Beneficial effect of adipokinetic hormone on ...

Comparative Biochemistry and Physiology, Part C 196 (2017) 11–18

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Beneficial effect of adipokinetic hormone on neuromuscular paralysis in insect body elicited by braconid wasp venom Haq Abdul Shaik a, Archana Mishra a, Dalibor Kodrík a,b,⁎ a b

Institute of Entomology, Biology Centre, CAS, Branišovská 31, 370 05 České Budějovice, Czech Republic Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic

a r t i c l e

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Article history: Received 19 January 2017 Received in revised form 21 February 2017 Accepted 23 February 2017 Available online 28 February 2017 Keywords: AKH Akh gene expression Habrobracon hebetor Metabolism Paralysis Q-PCR Stress Venom

a b s t r a c t The effect of Habrobracon hebetor venom and the role of the adipokinetic hormone (AKH) in poisoned adult females of the firebug Pyrrhocoris apterus were studied 24 and 48 h after treatments. Venom application elicited total neuromuscular paralysis in firebugs, but the co-application of venom and Pyrap-AKH significantly reduced paralysis (up to 3.2 times) compared to the application of venom only. Although the mechanisms of their action are unknown, both agents might affect neuromuscular junctions. Venom application significantly increased the expression of both P. apterus Akh genes (Pyrap-Akh 5.4 times and Peram-Cah-II 3.6 times), as well as the level of AKHs in the central nervous system (2.5 times) and haemolymph (3.0 times). In the haemolymph, increased AKH levels might have led to the mobilization of stored lipids, which increased 1.9 times, while the level of free carbohydrates remained unchanged. Total metabolism, monitored by carbon dioxide production, significantly declined in paralysed P. apterus individuals (1.4 times and 1.9 times, 24 and 48 h after the treatment, respectively), probably because of a malfunction of the muscular system. The results suggest an active role of AKH in the defence mechanism against the stress elicited by neuromuscular paralysis, and the possible involvement of this hormone in neuronal/neuromuscular signalling. © 2017 Elsevier Inc. All rights reserved.

1. Introduction The venom of parasitic wasps is a complex cocktail of proteinaceous and non-proteinaceous components that affect various biochemical reactions and physiological processes in the body of the attacked insects. Interestingly, despite the large number of parasitic wasp species, only a few venoms with biological activity have been identified and functionally evaluated thus far. One such venom is that produced by the minute Braconidae wasp Habrobracon hebetor, a polyphagous and gregarious species that primarily parasitizes lepidopteran and coleopteran larvae (Benson, 1974; Kovalenkov, 1984; Nay and Perring, 2005; Altuntas et al., 2010). This venom elicits complete neuromuscular paralysis in insects (Beckage and Gelman, 2004; Sláma and Lukáš, 2011) and strongly suppresses cell and humoral immunity (Kryukova et al., 2007, 2011; Pennacchio et al., 2014). Both actions aim to immobilize the host (‘a living can’) and suppress its defence immune response, providing optimal

Abbreviations: AKH, adipokinetic hormone; CNS, central nervous system; Peram-CAHII, Periplaneta americana cardio accelerating hormone-II; Pyrap-AKH, Pyrrhocoris apterus adipokinetic hormone; q-RT-PCR, quantitative real time PCR. ⁎ Corresponding author at: Institute of Entomology, Biology Centre, CAS, Branišovská 31, 370 05 České Budějovice, Czech Republic. E-mail address: [email protected] (D. Kodrík).

http://dx.doi.org/10.1016/j.cbpc.2017.02.011 1532-0456/© 2017 Elsevier Inc. All rights reserved.

