Journal of Insect Physiology 58 (2012) 1334–1342

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Journal of Insect Physiology

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Digestion, growth and reproductive performance of the zoophytophagous rovebeetle Philonthus quisquiliarius (Coleoptera: Staphylinidae) fed on animaland plant based diets

Matías García, Gema P. Farinós, Pedro Castañera, Félix Ortego ⇑Laboratorio de Interacción Planta-Insecto, Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:Received 16 May 2012Received in revised form 6 July 2012Accepted 16 July 2012Available online 25 July 2012

Keywords:ZoophytophagyPhilonthus quisquiliariusDigestive physiologyDietary mixingFeeding habits

0022-1910/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jinsphys.2012.07.007

⇑ Corresponding author. Tel.: +34 918373112; fax:E-mail address: ortego@cib.csic.es (F. Ortego).

a b s t r a c t

The zoophytophagous feeding habits of larvae and adults of the rove beetle, Philonthus quisquiliarius(Gyllenhal) (Coleoptera: Staphylinidae), are reported for the first time. This study evaluates the effectsof different feeding regimes on its growth and reproductive performance (i.e., larval growth, adult weightgain, consumption, fecundity and fertility) and digestive physiology. Larvae presented similar growthrates when fed on living animal or on green plant material for 48 h. However, higher consumption ratesand lower efficiencies of conversion of digested matter to body mass were obtained when leaves wereconsumed. Adults presented also positive weight gains regardless of the food consumed (plant or animalmaterial). Interestingly, the highest weight gain rate and efficiency of digestion resulted when adults fedon a rearing diet containing nutrients from both animals and plants. Moreover, we have found negativeeffects upon P. quisquiliarius fecundity and fertility when supplemental plant nutrients were removedfrom the optimum rearing diet. Physiological adaptations to allow trophic switching between predationand phytophagy have been found, such as the higher ratio of a-amylase activity to protease activity todeal with the inverted protein-carbohydrate ratio of plant versus animal tissues. Furthermore, this spe-cies has an arsenal of digestive proteases whose activity is affected by the type of diet ingested. Alltogether, our results suggest that P. quisquiliarius needs certain nutrients, which are obtained only fromplant material. This knowledge will help to understand the complex trophic interactions that occur inagroecosystems.

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1. Introduction

Zoophytophagous insects are described as predatory speciesthat are able to consume animal and plant tissue within a singlelife-stage (Coll and Guershon, 2002). In agroecosystems, zoophyto-phagous species are found within the orders Hemiptera, Neurop-tera, Thysanoptera and Coleoptera; and to varying degrees theyexploit leaf and fruit tissue, pollen, floral nectar and extra-floralsecretions (Coll and Guershon, 2002; Moser et al., 2008; Robinsonet al., 2008). There are two hypotheses regarding the consumptionof plant tissue by zoophytophagous insects: one that relates thisfeeding behavior with reduced prey or water availability, and an-other that is related with the need of specific nutrients that preda-tion alone does not provide (Moser et al., 2008; Torres et al., 2010).Because plant and prey diets differ greatly in their chemical com-position and nutritional value, combining both food source in thediet requires specific physiological and morphological adaptations(e.g., digestive enzymes) (Coll and Guershon, 2002). Knowledge

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about the digestive physiology in zoophytophagous insects hasprogressed significantly in the recent years, mainly due to studiesperformed on hemipteroids (Boyd et al., 2002; Guedes et al., 2007),which have been proven to be biochemically suited to digest ani-mal and plant material (Zeng and Cohen, 2000, 2001).

Staphylinidae is one of the largest beetle families with morethan 47,000 species distributed worldwide in a wide range of eco-systems (Vásquez-Vélez et al., 2010). Most rove beetles, such asthose species of the genus Philonthus, are primarily considered car-nivorous and have been reported to play an important role as pre-dators in agroecosystems (Hu and Frank, 1997; Balog et al., 2008;Vankosky et al., 2011). Other species of Staphylinidae have beendescribed as mycophagous or saprophagous (Bohac, 1999), thoughthere are a few species that have been reported as herbivorous(Bouchard et al., 2009; Klimaszewski et al., 2010). Yet, to ourknowledge there are no reports of zoophytophagous species withinthis group. However, while studying tritrophic interactions in ourlaboratory with Philonthus quisquiliarius (Gyllenhal) (Coleoptera:Staphylinidae), a predatory species present in Spanish maize (Zeamays L.) fields, we noticed that larvae and adults of this specieswere consuming maize green leaf material regardless of the

M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342 1335

presence of water or prey (personal observations). Often observedin zoophytophagous hemipterans (Zeng and Cohen, 2001; Guedeset al., 2007), green leaf tissue feeding by predatory beetles is notcommon and it is normally attributed to accidental ingestion andthe need for water, as reported for the coccinellids Harmonia axy-ridis Pallas and Coleomegilla maculata DeGeer (Coleoptera: Cocci-nellidae) (Moser et al., 2008). These authors studied thelikelihood of these predatory larvae to feed on maize seedlings,and the effects of plant exposure on development time and adultsize. Moreover, Ferry et al. (2003) studied the proteolytic activityof H. axyridis when fed on a prey. However, to the best of ourknowledge there are no studies that have analyzed the physiolog-ical adaptations of zoophytophagous coleopterans when consum-ing plant or animal based diets.

The main aims of this research were to evaluate the effects ofdifferent feeding regimes on P. quisquiliarius growth and reproduc-tive performance (i.e., larval growth, adult weight gain, consump-tion, fecundity and fertility), and to analyze the hydrolyticdigestive adaptations allowing trophic switching between preda-tion and phytophagy.

2. Materials and methods

2.1. Plant and insect cultures

Maize plants (variety DKC6040) were grown in plastic pots(30 cm diameter) using peat (Compo Sana Universal�, Compo Agri-cultura SL, Barcelona, Spain) as substrate and maintained in agrowth chamber (Sanyo MLR-350, Sanyo, Japan) at 25 ± 0.3 �C,70 ± 5% RH and L:D 16:8 h photoperiod. Maize plants were usedwhen reached the five-leaf stage.

