliva forms the sheath materialsurrounding stylets during its passage through the leaf (Peng andMiles, 1988; Miles and Peng, 1989).

A paracrine hormonal regulatory mechanism is proposed tocontrol the synthesis and release of insect digestive enzymesproduced in the midgut (Lehane et al., 1996). The available dataindicate that most of the midgut digestive processes are undercontrol of specialized midgut endocrine cells expressing genes for

Fig. 1. Scanning electron microphotograph of salivary glands from 3-day old P.apterus female. (1) Anterior lobe; (2) medial lobe; (3) posterior lobe; (4) accessorygland; (5) salivary duct.

K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67 59

allatostatin, leucomyosuppessin, neuropeptide F and some otherneurohormones which probably control enzyme production inthe main midgut columnar cells by paracrine signalling (Audsleyand Weaver, 2009). Also adipokinetic hormone (AKH; for detailssee below) stimulates food digestion and increases peptidase andglucosidase activities in midgut as was recently demonstrated inthe firebug, P. apterus (Kodrík et al., 2012), however, this activationappeared to be secondary rather, or indirect.

Unlike in the midgut, the SG secretion is mostly controlled neu-rally via direct innervation from suboesophageal ganglion and/orstomatogastric nervous system. Nevertheless, this control alsoinvolves the participation of neuroactive substances such as sero-tonin, dopamine and octopamine (Trimmer, 1985; Novak et al.,1995; Ali, 1997). FMRFamide-like peptides were also identified inthe nerves innervating the SGs of the locust Locusta migratoria(Fusé et al., 1996) and the blowfly Calliphora vomitoria (Duveet al., 1992).

Salivation is probably regulated by serotonin and dopamine viaprocesses involving cyclic AMP and inositol-3-phosphate secondmessenger systems (Berridge, 1970, 2005). Both amines activatecorresponding receptors in the SGs resulting in elevated cAMP lev-els, and in ion and water transport by activation of vacuolarH+-ATPase (V-ATPase) and cation/proton exchanger in the apicalmembrane of SGs (Baumann and Walz, 2012). Dopamine has beenshown to stimulate the production of watery saliva, whereasserotonin triggers secretion of proteinaceous saliva in a dose-dependent manner in cockroaches (Just and Walz, 1996), however,the transduction mechanisms that mediate this effect are as yetunclear. Different modes of action are probably involved in suchregulations controlled by octopamine and FMRFamide-likepeptide, as neither of these substances was capable of directly ele-vating cAMP/cGMP levels although the salivation was stimulated(Ali and Orchard, 1994; Fusé et al., 1996).

No data are available to date on whether the SG function ininsects is regulated by adipokinetic hormones (AKHs). AKHs belongto the AKH/RPCH peptide family and are synthesized, stored andreleased from the neurosecretory cells of the corpora cardiaca(CC), an otherwise neurohemal organ connected to the brain. AKHscomprise of 8–10 amino acids (Gäde et al., 1997) and their signaltransduction at the cellular level is well documented in the fatbody (Gäde and Auerswald, 2003). Since AKHs are peptidic hor-mones, they are not able to freely penetrate the cell membrane,however, their message is transduced via specific membranereceptors (Park et al., 2002; Staubli et al., 2002; Kaufmann andBrown, 2006; Hansen et al., 2006). These receptors are typicalG protein-coupled proteins with seven membrane spanningdomains, and they are structurally related to receptors of the ver-tebrate gonadotropin releasing hormone. The mode of AKH actionvia these receptors is well characterised for biochemical pathwaysleading to the production of energy-rich substrates (Gäde andAuerswald, 2003). AKH receptors have been cloned or deducedfrom the genomic sequences of several insect species includingthe fruit fly, Drosophila melanogaster and silkworm, Bombyx mori(Staubli et al., 2002; Park et al., 2002). The AKH peptides restorehomeostasis, when it is disrupted in stress situations, by stimulat-ing catabolic reactions (mobilize lipids, carbohydrates and proline),making energy substrates more available (Gäde et al., 1997). How-ever, these peptides are pleiotropic, with a number of actions thatboost their main roles in stress energy metabolism (Kodrík, 2008).Among other actions, the AKHs stimulate heart beat (Scarboroughet al., 1984) and general locomotion (Socha et al., 1999; Kodríket al., 2000), enhance immune responses (Goldsworthy et al.,2002), regulate starvation-induced foraging behaviour of Drosoph-ila (Lee and Park, 2004), participate in the activation of the anti-oxidative mechanisms (e.g. Kodrík et al., 2007; Krishnan andKodrík, 2012; Bednárová et al., 2013b), and as found recently

(see also above), enhance food uptake and digestive processes ininsect gut (Kodrík et al., 2012).