conditions for the laid eggs and for the new generation of H. hebetor. Venom components interact with receptors on the presynaptic membranes of neuromuscular junctions, probably blocking glutaminergic transmission (Slavnova et al., 1987; Pennacchio and Strand, 2006). Studies on the cellular and humoral immune responses of insects parasitized by H. hebetor are scarce (Hartzer et al., 2005). Nevertheless, venom components might have a potential function in targeting the two major host defence effectors, i.e., phenoloxidase cascade, and coagulation (Beckage and Gelman, 2004; Andrew et al., 2006). Phenoloxidase activity was suppressed in wax moth Galleria mellonella larvae after venom application, but other components of the cell and humoral immunity were also affected (Kryukova et al., 2011, 2015). Baker and Fabrick (2000) also found some changes in the composition of haemolymph proteins in parasitized insects, which was not surprising because defence proteins are key players in insect immunity (Shaik and Sehnal, 2009). The several attempts to isolate and characterize the structure of the toxins contained in H. hebetor venom revealed that it probably contains three active protein components, ranging from 18 to 73 kDa in molecular weight. However, data found in the literature are not consistent. Whereas one of the components responsible for the specific action upon the glutamatergic neuromuscular synapses was reported as a 18 kDa protein, approximately (Slavnova et al., 1987), other studies

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H.A. Shaik et al. / Comparative Biochemistry and Physiology, Part C 196 (2017) 11–18

reported two proteins weighing 43.7 kDa, and 56.7 kDa (Visser et al., 1983) or 73 kDa, approximately; the latter was partly sequenced (Quistad et al., 1994; Quistad and Leisy, 1996). Another venom, which is relatively well studied, comes from the wasp Nasonia vitripennis. This venom is known to contain about 80 different proteins and possibly also other compounds (De Graaf et al., 2010). They are active also in mammalian cells where they induce biochemical pathways related to early stress response (Danneels et al., 2015); a suppressive action on the nuclear factor kappa light chain enhancer of the B cells (NF-κB) was recorded as well (Danneels et al., 2014). Application of the wasp venom into the body represents a severe stress for the attacked insect, which has to activate anti-stress responses towards the venom while trying to reduce the impact of the stress and restore body homeostasis. Insect anti-stress response is a complex reaction controlled by the nervous and endocrine systems, with adipokinetic hormones (AKHs) playing the primary role (Kodrík, 2008). These peptides functionally resemble mammalian glucagon (Alquicer et al., 2009; Bednářová et al., 2013), and are predominantly synthesized, stored, and released by the neurosecretory cells of the corpora cardiaca, which are connected to the brain. Adipokinetic hormones comprise eight to 10 amino acids (Gäde et al., 1997) and their signal transduction at the cellular level is well documented in the fat body (Gäde and Auerswald, 2003). Generally, AKHs are a typical example of neuropeptides with complex functions in the control of insect metabolism, including the mobilization of different kinds of energy reserves such as lipids, carbohydrates, and/or certain amino acids (Gäde et al., 1997; Gäde and Goldsworthy, 2003). Additionally, AKHs are pleiotropic in nature with many activities associated to their metabolic role, including the stimulation of neuronal signalling (Milde et al., 1995), the increase of muscle tonus (O'Shea et al., 1984), and the stimulation of general locomotion (Socha et al., 1999). These hormones are also known to: regulate the starvation-induced foraging behaviour of Drosophila (Lee and Park, 2004); participate in the activation of antioxidant mechanisms (Kodrík et al., 2007, 2015b); enhance food intake and digestive processes in insect's gut (Kodrík et al., 2012; Bil et al., 2014; Bodláková et al., 2017); and interact with the cellular and humoral immune system (Goldsworthy et al., 2002a). Especially, the latter AKH function in immune system is remarkable in connection with the Habrobracon venom action. As mentioned above the venom supresses phenoloxidase activity (Kryukova et al., 2011); on the other hand AKH, if applied with suitable immunogen, activates the prophenoloxidase cascade (Goldsworthy et al., 2003; Mullen and Goldsworthy, 2006). Nevertheless, no comparative study between these two opposed actions is available. Interestingly, AKHs are able to penetrate the insect cuticle (e.g., Kodrík et al., 2002a; Lorenz et al., 2004). Indeed, several recent studies reported that the co-application of AKH, by injection or topically, with insecticides enhances their efficacy (Kodrík et al., 2010; Velki et al., 2011; Plavšin et al., 2015; reviewed by Kodrík et al., 2015c). Although this action mechanism is unknown, it is hypothesised that AKHs might intensify insecticide action by accelerating the rate of metabolites' exchange (documented by increased carbon dioxide production), accompanied by the faster penetration of toxins into tissues. One excellent model where the function of AKHs has been intensively studied is the firebug Pyrrhocoris apterus (Kodrík, 2008; Kodrík et al., 2015a). Its two AKHs, Pyrap-AKH (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-TrpNH2; Kodrík et al., 2000) and Peram-CAH-II (pGlu-Leu-Thr-Phe-ThrPro-Asn-Trp-NH2; Kodrík et al., 2002b), are well characterized, including cDNA sequences and the amino acid composition of their pre-prohormones (Kodrík et al., 2015a). Thus, the present study aimed to explore the putative role of AKHs in the neuromuscular paralysis elicited by H. hebetor venom in the P. apterus model, and to characterize its impact on other physiological and biochemical processes. Accordingly, this study might reveal new aspects of the biological mechanism leading to venom-induced paralysis and of the defence mechanisms against it.