A laboratory colony of P. quisquiliarius was established in 2008from adults collected in a experimental (variety DKC6040) maizefield in Madrid, Spain. They were reared according to Carneyet al. (2002) as follows: adults and larvae were reared in plasticboxes (23.5 � 23.5 � 5.5 cm) containing the rearing substrate,which consisted of a mix of peat, coconut fiber and vermiculite(at a 4:2:1 volume ratio). The moisture level was achieved by add-ing 200 ml of water weekly. Rearing food (RF) was a mixture of dogfood (25% protein, 12% fat, 3.4% cellulose) (Brekkies Excel Tenderand Delicious�, Affinity Petcare SA, Barcelona, Spain) and oatmeal(13.8% protein, 6.4% fat, 56.1% carbohydrate) (Kllön�, Peter KllönKGaA, Elmshorn, Germany), at a 4:1 weight ratio, ground to a thinpowder. Once a week, 50 g of this mixture was added into each cul-ture. This setting permitted the development and maintenance ofother organisms like nematodes and sciarid larvae present in thepeat. Adults and larvae of P. quisquiliarius feed on these organismsas well as on the rearing diet. Rearing and all experiments wereconducted at 20 ± 0.3 �C, 80 ± 5% RH and 16:8 h L:D photoperiodin a growth chamber (Sanyo MLR-350 H, Sanyo, Japan).

The red spider mite, Tetranychus urticae Koch (Acari: Tetrany-chidae), came from a permanent laboratory colony, maintainedon maize plants at 25 ± 0.3 �C, 70 ± 5% RH and L:D 16:8 h (SanyoMLR-350) photoperiod for several generations.

Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) larvaewere obtained from a permanent laboratory colony, maintained ona meridic diet for several generations as described in Farinós et al.(2004).

2.2. Enzymatic activities in P. quisquiliarius

Characterization of P. quisquiliarius hydrolytic enzymes wasdone using decapitated adults (2–5 days old) and whole third in-stars larvae (L3) obtained from the laboratory colony. Insects werehomogenized in 0.15 M NaCl (600 ll NaCl), centrifuged at 12,000g

for 5 min, and the supernatants pooled and stored frozen (�20 �C)until needed. All enzymatic activities were performed at 30 �C, as-says were carried out in triplicate and blanks were used to accountfor spontaneous breakdown of substrates. A series of overlappingbuffers were used to generate a pH gradient from 2 to 11: 0.1 Mcitric acid–NaOH (pH 2.0–5.0), 0.1 M sodium citrate (pH 3.0–5.0),0.1 M Tris–HCl (pH 6.5–9.0), 0.1 M sodium phosphate (pH 6.0–8.0) and 0.1 M glycine–NaOH (pH 9.0–11.0). All buffers contained0.15 M NaCl and 5 mM MgCl2. Reaction volumes were 200 ll forall enzymatic assays, except for cathepsin D-like activity that wasperformed in 1 ml. All substrates and protease inhibitors and acti-vators were purchased from Sigma Chemical Co. (St. Louis, MO,USA). Spectrophotometric measurements were made using amicrotiter plate reader (VersaMax™ Microplate Reader, MolecularDevices Inc., Sunnyvale, CA, USA), except for cathepsin D-like activ-ity that was recorded using a Hitachi U-2000 spectrophotometer(Hitachi High-Technologies Corporation, Tokyo, Japan).

Trypsin-like activity was measured using 1 mM BApNa (Na-ben-zoyl-DL-arginine-p-nitroanilide), 10 ll extract and incubating for1.25 h; chymotrypsin-like activity using 0.25 mM SA2PPpNa(N-succinyl-(alanine)2-proline-phenylalanine-p-nitroanilide), 10 llextract and incubating for 1 h; elastase-like activity using 0.5 mMSA3pNa (N-succinyl-(alanine)3-p-nitroanilide), 10 ll extract andincubating for 24 h; carboxypeptidase A-like activity with 1 mMHPA (hippuryl-phenylalanine), 10 ll extract and incubating for4 h; carboxypeptidase B-like activity with 1 mM HA (hippuryl-L-arginine), 10 ll extract and incubating for 4 h; and leucine amino-peptidase-like activity with 1 mM LpNa (L-leucine-p-nitroanilide),10 ll extract with an incubation time of 90 min, as described byOrtego et al. (1996). Cathepsin D-like activity was measured with0.2% hemoglobin solution, 20 ll extract and incubating for 24 h;and cathepsin B-like activity with 50 mM ZAA2MNA (N-carboben-zoxy-alanine-arginine-arginine 4-methoxy-b-naphthyl amide),20 ll extract and incubating for 4 h, as described by Novillo et al.(1997a). Assay of a-amylase activity (a-1,4-glucan-4-glucanohy-drolases) was performed according to Valencia et al. (2000) withsome modifications. Insect extracts (20 ll) were added in 50 mMsodium citrate with 10 mM NaCl and 20 mM CaCl2 (80 ll) buffer.The mixture was added to 0.5% starch (100 ll). Samples were incu-bated at 30 �C for 4 h. Iodine reagent (0.02% I2 and 0.2% KI) (1 ml)was then added and the sample was centrifuged 6000g for 5 min.The absorbance was read at 580 nm. Total protein was determinedaccording to the method of Bradford (1976) using bovine serumalbumin as the standard.

The proteolytic activities were assayed in the presence of thefollowing specific protease inhibitors: the serine protease inhibitorSBBI (Soybean Bowman-Birk inhibitor); the cysteine proteaseinhibitor E-64 (L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane); the aspartic protease inhibitor pepstatin-A; and the heavymetal ion CuCl2, inhibitor of aminopeptidases and carboxypeptid-ases. The cysteine protease activator L-cysteine was also tested.Protease inhibitors were incubated at 30 �C with the extract for15 min, prior to adding the substrate. All compounds were addedin 20 ll of 0.15 M NaCl, except pepstatin-A, which was added in4 ll of DMSO. The inhibitors and concentrations tested were se-lected according to the effective concentrations recommended byBeynon and Salvesen (1989).