Here, the effect of AKH on the activity of insect SGs, and the sub-sequent effect of the saliva on enzyme activities in the food mate-rial in a species using (partial) extra-oral digestion is reported. Thefirebug P. apterus is an appropriate model species for such studiessince it has well developed SGs which respond to Pyrap-AKH(Kodrík et al., 2000) treatment. The main aim of the study wastherefore a characterization of the effect of Pyrap-AKH on activa-tion of SG functions linked to the initiation of digestion of foodmaterial (linden seeds).

2. Materials and methods

2.1. Experimental animals

The stock culture of the firebug, P. apterus (L.) (Heteroptera)used in the present study, originated from specimens collected atCeské Budejovice (Czech Republic, 49�N). All stages, from egg toadult, were kept in small glass jars at a constant temperature of26 ± 1 �C under long-day conditions (LD 18:6 h) with an ad libitumsupply of linden seeds (Tilia cordata) and water. Three-day oldadult females (for specifics see Socha et al., 1997 and Kodríket al., 2012) were used in the experiments.

2.2. Chemicals

The P. apterus adipokinetic hormone Pyrap-AKH (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH2) (Kodrík et al., 2000) was commerciallysynthesized by Dr. Lepša, the Vidia Company (Praha, Czech Repub-lic). All chemicals and reagents used in the study were procuredfrom Sigma–Aldrich Co. (Praha, Czech Republic or St. Louis, USA)unless otherwise specified.

2.3. Hormonal treatment and sample preparation

A dose of 10 pmol Pyrap-AKH (for details of the selected dose,see Kodrík et al., 2000) dissolved in 2 ll 20% methanol in Ringersaline was injected through the metathoracic-abdominal interseg-mental membrane into the thorax of the experimental bugs kept instandard conditions (see Section 2.1.). Control bugs were injectedwith 2 ll of solvent only. The SGs (principal plus accessory glands)(Fig. 1) dissected at 24, 48 or 72 h (unless otherwise specified) afterthe treatment were collected in Eppendorf tubes (10 pairs/tubeunless otherwise specified), weighed and stored at �80 �C untilfurther use. In one experiment (see below) the midgut was

60 K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67

dissected from the bugs. The entire midgut (anterior, middle andposterior parts together) was washed in Ringer saline, placed sep-arately from individuals in eppendorf tubes, weighed and stored at�80 �C before further processing (Kodrík et al., 2012).

The linden seeds used in the experiments were carefullycleaned and then offered to the bugs for sucking (1 seed per 3 bugs)or immersed into water for 24, 48 or 72 h (see also below). Also,the effect of SG extract prepared by homogenization of the glandsin 0.2 M tris, pH 7.8 (3 SG pairs per 200 ll) on PG activity (seebelow) in linden seed was tested 24 h after injection of 2 ll(=0.03 SG pair equiv.) of the extract into an aperture made by a tinydrill into the seed. For subsequent processing, the testa (seed coat)of each of the seed was removed and the content dried in a vacuumdrier (Alpha 1–2 LD Plus, Christ). The dried tissue was weighed andstored at �80 �C until used.

2.4. Triacylglycerol determination

The triacylglycerols (TGs) from the linden seeds were extractedwith chloroform: methanol (2:1) solution following the method ofFolch et al. (1957) as modified by Koštál and Šimek (1998). Eachseed was homogenized in the solution and appropriate aliquotswere used for the TG determination according to Tomcala et al.(2010) using high performance liquid chromatography (HPLC)combined with the electrospray ionization mass spectrometry(ESI-MS) and ion trap LCQ FLEET mass spectrometry (Thermo,San Jose, CA, USA) coupled to a Rheos Allegro ternary HPLC system(Flux instruments, Basel, Switzerland), equipped with an Accelaautosampler (Thermo, San Jose, CA, USA).

2.5. Enzyme activity determinations

The lipase, peptidase, amylase, glucosidase and PG activitieswere determined in the SG and entire midgut extracts (used onlyfor PG – see Section 2.5.5.) as well as in samples of the linden seeds.The SGs or seed samples were homogenized (sonicated) in 0.2 Mtris pH 7.8, centrifuged and an appropriate aliquot (usually 3 SGpairs or 1/30 of the seed – unless otherwise specified) was testedfor enzyme activity.

2.5.1. Lipase activityLipase activity in salivary glands or linden seeds extracts was

assessed with 4-methylumbelliferyl butyrate (4-MU Butyrate) aspreviously described (Roberts, 1985; modified by Kodrík et al.,2012). Activity was expressed in pmol of 4-MU/min/mg of freshSG or dry seed weight.