2. Materials and methods 2.1. Insects and rearing A stock culture of the firebug P. apterus (L.) (Heteroptera), established from wild populations collected at České Budějovice (Czech Republic, 49°N), was used for the present study. Larvae and adults of the reproductive (brachypterous) morph were kept in 0.5 L glass jars in a mass culture (approximately 40 specimens per jar) and reared at constant temperature of 26 ± 1 °C under long-day conditions (18:6 h light:dark). They were supplied with linden seeds and water ad libitum, which were replenished twice weekly. Freshly ecdysed adults were transferred to small 0.25 L glass jars (females and males separately) and were kept under the same photoperiodic, food and temperature regimes as those under which they had developed (Socha and Kodrík, 1999). To have uniform material only 3-day old females were used for the experiments. Cohorts of H. hebetor (Say; Hymenoptera) adults (10 females and 5 males) were placed into 500 mL conical flask and supplied with honey as food. Each flask was also supplied with last instar Ephestia kuehniella larvae as hosts for oviposition. Individual flask containing H. hebetor were reared under the controlled conditions as described for P. apterus. Live female wasps were collected into tubes and frozen (−20 °C). As needed, they were retrieved, kept on ice and the venom glands were dissected under a dissection microscope. The dissected glands were washed with Ringer saline, and placed into a tube with a fresh portion of Ringer saline (1 mL) on ice. 2.2. Venom extraction Venom sacs from 100 H. hebetor adults were crushed in 1 mL Ringer saline by sonication and filtered through 0.22 μM filters. Afterwards the venom was separated into aliquots of 10 glands equivalent and stored at −20 °C until used. 2.3. Venom and AKH treatments, determination of paralysis effectivity The crude venom extract −0.2 gland equivalent in 2 μL volume of Ringer saline was injected with help of a Hamilton syringe through the metathoracal–abdominal intersegmental membrane to the thorax of experimental 3-day old P. apterus females. The dose of 0.2 gland equivalent was estimated from the dose response curve (Fig. 1) as minimal dose causing maximal effect i.e. 100% paralysed insects within 24 h. For controls the same volume of Ringer saline was injected. The experiments were run in three replicates with 16 bugs in each groups under the same conditions as for the stock culture. The paralysis was defined as total immobilization accompanied by limited or no movement of legs and antenna upon intensive shaking of the jar containing insects.

Fig. 1. Dose response curve of paralysis of 3-day old P. apterus adult females 24 h after injection of increasing doses of the venom gland equivalent.