2.3. Feeding bioassays

2.3.1. Effect of different diets on consumption, larval growth and adultweight gain

Feeding bioassays were performed with virgin males and fe-males 2–5 days old (used indistinctly). They were singly placedin a plastic arena (38 mm diameter � 19 mm height) that con-tained one piece of moistened filter paper (to ensure a constant

Table 1Abbreviations and composition of the different diets tested.

Diet Composition

RF (Rearing Food) A mixture of dog food and oatmeal at a 4:1 weightratio, ground to a thin powder

T (Tetranychus urticae) Adults of T. urticae bred on cornP (Plant) Corn leaf piecesS (Sesamia nonagrioides) Neonate larvae of S. nonagrioides (less than 24 h)PT (Plant + Tetranychus

urticae)Corn leaf pieces for 2 days, and subsequently fedwith T. urticae for 2 h

RFWO (Rearing FoodWithout Oatmeal)

Dog food alone

1336 M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342

water supply) and maintained in an environmental chamber as de-scribed above. Adult rove beetles were fed for 2 days with one ofthe following four different diets: rearing food (RF), a mixture ofanimal and vegetal tissue described above, represents a balanceddiet that fulfils nutritional requirements of Staphylinids under lab-oratory conditions (Carney et al., 2002; García et al., 2010, 2012);adults of T. urticae (T) bred on Z. mays, representing a diet formedby an herbivorous prey fed on plant material; a plant (P) based dietformed only by corn leaf pieces; and neonate larvae (less than24 h) of S. nonagrioides (S), that had not fed on any plant material,representing a diet formed only by animal living tissue (Table 1).Immature stages were also tested by feeding L3 larvae for 2 dayswith the diets 3 (P) or 4 (S), representing the ends of the feedingcontinuum from phytophagy to zoophagy. Fifteen larvae andadults were used with each diet.

Fresh weight (FW) of P. quisquiliarius larvae and adults was re-corded in an analytical balance with a sensitivity of 0.01 mg (Met-tler Toledo AX205, Mettler-Toledo International Inc., Columbus,OH, USA) at the beginning and at the end of the experiment, andtheir dry weight (DW) was estimated by calculating a fresh-dryweight conversion factor from thirty adults and thirty L3 larvaefed on normal rearing diet that were measured separately, driedat 60 �C for 72 h and then weighed to determine DWs. Consump-tion was estimated as the difference between the initial DW ofthe diet and the DW of the uneaten remains (dried at 60 �C for72 h and then weighed). To estimate initial DW of the diets, thirtysamples from each diet were weighed separately, dried at 60 �C for72 h and then weighed to determine DWs. The nutritional indiceswere calculated using DWs as follows: relative consumption rate[RCR = mg ingested/(insect initial DW � days)], relative growthrate [RGR = DW gained in feeding period/(insect initialDW � days)] for larvae, relative weight gain rate [RWGR = DWgained in feeding period/(insect initial DW � days)] for adults,and efficiency of conversion of ingested matter to body mass[ECI = (RCR/RGR) � 100 in larvae; and ECI = (RWGR/RGR) � 100]in adults, according to Farrar et al. (1989).

2.3.2. Effect of different diets on enzymatic activityAt the end of the above described bioassay, fifteen larvae and

adults from each diet were frozen at �20 �C until used. Adults ofP. quisquiliarius fed on maize for 2 days, and subsequently fed withT. urticae for 2 h (PT, Table 1), were also analyzed to determine ifthey were able to enzymatically respond to the switch in diet inthis short period of time. The hydrolytic activities of whole larvaeand decapitated adults were determined as described above. Inaddition, the inhibition of trypsin and chymotrypsin-like activitiesby proteinaceous (SBBI; STI, soybean trypsin inhibitor and LBI, limabean protease inhibitor) and non-proteinaceous (benzamidine;E-64 and HgCl2) inhibitors was carried out. A range of concentra-tions were tested for each inhibitor, as previously described, to cal-culate by linear regression the concentration that inhibits 50% ofthe protease activity (IC50).

Electrophoretic detection of proteolytic forms was performedby 0.1% (w/v) gelatine-containing, 0.1% (w/v) SDS, 12% (w/v) poly-acrylamide gel electrophoresis under non-denaturing conditionsusing a Bio-Rad Mini-Protean II Electrophoresis Cell system. Theratio of acrylamide to bis-acrylamide was 36.5:1. Samples of L3 lar-vae extracts contained 4.2 lg of protein, whereas adult extractscontained 5 lg of protein approximately. Gels were run at 150 Vand 4 �C. After migration, gels were transferred to a 2.5% (v/v)aqueous solution of Triton X-100 for 20 min at room temperature,to allow renaturation of the proteases. Gels were then placed in thefollowing activation buffers for 24 h at 30 �C: 0.1 citrate, pH 3.0;0.1 M Tris–HCl, pH 7.0; 0.1 M Tris–HCl and 10 mM L-cysteine, pH7.0 and 0.1 M glycine–NaOH, pH 9.0. Proteolysis was stopped bytransferring the gels into a staining solution [0.3% (w/v) CoomassieBlue R-250 in 40% (v/v) methanol and 10% (v/v) acetic acid]. Bandsof proteolytic activity were visualized against the blue backgroundof the gel.

2.3.3. Gut pH determinationTo prove if there is variation of the pH in the luminal content

depending on the diet ingested, we determined the pH inside gutsfrom adults of P. quisquiliarius fed with different diets (RF, T, P, PTand S) during 2 days. The gut was carefully removed from the bodyand placed on a piece of universal pH indicator paper (pH range1–10). By pressing gently, the luminal content of the gut wasreleased and the change in coloration of the pH paper was scanned(Shanbhag and Tripathi, 2005). Colours generated by pH standardsof 1–10 on the same pH paper were also scanned for calibration.The resulting coloured solutions were compared with standards.Three replicates were performed with each diet.