2.5.2. Peptidase activityThe total peptidase activity was assessed with the resorufin-

casein kit (Roche) according to manufacturer’s instructions. Briefly,20 ll sample aliquots in 0.2 M tris pH 7.8 were mixed with 20 ll of0.4% substrate (resorufin-casein) and 20 ll of 0.02 M calcium chlo-ride solution, and adjusted up to 100 ll by the same tris buffer inthe microtubes. The mixture was subsequently subjected to gentleshaking for 1 h at 37 �C. The reaction was terminated by addition of240 ll of 5% trichloroacetic acid, and after 10 min of subsequentincubation at 37 �C, centrifuged to remove the non-hydrolyzedcasein. The absorbance was measured at 490 nm in 300 ll aliquotspipetted into 96-well microplates in a Spectra Max 340 PC reader(Molecular Devices). Appropriate controls without the sampleswere assayed simultaneously. Peptidase activity was expressedas a relative absorbance per mg of fresh SG or dry seed weight.

2.5.3. Amylase activityAmylase activity assay was performed with 3,5-dinitrosalicylic

acid reagent (DNS) according to Bernfeld (1955) modified by

Kodrík et al. (2012) and results were expressed in lmol maltose/min per mg of fresh SG or dry seed weight.

2.5.4. Glucosidase activity4-methylumbelliferyl a-D-glucopyranoside derivative was used

as a substrate for a-glucosidase assay as described previously(Kodrík et al., 2012). Glucosidase activity was expressed in pmolof 4-MU/min/mg of fresh SG or dry seed weigh.

2.5.5. Polygalacturonase activityPolygalacturonase (PG; EC 3.2.1.15) activity in SGs, midgut and

seeds was measured according to the rate of increase of reducingsugars using arsenomolybdate-copper reagent method (Nelson,1944; Somogyi, 1945) with some modifications by Torres et al.(2011) using D-galacturonic acid as standard. In brief, SGs (5 pairsper sample), midgut (2 midguts per sample) or seeds (5 seeds persample) were homogenized in 50 mM phosphate buffer and centri-fuged at 16,000g and the supernatant was used for the assay. Theprotein content of the supernatant was estimated using bicinchon-inic acid reagent (Pierce, Rockford, IL) and bovine serum albuminwas used as standard. In microfuge tubes enzyme supernatant wasmixed with 0.5% polygalacturonic acid solution (in 50 mM sodiumacetate buffer, pH 5.0) followed by incubation at 30 �C for 10 min.Negative controls contained 0.05% bovine serum albumin. The reac-tion was stopped by adding Cu–alkaline reagent and heated for15 min at 95 �C. Finally, arsenomolybdate reagent was added todevelop and stabilize the colour, mixed by vortexing and centrifugedat 6000g. The supernatant was pipetted into wells of a microtitreplate and absorbance was read at 540 nm in a BioTek SynergyH1 M plate reader. PG activity was expressed in units representinglmoles of D-galacturonic acid released per minute per mg protein.

2.6. Polygalacturonase mRNA expression

For PG expression studies, RNA from SGs (5 pairs per sample)and midgut tissue (2 midguts per sample) was extracted in TriZolfollowed by removal of genomic DNA by using recombinant DNaseI (TaKaRa Bio Inc, Japan). The RNA was further purified by precip-itation with 3 M sodium acetate and quantified using Take 3 nano-drop plate in a BioTek Synergy H1M plate reader. cDNA wassynthesized using iScript cDNA synthesis kit (BioRad, USA). Geneexpression was conducted in an Eppendorf realplex2 Mastercyclerusing Power SYBR Green PCR Mastermix (Applied Biosystems, USA)with the following primer sequence for PG- Forward: 50GGG GAGAGA CTG GGG CGA TT 30 Reverse: 50GAA CGG GGT CGC ATC ACCTG 30. The gene Rpl6 coding for 60S ribosomal protein L6 (Forward:50TGC TGG CCG GTC TTC ACA AAG G 30 Reverse: 50AGG TGG GGATGA CGA AGT TGG G 30) was used as normaliser. The primersequences were designed based on L. lineolaris PG primers (Walkerand Allen, 2010). Gene expression was quantified using 2�DDCT

method (Livak and Schmittgen, 2001).

2.7. cAMP/cGMP level determinations

The experimental bugs were treated by Pyrap-AKH as men-tioned above (see Section 2.3.). The SGs were dissected 5 min afterthe treatment in ice-cold Ringer saline solution, weighed andstored at �80 �C until needed. Three pairs of SGs per sample wereused for the cAMP and ten pairs for the cGMP determination.

– cAMP determination (cAMP-Screen System; Applied Biosystems) –the SGs were thawed on ice and gently sonicated in 60 ll ofassay/lysis buffer. The lysate was incubated for 15 min at roomtemperature and used for the reaction based on a chemilumines-cent immunoassay according to the manufacturer’s instructions.This immunoassay – direct ELISA – utilizes chemiluminiscent

Fig. 3. Temporal changes of polygalacturonase mRNA transcription (A) andenzymatic activity (B) in salivary glands of control (open columns) and Pyrap-AKH treated (10 pmol; hatched columns) 3-day old P. apterus females. Statisticallysignificant differences at the 5% level between the control and AKH treated SGs areevaluated using two-way ANOVA with the Bonferroni’s post test (variant ofcomparison of means within the relevant hour) and indicated by asterisks (n = 4).