Only live bugs were counted into the results; interestingly, no or only negligible mortality comparable to controls was recorded in the experimental bugs within 120 h after the venom treatment (data not shown). In some experiments, the effect of Pyrap-AKH on the course of paralysis was tested, and both peptides Pyrap-AKH and Peram-CAH-II were used as HPLC standards (for details see Section 2.6). The peptides were commercially synthesized by Dr. L. Lepša from Vidia Company (Praha, Czech Republic). A dose of 80 pmol Pyrap-AKH was applied topically as described previously (Kodrík et al., 2002a): 2 μL of the hormonal solution in 20% methanol in Ringer saline was applied using a pipette onto the thorax and abdomen under the wings. Similarly, control bugs were treated with the solvent only in the same way.

2.4. RNA isolation and cDNA synthesis Total RNAs were isolated from the CNS using RiboZol™ RNA Extraction Reagents (AMRESCO, LLC. Solon, Ohio, USA) following the manufacturer's protocol. RNA isolates were treated with TURBO DNAfree™ DNase (AMBION® by Life Technologies™, Carlsbad, California, USA) to remove traces of contaminant DNA. Reverse transcription was carried out using the Superscript FirstStrand Synthesis System for RT-PCR (Invitrogen) on 1 μg of template RNA with and random hexamers. The resulting cDNA was amplified by a subsequent PCR reaction using a 1 μL aliquot of the RT reaction as a template. The forward and reverse primers used for PCR amplification were designed based on the known amino acid structure of Pyrap-AKH (QLNFTPNW) and Peram-CAH II (QLTFTPNW) (see Table S1 for complete DNA sequences), and ribosomal protein RP49 as described previously (Kodrík et al., 2015a). The PCR profile included initial denaturation (3 min at 95 °C), 35 cycles of denaturation (95 °C for 30 s), primer annealing (59 °C for 30 s) and extension (72 °C for 30 s), followed by final extension at 72 °C for 7 min. The RT-PCR products were analysed by agarose gel electrophoresis.

2.5. Quantification of Pyrap-Akh and Peram-Cah-II gene expressions Expression of Akh genes was quantified by technique. Total RNA and cDNA synthesis was performed as described in previous Section 2.4. To monitor genomic DNA contamination we performed RT- control reactions. One q-RT-PCR reaction mixture contained 7 μL SYBR® Premix Ex Taq™II (TaKaRa), 3 μL of 10 × diluted cDNA template, 500 nM forward and reverse primers, and water in total volume of 14 μL. The gene specific primers, forward (5′- GCA TCC CAG AGG ACA ACT ACA -3′) and reverse (5′- TTT ACA TTC GTC CTG GGT CA -3′) to amplify Pyrap-Akh, and forward (5′- GCA TCC CAG AGG AGA ACTA -3′) and reverse (5′- GGC TCA CGG TCA TAC GTT -3′) to amplify Peram-Cah-II were used, respectively (Table S1). The sequence encoding ribosomal protein RP49 was amplified with forward (5′- CCG ATA TGT AAA ACT GAG AAA C -3′) and reverse (5′- GGA GCA TGT GCC TGG TCT TTT -3′) primers and it was used as a reference gene (Doležel et al., 2007). The reaction was performed on the Light Cycler (Bio-Rad, USA) and the program was as follows: initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 94 °C for 15 s, annealing at 59 °C for 30 s and elongation at 72 °C for 30 s. A final melt-curve step was included post-PCR (ramping from 65 °C to 95 °C by 0.5 °C every 5 s) to confirm the absence of any non-specific amplification. The efficiency of each primer pair was assessed by constructing a standard curve through four serial dilutions. Each q-RT-PCR experiment consisted of three independent biological replicates (11 respective tissues per replicate) with three technical replicates for each parallel group. The reaction efficiency and Ct values were analysed using Bio-Rad CFX Manager software. Relative gene expression was determined using the method described by Pfaffl (2001).