2.4. Fecundity and egg fertility of P. quisquiliarius fed on a plantnutrient-depleted diet

To evaluate if the absence of plant nutrients in diets producesany effect on reproduction, eggs from P. quisquiliarius were ran-domly collected from the laboratory colony and placed in plasticboxes prepared for rearing (see above). Two treatments that dif-fered in the food that rove beetles received during their completeimmature development (from first instar to adult emergence)and sexual maturation (from adult emergence to 30 days old) weretested. One diet was the regular rearing food (RF), formed by dogfood and oatmeal at a 4:1 weight ratio and the second diet wasdog food without the oatmeal supplement (RFWO) (Table 1).Adults emerged from each treatment were weighed and sexed,and adult fecundity and egg fertility evaluated as follows: one fe-male and two males randomly selected from each treatment wereplaced in a new test arena (Petri dish 89 � 23 mm) containing thesame rearing conditions (substrate and diet). The dish was moist-ened weekly with 1 ml of water to ensure an appropriate level ofhumidity. Adults were fed ad libitum with each diet, and the num-bers of eggs laid were recorded daily for 30 days. Eggs from eachtreatment were collected daily and individually placed in the testarena containing a piece of filter paper moistened with 200 ll ofwater. Egg hatching was checked daily. Ten replicates per treat-ment were performed.

2.5. Data analysis

Homogeneity of variances (Levene test) and normal distribution(Kolmogorov–Smirnov test) were tested in all variables before sta-tistical analysis. When these requirements were not fulfilled, datawere transformed using logarithmic or Box–Cox transformation tonormalize distributions and stabilize variances. Data from adultsand larvae were analyzed separately. Comparisons among RWGR,RGR, RCR and ECI were made with ANCOVA followed by Tukey’s

M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342 1337

multiple comparisons test using initial DWs as the covariate (Rau-benheimer and Simpson, 1992). Proteolytic activities of P. quisquil-iarius extracts fed on different diets were compared by ANCOVAfollowed by Tukey’s multiple comparisons test, using protein con-tent as covariate (Ortego et al., 1999). Differences in IC50 for differ-ent inhibitors were compared by Student’s t-test for larvae and byone way-ANOVA, followed by Tukey’s multiple comparisons testsfor adults. Student’s t-test was used to detect differences betweenRF and RFWO treatments in female and male initial weights, fecun-dity and fertility. A significance level of p < 0.05 was considered forall tests.

3. Results

3.1. Hydrolytic activities in P. quisquiliarius

Hydrolytic activities in P. quisquiliarius adult extracts were char-acterized using specific substrates and inhibitors (Table 2). Maxi-mal hydrolysis of trypsin substrate BApNa was at pH 9.0 and ofthe chymotrypsin substrate SA2PPpNa at pH 10.0, being both spe-cifically inhibited by SBBI. Hydrolysis of ZAA2MNA, a substrate forcysteine proteases, was optimum at pH 7.0, but this activity wasnot inhibited by E-64, whereas inhibition was found with SBBIand CuCl2. The hydrolysis of hemoglobin reached a maximum atpH 3.0 and was inhibited by pepstatin-A. Leucine aminopeptidasesubstrate, LpNa, presented its maximum activity at pH 7.0, and itshydrolysis was inhibited by the divalent heavy metal ion, CuCl2.The optimum pH for the hydrolysis of HPA and HA, substrates ofcarboxypeptidase A and carboxypeptidase B-like activities, were7.0, and their hydrolysis were inhibited by CuCl2 in both cases.Maximum hydrolysis of starch, used as substrate for a-amylaseactivity, was at pH 7.0. Same enzymatic activities were found inlarvae (data not shown). The hydrolysis of the elastase substrateSA3pNa was also tested but showed no activity, eliminating theelastase-like protease activity as part of P. quisquiliarius digestiveenzymes.

3.2. Feeding bioassays

3.2.1. Effect of different diets on consumption, larval growth and adultweight gain

Adults presented positive weight gains after 2 days eating thedifferent diets (Table 3). The highest RWGR corresponded to adultsfed with RF, however no differences were found among adults fedwith the other diets (T, P or S). Larvae fed on plant material (P) pre-sented also similar RGR than those larvae fed with animal material(S). With respect to consumption, adults and larvae fed on a diet

Table 2Hydrolytic activities of Philonthus quisquiliarius adults extracts against general and specifi

Substratea Optimum pH Specific activityb % Rel

SBBI(1

BApNa 9.0 32.7 ± 1.4 6 ± 2SA2PPpNa 10.0 11.7 ± 0.7 15 ± 2ZAA2MNA 7.0 14.5 ± 0.4 63 ± 5Hemoglobin 3.0 6.6 ± 0.5 neLpNa 7.0 24.9 ± 0.4 neHPA 7.0 312 ± 72 neHA 7.0 531 ± 54 neStarch 7.0 253 ± 4 –

a Substrates: BApNa, (Na-benzoyl-DL-arginine p-nitroanilide); SA2PPpNa, (N-succinalanine-arginine-arginine 4-methoxy-p-naphthyl amide); LpNa, (L-leucine-p-nitroanilide

b Specific activities as nmoles of substrate hydrolyzed/(min * mg protein), except for pfor a-amylase activity as mg of starch hydrolyzed/(min * mg protein). Values are mean

c Values are means ± S.E. of triplicate measurements from a pool of decapitated adultseffect (ne) was considered for activities between 80% and 120%.

formed by plant material (P) consumed significantly more thanthose fed on the other diets (RF, T or S) (Table 3). As a consequence,the efficiency of conversion of ingested matter to body mass wasbelow 2% when larvae or adults fed on plant material, whereasranged between 22% and 55% when fed on any of the other diets(Table 3).