Fig. 4. Composition of the linden seed. The analysis done and results obtained bycourtesy of Dr. Jirí Horácek (Agritec Šumperk, Czech Republic).

K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67 61

1,2-dioxetane enzyme substrates which provide an ultrasensi-tive assay with a wide dynamic assay range of cAMP quantifica-tion. Light signal intensity is inversely proportional to the cAMPlevel in the sample in this assay, and is measured in a lumino-meter 30 min after substrate addition (Chiulli et al., 2000).

– cGMP determination (cGMP Enzyme Immunoassay Kit; Sigma) –the SGs were thawed on ice, mechanically homogenized andgently sonicated in 100 ll of 5% trichloroacetic acid. The lysatewas centrifuged at 12,000g for 10 min at 4 �C, the supernatantmixed with 300 ll of water saturated ether (1:1) and evapo-rated in a vacuum concentrator. The residue dissolved in100 ll of assay buffer (containing proteins, detergents andsodium azide) was directly used for the cGMP level detection.The competitive immunoreactive assay using the acetylationmodification (due to the low cGMP level in the samples) wasdone according to the manufacturer’s instructions. The reactionuses a polyclonal antibody to cGMP to bind, in a competitivemanner, to the cGMP in the sample. After 2 h incubation a yel-low colour is generated in the reaction mixture and is read in areader at 405 nm; the colour intensity is inversely proportionalto the concentration of cGMP (Chard, 1990).

2.8. Electron microscopy analysis

For a better understanding of the function of SGs their anatomywas documented using conventional scanning electron micros-copy. For the study, P. apterus SGs were dehydrated in a series ofethanol solutions, dried, glued on aluminium holder, sputter-coated with gold and observed and photographed using a Jeol6300 scanning electron microscope.

2.9. Data presentation and statistical analysis

The results were plotted using the graphic program Prism(GraphPad Software, version 6.0, San Diego, CA, USA). The bargraphs and the points in one linear graph (Fig. 5) represent themean ± SD. Statistically significant differences at the 5% level wereevaluated by Students t-tests (Figs. 2, 5, 8 and 9), by two-wayANOVA with the Bonferroni’s post test (Figs. 3 and 7) and byone-way ANOVA with the Dunnett’s (Fig. 5) and Tukey’s post tests(Fig. 6). For the number of repetitions (n) see the figure legends.

3. Results

3.1. Enzymatic characterization of P. apterus salivary glands

The attempts to determine lipase, peptidase, amylase andglucosidase activities in SGs revealed that this organ contains no

Fig. 2. Polygalacturonase mRNA transcription (finely hatched columns) and enzy-matic activity (coarsely hatched columns) in salivary glands (SG) and midgut of 3-day old P. apterus females. Statistically significant differences at the 5% levelbetween the SGs and midgut are evaluated using the Student’s t-test and indicatedby asterisks (n = 5–12).

Fig. 5. Temporal changes in content of triacylglycerols (TGs) in linden seedsimmersed into water (dashed line), or pierced and sucked by 3-day old P. apterusfemales (solid line). Statistically significant differences at the 5% level (1) betweenthe both groups within the relevant time (water vs. bugs) were evaluated using theStudent’s t-test and indicated by asterisks; and (2) between the control (time 0) andboth experimental groups (effect of time) were evaluated using one-way ANOVAwith the Dunnett’s post test and are indicated by @ (n = 4–8).

or just trace levels of this conventional set of digestive enzymes(data are not shown). On the other hand, expression of the polyga-lacturonase (PG) gene and activity of the corresponding enzymewas documented in P. apterus SGs (Fig. 2) suggesting the secretionof this enzyme into the saliva. Furthermore, the transcription of PG

Fig. 6. Temporal changes of lipase (A) and amylase (B) activities in linden seedsimmersed into water. Statistically significant differences at the 5% level betweenthe columns are evaluated using one-way ANOVA with the Tukey’s post test and areindicated by different letters (n = 9).

62 K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67

mRNA in SGs was about 2.6-fold higher than that in the midgut,while the PG activity in SGs was 2.2-fold lower than in the midgutindicating activation of the enzyme outside the SGs.

Fig. 7. Temporal changes of lipase (A), peptidase (B), amylase (C) and a-glucosidase (D)AKH (10 pmol) treated (hatched columns) 3-day old P. apterus females. The time of expos0 (full columns) represents untreated seeds. The numbers above the columns representbetween the control and Pyrap-AKH treated groups were evaluated using two-way ANrelevant hour) and indicated by asterisks (n = 4–9).