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2.6. AKH extraction from CNS and haemolymph, AKH titre estimation in haemolymph Central nervous system containing the brain with corpora cardiaca and corpus allatum attached was dissected from the firebug head cut off from the body under the Ringer saline. The AKHs were extracted from the CNS using 80% methanol, the solution was evaporated in a vacuum centrifuge and the resulting pellet stored at −20 °C until needed. For semi-quantitative estimation of the endogenous AKH titre in the haemolymph some pre-purification steps described in our previous paper (Goldsworthy et al., 2002b) were essential. Briefly, haemolymph samples collected from several dozens (80–100 individuals) of firebugs by cutting off their antennae (volumes 100 μL – see the Results section) were extracted in 80% methanol and after centrifugation the supernatants were evaporated to dryness. Then the pellets were dissolved in 0.11% trifluoroacetic acid, applied to a solid phase extraction cartridge Sep Pak C18 (Waters), and eluted by 60% acetonitrile. The eluent was analysed on a Waters HPLC system operated by Clarity software (DataApex version 6.2) with a fluorescence detector Waters 2475 (wave length λEx – 280 nm; λEm – 348 nm) using a Chromolith Performance RP-18e column 150–4.6 mm (Merck), solutions A and B (A – 0.11% trifluoroacetic acid in water; B – 0.1% trifluoroacetic acid in 60% acetonitrile) and a flow rate 2 mL/min. Retention times of the two Pyrrhocoris synthetic adipokinetic peptides Pyrap-AKH and PeramCAH-II were identical under the used conditions - 7.54 min. Relative titre of AKHs in the haemolymph samples was estimated using areas of the corresponding HPLC peaks identified by applying the synthetic standards. 2.7. ELISA determination of AKH level in CNS A competitive ELISA was used for determination of total AKH content in P. apterus CNS (antibody dilution 1:5000, 0.5 CNS equiv. per well, detection limit 20 fmol per well according to our protocol published earlier (Goldsworthy et al., 2002b). Briefly, rabbit antibodies were raised commercially against Cys1Pyrap-AKH (Sigma Genosys, Cambridge, UK) and the resulting antibody recognized well both the Pyrap-AKH and the Peram-CAH-II. A biotinylated probe was prepared from Cys1-Pyrap-AKH using Biotin Long Arm Maleimide (BLAM, Vector Laboratories, Peterborough, UK). The ELISA comprised pre-coating of the 96-well microtiter plates (high binding Costar, Corning Incorporated, Corning, NY, USA) overnight with the antibody preparation in coating buffer. After blocking (with non-fat dried milk), test samples were added to specific wells, followed by the biotinylated probe, both in an assay buffer. After the competition for the binding sites on the antibody bound to the plates a streptavidin conjugated with horseradish peroxidase solution (Vector Laboratories) diluted (1:500) in PBS-Tween was added to each well. All of the above-mentioned steps were terminated by washing. Finally, the ELISA substrate (3,3′,5,5′-tetramethylbenzidine, Sigma Aldrich) was added and then the reaction was stopped by adding 0.5 M sulphuric acid. The absorbance values were determined in a microtiter plate reader at 450 nm. One row of each plate always contained a dilution series of synthetic Pyrap-AKH, which allowed the construction of a competition curve and estimation of the AKH content of unknown samples. 2.8. Metabolic rate measurement The Li-7000 CO2/H2O analyser (Li-COR Biosciences, Lincoln, NE, USA) was used to measure the rate of carbon dioxide production by experimental bugs as previously described (Kodrík et al., 2010). Eight individual bugs were measured separately in eight measuring chambers 24 h after the treatment by venom for a period of 40 min. Data were analysed by data acquisition software (Sable System, Las Vegas, Nevada, USA). The carbon dioxide production (VCO2) was calculated from fractional concentrations of carbon dioxide going in (FI) and coming out

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(FE) of the respirometry chamber using an equation according to Withers (1977) and expressed in μL min− 1 bug− 1 units: VCO2 = (FECO2 − FICO2) f, where f is the flow rate in μL h−1. The experiment was repeated three times.