3.2.2. Effect of different diets on enzymatic activityBiochemical analyses were carried out on larvae and adults fed

for 2 days with different diets (Table 4). Total protein content andtrypsin-like activity were similar among treatments for adults.However, the biochemical analysis showed that chymotrypsin-,carboxypeptidase A- and carboxypeptidase B-like activities weresignificantly lower when fed on plant material (P), compared withfeeding on animal based diets (T and S) and rearing food (RF).Adults fed on plant material for 2 days and then switched to ananimal based diet for 2 h (PT) recovered their chymotrypsin- andcarboxypeptidase A-like activities, whereas carboxypeptidaseB-like activity remained at low levels. Leucin aminopeptidase-likeactivity was significantly higher when adults were fed withneonates of S. nonagrioides (S). Similarly, values of cathepsinD-like activity were also significantly higher in adults fed withS. nonagrioides (S) than in those fed on leaf pieces (P). On the con-trary, the lowest a-amylase-like activity corresponded to adultsfed on neonates of S. nonagrioides (S). When adults fed on plantmaterial were switched to an animal based diet for 2 h (PT) theira-amylase-like activity was also significantly reduced. Larvae fedwith plant material (P), presented also lower protein content,trypsin-, chymotrypsin-, cathepsin D-, carboxypeptidase A- andcarboxypeptidase B-like activities than larvae fed with animalmaterial (S) (Table 4). However, no differences on leucinaminopeptidase- and a-amylase-like activities were observed be-tween larvae fed with plant (P) or animal material (S).

Trypsin and chymotrypsin-like activities present in the extractsof rove beetles fed on different diets were further analyzed in orderto compare their sensitivity to protease inhibitors (Table 5). Gen-eral serine protease inhibitors (SBBI, STI and LBI), and the specifictrypsin inhibitor benzamidine were tested. The cysteine proteaseinhibitor E-64 and HgCl2 were also tested because of their abilityto inhibit specific trypsin forms in insects (Novillo et al., 1997b;Díaz-Mendoza et al., 2005). Similar IC50 were obtained for tryp-sin-like activity in adults, except for the higher susceptibility ofthose fed on plants (P) to benzamidine and E-64, the lower suscep-tibility of adults fed on T. urticae (T) to HgCl2, and the higher sus-ceptibility to E-64 of those fed on rearing food (RF).Chymotrypsin-like activity inhibition by SBBI and STI was lowerin adults that ate T. urticae (T). Adults fed on plants and subse-

c substrates and effects of protease inhibitors.

ative activityc

0lm) E-64(10lm) CuCl2(1mM) Pepstatin-A(10lm)

ne ne nene ne nene 11 ± 7 nene 77 ± 2 9 ± 4ne 11 ± 0 nene 3 ± 2 nene 16 ± 12 ne– – –

yl-(alanine)2-proline-phenylalanin-p-nitroanilide); ZAA2MNA, (N-carbobenzoxy-); HPA, (hippuryl-phenylalanine); HA, (hippuryl-L-arginine).roteolytic activity against hemoglobin as mU DAbs 280 nm/(min * mg protein) and± S.E. of triplicate measurements.extracts treated with an inhibitor vs. their corresponding control without them. No

Table 3Nutritional indices of Philonthus quisquiliarius adults and L3 larvae fed during 2 days on different diets.

Stage Dietsa Nutritional indicesb

RWGR (mg/mg � day) RCR (mg/mg � day) ECI (%)

Adult RF 0.08 ± 0.0b 0.23 ± 0.0b 55.0 ± 21.0b

T 0.02 ± 0.0a 0.07 ± 0.0a 41.2 ± 8.15b

P 0.03 ± 0.0a 2.01 ± 0.1c 1.69 ± 0.16a

S 0.03 ± 0.0a 0.08 ± 0.0a,b 46.3 ± 7.69b

p < 0.00 p < 0.00 p < 0.00

Larva P 0.07 ± 0.0a 6.37 ± 0.7a 1.77 ± 0.66a

S 0.06 ± 0.0a 0.21 ± 0.0b 21.7 ± 4.08b

p = 0.45 p < 0.00 p < 0.00

For each stage, means were compared by ANCOVA using as covariate initial dry weights, followed by Tukey’s multiple comparisons tests.Values are means ± S.E. of 15 measurements for each stage.Means followed by different letters within columns for each stage indicate significant differences (p < 0.05).

a RF (rearing food); T (adults of Tetranychus urticae); P (corn leaf pieces) and S (larvae of Sesamia nonagrioides).b RWGR, relative weight gain rate; RGR, relative growth rate; RCR, relative consumption rate; ECI, efficiency of conversion of ingested matter to body

mass.

Table 4Hydrolytic activities of Philonthus quisquiliarius larvae and adults fed on different diets.

Stage Dietsa Total protein (lg protein/insectb) Specific activityc

TRY CHY CTD LAP CPA CPB a-AMY

Adult RF 128 ± 11a 70 ± 7.4a 158 ± 21b 17 ± 3.0a,b 61 ± 5.9a 373 ± 59b 931 ± 11b 974 ± 75b,c

T 119 ± 8.3a 77 ± 9.9a 209 ± 27b 18 ± 1.8a,b 64 ± 5.6a 586 ± 84b 839 ± 60b 1044 ± 63b,c

P 108 ± 7.8a 54 ± 3.6a 100 ± 15a 14 ± 1.5a 59 ± 5.2a 193 ± 45a 585 ± 42a 1166 ± 77c

PT 123 ± 7.4a 76 ± 6.7a 214 ± 38b 15 ± 1.2a,b 56 ± 3.2a 429 ± 50b 541 ± 48a 961 ± 493b

S 138 ± 8.3a 71 ± 10a 198 ± 30b 17 ± 1.4b 78 ± 6.3b 617 ± 40b 790 ± 45b 788 ± 40a

p = 0.19 p = 0.15 p < 0.00 p = 0.03 p < 0.00 p < 0.00 p < 0.00 p < 0.00

Larva P 93 ± 6.0a 43 ± 16a 35 ± 11a 2 ± 1.3a 59 ± 10a 180 ± 30a 552 ± 10a 621 ± 33a