3.2. The effect of Pyrap-AKH on P. apterus salivary gland activities

The activity of PG determined in SGs (see above) seems to beunder AKH control. The application of 10 pmol Pyrap-AKH signifi-cantly stimulated the PG gene expression in SGs 6 h after the hor-monal treatment (Fig. 3A). This stimulation was followed by aconcomitant significant elevation of enzyme activity (2.2-fold)reaching a maximum at 24 h post treatment (Fig. 3B).

As mentioned above, P. apterus saliva does not contain an effec-tive level of its own classical digestive enzymes which wouldensure a liquefaction of the solid content of the linden seeds. Theseseeds are rich primarily in lipids and proteins (Fig. 4) accountingfor about two thirds of the nutrient content; on the other hand,the level of free sugars is lower while the starch content, especiallyits soluble fraction, is negligible (2%).

In subsequent experiments it was tested whether the salivaserves as an inducer of the seeds own enzymatic apparatus. Thetriacylglycerol (TG) degradation was monitored in linden seedsimmersed for 24–72 h in water or in the seeds offered to the exper-imental bugs as food (Fig. 5). The results showed a significantdecrease of TG content in the both groups of seeds, however, theeffect of saliva was more intensive: 72 h after the treatments theTG level in the seeds pierced-sucked by the bugs was significantlylower than in the seeds just immersed in water. Interestingly, sim-ple immersion of seeds in water indeed significantly activated notonly lipase, but amylase activity as well (Fig. 6).

In another set of experiments it was tested whether the stimu-lation of seed hydrolases by saliva from piercing-sucking bugscould be enhanced when the Pyrap-AKH treated bugs areemployed. The results showed a significantly more intensive stim-ulation of all tested enzymes – lipase, peptidase, amylase andglucosidase (Fig. 7) – in the seeds injected by saliva from thehormonally treated bugs. Despite only a low level of starch in thelinden seeds (see Fig. 4), the highest stimulation was recoded foramylases (Fig. 7C). Further, 1.4–4.3-fold significant stimulation

activities in linden seeds pierced and sucked by control (opened columns) or Pyrap-ure represents a period from the giving to taking away the seeds from the bugs; timefold-change by AKH stimulation. Statistically significant differences at the 5% levelOVA with the Bonferroni’s post test (variant of comparison of means within the

Fig. 8. Polygalacturonase activity in linden seeds 24 h after the experimentaltreatment. The seeds were immersed into the water (open column); pierced andsucked by control or Pyrap-AKH (10 pmol) treated 3-day old P. apterus females(finely hatched columns); and injected by salivary gland (SG) extract from controlor Pyrap-AKH (10 pmol) treated 3-day old P. apterus females (coarsely hatchedcolumns). The numbers above the columns represent fold-change by AKH stimu-lation. Statistically significant differences at the 5% level between the experimentalgroup and relevant control are evaluated using the Student’s t-test and indicated byasterisks (n = 6).

Fig. 9. Levels of cAMP and cGMP in salivary glands of control and Pyrap-AKHtreated 3-day old P. apterus females. The levels were determined 5 min after theRinger saline (control) or Pyrap-AKH (AKH) injection. The number above thecolumn represents fold-change by AKH stimulation. Statistically significant differ-ences at the 5% level between the control and Pyrap-AKH treated groups areevaluated using the Student’s t-test and indicated by asterisks (n = 4–9).

K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67 63

was recorded also for lipase (Fig. 7A) and a-glucosidase (Fig. 7D)activities. Peptidase activity (Fig. 7B) although lower, was signifi-cantly stimulated at least at 72 h post treatment.

The immediate Pyrap-AKH effect on SG enzymatic action wasrecorded when the activity of PG was monitored in similar exper-iments (Fig. 8). The PG activity was 3.7 times higher in the seedssucked by the bugs treated with 10 pmol of Pyrap-AKH (24 h treat-ment) as compared with the control bugs; the increase of PG activ-ity in seeds injected by SG extracts from hormonally treated bugscompared to control ones was almost 9-fold. There were no differ-ences recorded among the PG activities in seeds immersed inwater, sucked by control bugs or injected by the SG extract fromcontrol bugs.

3.3. The effect of Pyrap-AKH on the cAMP/cGMP level in salivary glands

The level of cAMP and cGMP was determined in SGs from con-trol and Pyrap-AKH treated bugs to elucidate the possible mecha-nism of AKH action in the SGs. The results revealed (Fig. 9) asignificant increase (about 2-fold) of cAMP level in SGs after

hormonal treatment. On the other hand, no hormonal effect onthe cGMP level was documented in this experiment. Additionally,cGMP level was almost 700 times lower than the level of cAMPin SGs.

4. Discussion

4.1. Role of salivary glands and initiation of food degradation

Enzymatic degradation of nutrients immediately inside the foodsource, prior to ingestion, is a characteristic feature of insects uti-lizing extra-oral digestion. The classical model supposes that thereis an enzymatic cocktail present in the saliva that liquefies the foodto facilitate ingestion and initiates its preliminary chemical degra-dation before the food is delivered into specialized organs insidethe body. The use of an appropriate suite of enzymes for utilizationof the content of linden seeds is certainly essential also for P. apte-rus. However, the absence of conventional digestive enzymes –lipases, peptidases, amylases and glucosidases – in the bug’s SGsis shown in this work. Importantly, P. apterus individuals, used inour experiments were fed exclusively on linden seeds and didnot have any access to alternative food. Moreover, the 3rd day afteradult emergence is characterized by a peak of feeding activityaccompanied by a considerable increase of the weight of the gut(Socha et al., 1997) and a rise of digestive hydrolase levels (Sochaet al., 1998; Kodrík et al., 2012). To clarify the role of such digestivehydrolase-free saliva in utilization of the linden seeds nutrients itwas hypothesized that injection of P. apterus saliva activates theenzymatic apparatus of dry linden seeds. The mechanism of activa-tion remains unknown; however, its pattern seems to resembleprocesses occurring at seed germination induced by water uptakeas described in detail in relevant literature (see e.g. Heldt, 2005).The results of the experiments where the seeds were immersedin water and/or offered to the bugs for piercing and sucking sup-port this assumption. In both cases, the activity of seed lipasesand amylases was significantly stimulated. It is interesting to notethat the level of seed lipase and amylase activities elicited by thebug saliva (see Fig. 7) was about 1–2 order of magnitude higherthan those in seeds stimulated by water only (see Fig. 6) as it prob-ably occurs during the ordinary seed germination. One can proposethat saliva might intensify the enzyme activity also by more suit-able ionic or pH milieu. Also, the enzyme polygalacturonase couldplay a role: it might degrade cell walls and thereby trigger enzyme/substrate decompartmentalisation ending in higher enzyme activ-ities. Nevertheless, in the absence of supporting data, this remainsa speculation only.

It seems that primarily lipases play an important role in P. apte-rus digestion (Socha et al., 1997, 1998; Šula et al., 1998) becausethe linden seeds are rich mainly in lipids (this work) representinga substantial part of the bug food (Bártu et al., 2010). It is also verywell documented that energy metabolism of P. apterus uses pri-marily lipids while consumption of carbohydrate sources is minor(Kodrík et al., 2000; Kodrík et al., 2002; Socha et al., 2004).

A proof of utilization by pyrrhocorid bugs of digestive enzyme-free saliva for activation of plant hydrolases is provided here forthe first time. It is interesting that salivary secretion of other pyr-rhocorid Dysdercus peruvianus also lacks digestive enzymes (Silvaand Terra, 1994), whereas an array of digestive hydrolases wasfound in saliva of mirid and lygaeid bugs (Laurema et al., 1985;Woodring et al., 2007). One can only speculate that utilization ofhydrolase-free saliva by some pyrrhocorids might be an adaptationto feeding on seeds of mallows plants (Malvaceae) – linden seeds(T. cordata) by P. apterus and cotton seeds (Gossypium sp.) by D.peruvianus. The composition of cotton and linden seeds is very sim-ilar – besides lipids, both are relatively rich in proteins and soluble

64 K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67

reducing sugars, but contain a low level of starch (Silva and Terra,1994).

The presence of the enzyme polygalacturonase (PG) was provenin the P. apterus SGs (first reported by Courtois et al. (1968)) whichis apparently both expressed and synthesized in this organ. Thepresence of PG, known mostly from fungi, bacteria and plants,has been also recorded within insects especially in the speciesfeeding by piercing-sucking (e.g. Shen et al., 1996; Cohen, 1998;Shackel et al., 2005), but also by chewing (Girard and Jouanin,1999; Shen et al., 2003). This enzyme is typically present in theSGs (only traces were identified in the midgut lumen) of Heterop-tera representatives especially in the Miridae family where itdegrades pectin from the plant cell walls facilitating to overcomethis physical protection barrier (Laurema et al., 1985; Cohen,1998; Shackel et al., 2005; Frati et al., 2006).