S 154 ± 20b 105 ± 25b 90 ± 23b 13 ± 2.5b 66 ± 15a 761 ± 91b 724 ± 77b 477 ± 54a

p < 0.00 p < 0.00 p < 0.00 p < 0.00 p = 0.22 p < 0.00 p = 0.01 p = 0.96

For each stage, means were compared by ANCOVA using as covariate protein content, followed by Tukey’s multiple comparisons tests.Values are means ± S.E. of 15 measurements for each stage.Means followed by different letters within columns for each stage indicate significant differences (p < 0.05).

a RF (rearing food); T (adults of Tetranychus urticae); P (corn leaf pieces) and S (larvae of Sesamia nonagrioides) for 2 days. PT (leaf pieces for 2 days and T. urticae for 2 h).b Decapitated adults and whole L3 larvae.c Specific activities as nmoles of substrate hydrolyzed/(min * mg protein), except for proteolytic activity against hemoglobin as mU DAbs 280 nm/(min * mg protein) and

for a-amylase activity as lg of starch hydrolyzed/(min * mg protein). Protease type abbreviations: TRY, trypsin; CHY, chymotrypsin; LAP, leucine aminopeptidase; CTD,cathepsin D; CPA, carboxypeptidase A; CPB, carboxypeptidase B and a-AMY, a-amylase.

Table 5Inhibition of trypsin and chymotrypsin-like activities by different proteinaceous and non-proteinaceous inhibitors.

Stage Dietsa IC50b

TRY CHY

SBBI(nM) STI(nM) LBI(nM) Benzamidine(lM) E-64(lM) HgCl2(lM) SBBI(nM) STI(nM) LBI(nM)

Adult RF 11.5 ± 0.6a 17.5 ± 5.1b 78.5 ± 12.8a 36.0 ± 1.8b 190.8 ± 33.6a 77.2 ± 5.7a 42.1 ± 10.0a 62.0 ± 4.7a 439.8 ± 17.2b

T 15.6 ± 5.4a 8.5 ± 3.7a,b 93.4 ± 13.2a 45.4 ± 3.6b 448.2 ± 74.9b 2460 ± 461b 123.4 ± 33.6b 272.0 ± 67.2b 469.0 ± 78.0b

P 7.5 ± 2.9a 3.1 ± 0.6a 79.2 ± 20.4a 11.1 ± 2.6a 41.5 ± 9.8a 34.1 ± 3.1a 56.5 ± 12.5a 65.0 ± 9.4a 232.0 ± 94.0a,b

PT 8.7 ± 2.2a 5.3 ± 0.8a,b 110.1 ± 8.0a 51.2 ± 9.2b 424.4 ± 36.7b 167.5 ± 37.1a 40.0 ± 11.7a 44.7 ± 5.4a 151.4 ± 12.0a

S 11.1 ± 4.1a 2.9 ± 0.5a 77.5 ± 11.3a 41.0 ± 5.6b 520.9 ± 30.6b 145.3 ± 27.4a 38.8 ± 11.4a 60.0 ± 7.4a 155.0 ± 33.0a

p = 0.55 p = 0.02 p = 0.43 p < 0.00 p < 0.00 p < 0.00 p = 0.03 p = 0.00 p = 0.00

Larva P 27.0 ± 6.4a 5.3 ± 1.2a 44.4 ± 9.3a 12.0 ± 0.4a 62.0 ± 12.2a 51.5 ± 8.0a 9.6 ± 5.5a 35.8 ± 23.3a 103.6 ± 14.8a

S 4.6 ± 0.4b 166.5 ± 16.6b 414.3 ± 58.0b 40.6 ± 5.3b 135.4 ± 22.6b 138.4 ± 7.9b 125.2 ± 1.3b 77.8 ± 52.1a 127.4 ± 37.0a

p = 0.01 p < 0.00 p < 0.00 p < 0.00 p = 0.02 p < 0.00 p < 0.00 p = 0.50 p = 0.58

Adult’s means were compared by one-way ANOVA, followed by Tukey’s multiple comparisons tests.Comparisons between larvae were done by Student’s t-test.Values are means ± S.E. of three replicates from extracts treated with an inhibitor vs. their corresponding control without them.Means followed by different letters within columns for each stage indicate significant differences (p < 0.05).

a RF (rearing food); T (adults of Tetranychus urticae); P (corn leaf pieces) and S (larvae of Sesamia nonagrioides) for 2 days. PT (leaf pieces for 2 days and T. urticae for 2 h).b Concentration of inhibitors that inhibit 50% of the protease activity. Protease type abbreviations: TRY, trypsin (using BApNA as substrate at pH 9.0) and CHY, chymo-

trypsin (using SA2PPpNa at pH 10.0). Inhibitor abbreviations: SBBI, soybean Bowman-Birk inhibitor; STI, soybean trypsin inhibitor; LBI, lima bean protease inhibitor and E-64(L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane).

1338 M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342

M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342 1339

quently with T. urticae for 2 h (PT) or with neonate larvae ofS. nonagrioides (S) presented higher inhibition by LBI. Extracts oflarvae fed on plant material (P) presented higher susceptibility oftrypsin-like activity to STI, LBI, benzamidine, E-64 and HgCl2, butlower to SBBI (Table 5). Chymotrypsin-like activity on larvae ex-tracts presented differential inhibition only when SBBI was evalu-ated; having those larvae fed with plant material (P) lower IC50

values.Gel assays were performed to look for qualitative differences in

protease forms in extracts of larvae and adults of P. quisquiliariusfed with different diets (Fig. 1). Very similar patterns were ob-

Fig. 1. Gelatin-containing SDS–PAGE gels of adults and L3 larvae extracts ofPhilonthus quisquiliarius fed on RF (rearing food); T (adults of Tetranychus urticae); P(leaf pieces); PT (leaf pieces and subsequently T. urticae for 2 h) and S (larvae ofSesamia nonagrioides). Gels were incubated for 24 h at 30 �C with (A) 0.1 M citrate,pH 3.0, (B) 0.1 M Tris–HCl, pH 7.0, or (C) 0.1 M glycine–NaOH, pH 9.0.

tained at the different pHs tested: 3.0 (Fig. 1a), 7.0 (Fig. 1b), 9.0(Fig. 1c), and 7.0 + 10 mM L-cysteine (data not shown), indicatingthat the proteolityc forms are stable over a wide range of pHs.The proteolytic profile in adults was not affected by the differentdiet regimes at three different pHs, 3.0, 7.0 and 9.0 (Fig. 1a–c),though the intensity of some bands were higher in adults fed onT, PT or S than when fed on RF or P. Similar results were obtainedwhen zymograms were performed at pH 7.0 + 10 mM L-cysteine(data not shown). Extracts from larvae fed on plant material (P)presented a faint banding pattern, more evident at pH 3.0 than atpH 7.0 or 9.0 (Fig. 1a–c).

3.2.3. Gut pH determinationOur results showed that the pH of the luminal content is around

7.0 and it is constant along the gut in P. quisquiliarius adults. Iden-tical results were obtained with rove beetles fed with the five dif-ferent diets evaluated herein, signifying that there is no variationin P. quisquiliarius gut pH related to the ingestion of plant or animaltissue (data not shown).

3.3. Fecundity and egg fertility of P. quisquiliarius fed on a plantnutrient-depleted diet

An experiment to measure fecundity and egg fertility was per-formed to evaluate the effect of removing plant material fromthe most efficient diet tested (RF: a mixture of dog food with oat-meal) according to our feeding bioassays (see Section 3.2.1). Fe-males and males emerged from larvae fed on the completerearing food (RF) or on the rearing food without the oatmeal sup-plement (RFWO) presented no differences in their initial weight(p > 0.05). However, the fecundity was significantly reduced from43.6 (±3.1) eggs per female in RF to 22.6 (±3.8) when fed on RFWO(Fig. 2a), and the fertility from 78.3% (±2.2) in RF to 63.3% (±7.3) inRFWO (Fig. 2b).

4. Discussion

We have shown that larvae and adults of the rove beetle P. quis-quiliarius can feed on animal and plant based diets, exhibiting zoo-phytophagous feeding habits. In fact, larvae presented similargrowth rates when fed on living animal (S) or on green plant mate-rial (P), and adults positive weight gains on plant or animal baseddiets, demonstrating their ability to exploit both animal or plantfood types. However, higher consumption rates were needed whenplant pieces were consumed. This could be explained because ofthe low protein/high carbohydrate contents of most plant tissuescompared to animal prey (Chown and Nicolson, 2004), or becausethey may increase their food intake when fed on plants to obtainadequate proportions of other vital nutrients (Boggs, 2009). As aresult, digestive efficiencies when fed with plant material (P) werelower than when fed with animal based diets (T or S); howeverpositive values indicate that P. quisquiliarius have certain abilityto utilize plant material for nutrition. Interestingly, the relativeweight gain rate was the highest in adults fed with the food em-ployed to breed the colony (RF). This could be indicating that RFrepresents a more efficient diet to P. quisquiliarius due to the pres-ence of nutrients from animals and plants. However, we cannotdiscern which aspect of the physiology is being affected (accumu-lation of reserve substances, development of the gonads, etc.).Nevertheless, we have found negative effects upon P. quisquiliariusfecundity and fertility when supplemental plant nutrients(oatmeal) were removed from the rearing diet (RFWO). Similarly,when the zoophytophagous hemipteroid Brontocoris tabidus(Signoret) (Hemiptera: Pentatomidae) was bred on a diet withoutplant material, deleterious effects were found on the ovarian and

Fig. 2. (A) Mean number of eggs laid per female by Philonthus quisquiliarius; and (B) percentage of egg hatching after 30 days. Rove beetles were fed on two different diets(RF: rearing food; RFWO: rearing food without oatmeal). Values are means ± S.E. of 10 replicates. Different letters on the bars indicate significant differences (p < 0.05).

1340 M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342

fat body development. These authors suggested that the presenceof plants in the diet is necessary for an adequate ovarian develop-ment of B. tabidus (Lemos et al., 2011).

A common explanation to the consumption of plant tissue byzoophytophagous predatory insects is the need of water or lowprey density (Coll and Guershon, 2002). However, P. quisquiliariusconsumed green plant material despite having constant access towater all the time. Moreover, this hypothesis does not explainthe larval growth and adult weight gain sustained when fed onplant material and the negative effects on fecundity and fertilitywhen a plant nutrient-depleted diet was given to larvae and adults.Taking into account this evidence, our results are aligned with analternative hypothesis, the one that relates plant consumption byzoophytophagous with the acquisition of specific nutrients, vita-mins, and minerals that are limited in its primary diet (Moseret al., 2008; Torres et al., 2010).

Plant feeding by predatory insects requires physiologicaladaptations to allow trophic switching between predation andphytophagy, and in particular to deal with the inverted protein-carbohydrate ratio of plant versus animal tissue (Coll andGuershon, 2002). Thus, the ability to use plant material as food isusually correlated with higher ratio of a-amylase activity to prote-ase activity, whereas the contrary occurs in those insects adaptedto zoophagy. Zeng and Cohen (2000) showed that the ratio ofa-amylase activity to protease activity was higher in the phytozoo-phagous Lygus lineolaris (Palisot de Beauvois) and Lygus hesperus(Knight) (Hemiptera: Miridae), two species that mostly feed onplants but eventually act as predators, than in the zoophytopha-gous Geocoris punctipes (Say) (Hemiptera: Geocoridae) whichusually acts as a predator but may feed on plants. Our results arein line with previous findings, since both larvae and adults of P.quisquiliarius fed on plants (P) presented high a-amylase activityand low proteolytic activity when compared to individuals fed ondiets with animal material. In fact, the regulation of these physio-logical changes may occur in short periods of time, since adults fedon plant material for 48 h and then fed on T. urticae for 2 h pre-sented similar proteolytic and a-amylase activities than those fedon T. urticae for 48 h. Zeng and Cohen (2001) found also that pro-tease activity in the zoophytophagous hemipteroid L. hesperuswas induced in response to food components. Variations in prote-ases activity were also found in the predator Podisus maculiventris(Say) (Hemiptera: Pentatomidae) when fed on different prey(Pascual-Ruíz et al., 2009).