Initially the function of PGs was thought to be related to thedigestion of plant material and it was also hypothesized that theseenzymes were synthesized by symbiotic microorganisms present inthe gut of insects (Shen et al., 1996; Doostdar et al., 1997; Girardand Jouanin, 1999). However, PG from Sitophilus oryzae (L.) isencoded in the insect’s genome and its expression is documentedin the midgut epithelium (Shen et al., 2003). Recent studies alsoindicate that beetle genomes also incorporate genes of enzymes in-volved in degradation of plant cell walls commonly belonging to thefamily of so-called plant cell wall degrading enzymes (PCWDEs)(Pauchet et al., 2010; Kirsch et al., 2012). Thus, PGs could be aninherent part of the genome of phytophagous insects and also a partof the offensive arsenal of these insects serving roles in digestiveprocesses as well as establishment on host plant. Nevertheless,the PGs originating in midgut found in chewing insects especiallyleaf beetles and grain weevils (Girard and Jouanin, 1999; Shenet al., 2003) consuming large pieces of plant tissues, help only toextract proteins and sugars from the food, not working as truedigestive hydrolases (Laurema et al., 1985). The gene structures ofthree PGs have been characterized in the bug L. lineolaris (Allenand Mertens, 2008; Walker and Allen, 2010). In the present worka relatively high expression of PG in SGs compared to the midgutwas found. The activity however, showed a reverse pattern: rela-tively low activity in SGs and significantly higher in the midgut.Considering the activity of other digestive enzymes and the mainrole of PG in insect–plant interaction and feeding strategies, it issupposed that the main site of the PG action will be in the lindenseed. A certain level of PG activity recorded in the seeds immersedin water (see Fig. 8) may be representative of activation of inherentPG enzyme activity of seeds when exposed to moisture, since plantsare also known to harbour PGs necessary primarily for cell wall re-modelling growth, ripening and seed germination (Kim et al., 2006).Some activity in the midgut comes probably partially from the ownPG expression, however, a substantial part could be delivered tothis organ from the SGs with saliva saturated liquefied seedmaterial (containing possibly also the seeds’ own PG) accumulatedin a large-scale in the widened anterior midgut part of P. apterus.

4.2. Hormonal control of salivary gland activities

It is generally accepted that hormones play a pivotal role in theregulation of feeding activities in insects (reviewed by Audsley andWeaver, 2009). However, such hormonal regulation is a complexphenomenon involving a number of mechanisms, the precise modeof action still poorly understood. The involved (neuro)hormonesare produced either directly in specialized midgut endocrine cellsor in other endocrine centres outside the digestive system; bothgroups control digestive enzyme synthesis and secretion, as wellas gut peristalsis. The direct neurohormonal effect on the insectdigestive enzymes was reported for allatostatin and leucomyosu-pressin (Fusé et al., 1999), crustacean cardioactive peptide (Sakai

et al., 2004), trypsin modulating oostatic factor (Borovsky et al.,1990) and others. Also AKH appears to affect the digestive func-tions: as demonstrated in P. apterus (Kodrík et al., 2012), the Pyr-ap-AKH treatment could stimulate food consumption (seebelow), because significantly increased level of lipids (both tri-and diacylglycerols) and proteins in the midgut was recorded afterhormonal treatment. Further, no hormonal effect on lipase activitywas recorded here, while peptidase and a-glucosidase activitieswere significantly increased. These results indicated that the Pyr-ap-AKH probably intensifies food turnover by stimulating foodingestion and metabolite absorption; the activation of digestiveenzymes seems to be secondary or controlled by othermechanisms.

The present results demonstrate that the SGs are likely alsopotential target organs for AKH activities. The hormonal treatmentsignificantly increased both the PG expression and its activity inthe SGs. It is interesting to note that the stimulatory effect ofAKH was recorded at 6 h after the treatment for the PG mRNA tran-scription and after 24 h for PG activity, respectively. This suggeststhe involvement of a more complicated pathway or cascade in themechanism. The delayed effect after the topical AKH treatment,although not so prolonged, has been recorded when the lipid mobi-lization and locomotory activity was monitored in P. apterus(Kodrík et al. 2002). Maximal lipid level and maximal locomotoryactivity were recorded 4 h and 8 h, respectively, after the AKHtreatment; nevertheless, the delayed response in this experimentcan probably be explained mostly by a slow penetration of the hor-mone through the integument. Moreover, the increased PG mRNAexpression at 6 h would eventually be translated to protein involv-ing several post-translational processes showing increased enzy-matic activity at 24 h post Pyrap-AKH treatment. The precisecause for this delayed response between stimulation of enzymemRNA expression and its activity by AKH, however, remains un-clear at present.

Further, a significant stimulation of linden seeds hydrolases wasrecorded when the seeds were pierced and sucked by AKH treatedbugs as compared to untreated controls. One can speculate that thesignalling pathway might include the stimulation of SG enzymeactivities (i.e. PG or maybe other enzymes not detected as yet)and also induction of mechanisms such as V-ATPase and cation/proton antiporter leading to increased water uptake from haemo-lymph. Thus, the higher volume of saliva produced could lead tohigher activation of the seed enzymes (see Fig. 10 summarizingthis hypothetical model). The precise subcellular mechanism is un-known; however, one can postulate that putative signal transduc-tion could include the G-protein coupled receptor (GPCR) knownfor AKHs in several insect species (e.g. Park et al., 2002; and manyothers) involving cAMP as a second messenger. This assumption issupported by our results because the Pyrap-AKH treatment signif-icantly increased the cAMP level in SGs. The potential involvementof cAMP is not surprising because a number of peptides regulatingwater transport in insect organs are reported to use cAMP as a sec-ond messenger (Coast et al., 1991). The involvement of cGMP isunlikely, because its level in the SG cells is very low and its in-crease after the hormonal treatment was not detectable. cAMPusually mediates activation of the protein kinase A pathway thatelicits gene transcription; the involvement of this pathway couldexplain the stimulation of PG mRNA expression in the SGs ofPyrap-AKH treated bugs. Alternatively, protein kinase A can alsostimulate various cellular enzymes through their phosphorylationor dephosphorylation. The involvement of this pathway has beendescribed for the AKH stimulation of lipid mobilization. Arreseet al. (1999) have shown that the endogenous AKH of Manducasexta rapidly increases the activity of protein kinase A which is fol-lowed by activation of triacylglycerol lipase and the release of lip-ids into the haemolymph. It cannot be excluded that AKH signal

Fig. 10. A hypothetical model of the stimulatory effect of Pyrap-AKH on P. apterussalivary glands leading to subsequent induction of enzyme activity in linden seedafter the injection of saliva. CC – corpora cardiaca, AKH – Pyrap-AKH. The photo ofthe bug on linden seed was obtained by courtesy of Dr. František Weyda (CeskéBudejovice, Czech Republic).

K. Vinokurov et al. / Journal of Insect Physiology 60 (2014) 58–67 65

transduction in the SGs also uses this enzyme activating protein ki-nase A pathway - both for the AKH activation of PG and for thestimulation of other SG activities. A similar pathway including pro-tein kinase A is also used when the glycogen phosphorylase is acti-vated by AKH action (van Marrewijk et al., 1983), but this might bea rare occurrence having been described only in locusts, so far;AKH mediated activation of glycogen phosphorylase usually use apathway employing phospholipase C and inositol triphosphate(Gäde and Auerswald, 2003). Both pathways – or at least cAMPand protein kinase C – seem to be involved in AKH signal transduc-tion during oxidative stress (Bednárová et al., 2013a; Bednárováet al., 2013c).

Serotonin has been reported to play an important role in regu-lating SG activity (Berridge, 1970; Orchard, 2006; Rein et al., 2008;Baumann and Walz, 2012). Serotonin identified both in the SGinnervation and in the intrinsic gland tissue, stimulated saliva pro-duction via vacuolar H+-ATPases by employing cAMP as a secondmessenger. Additionally, serotonin was reported to increase alsothe amplitude of phasic contractions of muscles surrounding theSGs (Orchard, 2006). It is not known if there is any relationshipbetween serotonin or any other biogenic amine and the describedeffect of Pyrap-AKH on SGs. However, it has been established thatcertain biogenic amines (octopamine, dopamine, norepinephrine,serotonin, tyramine) are involved in regulation of AKH production(Van der Horst et al., 2001; Orchard et al., 1993). In P. apterus it hasalso been shown that biogenic amine norepinephrine increased theAKH level in haemolymph and thus (indirectly) stimulated lipidmobilization and walking activity (Socha et al., 2008).

Previous work has also dealt with the effect of peptidic factors/hormones on the SG activity. Duve et al. (1991) reported that sev-eral neuropeptides isolated from nervous tissue stimulated fluidsecretion in isolated SGs of C. vomitoria. Shortly after, a similareffect was proven also in L. migratoria (Veelaert et al., 1995) wherea peptide originating from the SGs (mw 1779 Da) stimulated cAMPproduction and increased salivation.

In conclusion, the present work demonstrates a possible newphysiological function of AKH that is probably closely connectedwith the recently described stimulatory function of AKH on insectdigestion (Kodrík et al., 2012). In P. apterus the Pyrap-AKH (1) stim-ulated the expression and activity of PG in SGs; (2) increased activ-ity of enzymatic systems in the linden seeds mediated by salivausing an unknown mechanism; (3) the mechanism of Pyrap-AKH

action in SGs at the subcellular level probably involves cAMP asthe second messenger.

Acknowledgements

This study was supported by the Grant No. P501/10/1215 fromthe Grant Agency of the Czech Republic (DK), by the start-up fundsMSU#269110-151250 (NK) from NSF, EPSCOR, and by the Instituteof Entomology support RVO: 60077344. The authors thank Mrs. D.Rienesslová and Mrs. J. Pflegerová for their technical assistance, Dr.Jirí Horácek (Agritec Šumperk) for analysis of linden seeds and Dr.František Weyda (University of South Bohemia) for providing of P.apterus photo.

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