In coleopterans, protein digestion is usually performed by ser-ine and/or cysteine proteases (Terra and Cristofoletti, 1996; Terra

and Ferreira, 2005). Our results confirmed the presence of tryp-sin-, chymotrypsin-, cathepsin D-, leucine aminopeptidase-, car-boxypeptidase A-, and B-like proteases and a-amylase in P.quisquiliarius adults and larvae. Hydrolysis of ZAA2MNA was alsoobtained, but it was not inhibited by E-64 nor activated by L-cys-teine, indicating that cysteine proteases were not part of the P.quisquiliarius digestion enzymes. Digestive systems based on serineproteases for protein digestion have been also reported for otherpredatory beetles, such as the carabid Pheropsophus aequinoctialis(Coleoptera: Carabidae) (Ferreira and Terra, 1989), the elaterid Pyr-earinus termitilluminans Costa (Coleoptera: Elateridae) (Colepicolo-Neto et al., 1986) and another rove beetle, Atheta coriaria (Say)(Coleoptera: Staphylinidae) (García et al., 2010). However, thepresence of serine proteases is not distinctive for all predatory bee-tles, since coccinellids mostly rely on cysteine proteases (Walkeret al., 1998; Ferry et al., 2003) and some carabids present both ser-ine and cysteine proteases (Burgess et al., 2002).

Differences in the susceptibility of trypsin- and chymotrypsin-like protease activities to inhibitors were observed, being larvaeand adults of P. quisquiliarius fed on plants more susceptible toinhibitors in most cases. These results may suggest qualitativechanges in the expression of specific enzymes depending on thediet, since the susceptibility of different forms of trypsins andchymotrypsins to serine protease inhibitors vary within a givenspecies (Díaz-Mendoza et al., 2005), as reported for both phytoph-agous and zoophagous species (Patankar et al., 2001; Chouguleet al., 2005; Pascual-Ruíz et al., 2009). However, the proteolyticpattern on zymograms did not present conspicuous qualitative dif-ferences among P. quisquiliarius fed on different types of diet. Thisapparent contradiction may be explained because most proteolyticbands probably correspond to several isoenzymes with the samemobility but different susceptibility to inhibition. Thus, differencesin expression of particular isoenzymes may affect the intensity ofthe bands, but not the presence of novel bands. An alternativeexplanation is that the low protein concentration in the guts of lar-vae and adults fed on plants (protein poor diets) may be responsi-ble of their high susceptibility to inhibitors because there are fewerproteins that may interfere with the inhibitors.

Another factor influencing the optimal conditions for digestiveenzymes and the quality and quantity of nutrients that can be di-gested is the pH of gut content (Terra and Ferreira, 2005). Plantdiets have been shown to affect the midgut pH of some species(Schultz and Lechowicz, 1986), because of the differences in acid-base composition among plants species (Harrison, 2001). Our datashow that the gut content in adults of P. quisquiliarius is around 7.0

M. García et al. / Journal of Insect Physiology 58 (2012) 1334–1342 1341

and constant along the gut independently of the type of diet (ani-mal or plant based) that have consumed. These results suggest thatthere is an active regulation to stabilize the lumen pH after feeding,as reported for phytophagous insects (Harrison, 2001). Moreover,results from pH activity profiles showed that leucine aminopepti-dase-, carboxypeptidase A-, and B-like and a-amylase-like enzy-matic activities have their optimum activity at pH 7.0, which iscorrelated with the pH prevailing in the midgut of P. quisquiliarius.However, in vitro optimal pH values for serine proteases in thisspecies were in the alkaline range, and acidic for aspartyl prote-ases. According to Oppert et al. (2002) differences in values ofthe luminal pH and optimal pH activity could be attributed to sev-eral factors, like specific localization of proteases, interaction ofproteases with other proteins of other compounds in the luminalcontent, or because proteases are active over a range of pHs andmay be more stable at pH values other than their respective max-imum. Our results with zymograms support this last hypothesis,since similar banding pattern were found at pHs 3.0, 7.0 and 9.0.

In summary, in the present study we report the zoophytopha-gous feeding habits of larvae and adults of the rove beetle P. quis-quiliarius. We have demonstrated that this species has an arsenal ofdigestive enzymes suitable for zoophytophagy that are influencedby the animal or plant based diet ingested; and that scarcity of cer-tain plant material negatively affects its reproduction. All together,our results suggest that P. quisquiliarius needs certain nutrients,which are obtained only from plant material. The need to ingestand digest both plant and animal material has major consequencesin terms of an organism’ ecological role (Coll and Guershon, 2002).Zoophytophagy could be important for natural enemies’ perfor-mance, since it would allow the zoophytophagous to colonizecrops before the arrival of prey, and to persist during periods ofprey scarcity. These results will help to understand the complextrophic interactions that occur in agroecosystems, and in the de-sign of management strategies that could include zoophytopha-gous species in conservation biological control programs.

Acknowledgements

We thank Dr. Raimundo Outerelo Domínguez (UniversidadComplutense de Madrid, Spain) for the taxonomic classificationof Philonthus quisquiliarius. This work received financial supportfrom the Spanish Ministry of Environment (MMA) and CICYT(AGL2009-08813). Matías García’s research was supported by acontract from the Science and Innovation Ministry (JAE-Doc).

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