2.9. Spectrophotometric determination of lipids and carbohydrates The level of lipids and carbohydrates was determined in the firebug haemolymph 24 h after the venom treatment. To do that the haemolymph samples were obtained from cutting end of the antenna as described in Section 2.6. Further, haemocytes were removed from the samples by centrifuging at 13,000g for 2 min at 4 °C, and 1 μL of supernatant per sample was used for determination of the level of nutrients. - Lipid determination – was done by sulpho-phosho-vanillin method according to Zöllner and Kirsch (1962), as modified for Pyrrhocoris by Kodrík et al. (2000). The optical densities at 546 nm, measured in a spectrophotometer (UV 1601 Shimadzu), were converted to μg lipids per μL haemolymph with the aid of a calibration curve based on known amounts of oleic acid. - Carbohydrate determination – the haemolymph supernatant (1 μL) was diluted in 39 μL of distilled water and then used for quantification of carbohydrate level by the anthrone method (Carroll et al., 1956) that was modified for Pyrrhocoris by Socha et al. (2004). For the calibration curve known amounts of glucose were used.

Fig. 2. (A) Time course of paralysis of 3-day old P. apterus adult females after injection of venom (0.2 gland equiv.) and/or after topical application of Pyrap-AKH (80 pmol). Application of Pyrap-AKH (80 pmol) or saline only (control) elicited no paralysis (data not shown for clarity). Statistical analysis with the Chi-square test for trend (Chisquare = 31.04, df = 1, P = 0.0001) proved significant difference between both curves at the 0.01% level (n = 3). (B) The same effect 8 h after the treatments. The number above the venom + AKH column represents fold-difference of percentage of paralysed bugs as compared with venom treated bugs. Statistically significant differences among the experimental groups evaluated using one-way ANOVA with Tukey's post test at the 5% level are indicated by different letters (n = 3).

2.10. Data presentation and statistical analyses. The results were plotted and statistics were calculated using the graphic software Prism (Graph Pad Software, version 6.01 San Diego, CA, USA). Points in the graphs represent mean ± SD, the numbers of replicates (n) are depicted in the figure legends. The statistical differences were evaluated by Student's t-test (Figs. 3, 4, 6 and 7) and oneway ANOVA with the Tukey's post-test (Fig. 2B). Time course of paralysis (Fig. 2A) was evaluated with the Chi-square test for trend.

3. Results 3.1. The effects of venom and AKH on paralysis The impact of H. hebetor venom on the paralysis of 3-day-old P. apterus adult females was evaluated. Tests using 0.2 gland-equivalent venom per female revealed a time-response effect on the experimental bugs. The first paralysed individuals were recorded 6 h after the venom treatment, 50% of the individuals were paralysed (ET50) 7.9 h after the treatment, and all bugs were totally paralysed within 15 h after the treatment (Fig. 2A). Notably, the topical application of 80 pmol PyrapAKH reduced the impact of the venom on the experimental bugs and significantly slowed down the paralysing process. The first paralysed individuals in this group (venom + Pyrap-AKH) were recorded 7 h after the treatment, ET50 was observed 11.5 h after the treatment, and all bugs were paralysed within 20 h after the treatment. The largest difference between groups was observed about 8 h after the treatment (near ET50 for the venom only group), when the percentage venom + PyrapAKH-paralysed bugs was about 3.2 times lower than that in the group treated with venom only (Fig. 2B). During the experiment, no paralysis was recorded in the bugs treated with Ringer saline or 80 pmol PyrapAKH only.

Fig. 3. The relative expression of Pyrap-Akh (A) and Peram-Cah-II (B) genes in 3-day old P. apterus adult females 24 h after the venom (0.2 gland equiv.) treatment. The numbers above the venom columns represent fold-difference of relative gene expressions as compared with corresponding controls. Statistically significant differences between the experimental and control groups evaluated using the Student's t-test at the 5% level are indicated by asterisks (n = 3).


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Fig. 4. ELISA quantification of the Pyrap-AKH and Peram-CAH-II (together) levels in CNS of 3-day old P. apterus adult females 24 h after the venom (0.2 gland equiv.) treatment. The number above the venom column represents fold-difference of AKH level as compared with control. Statistically significant difference between the experimental and control groups evaluated using the Student's t-test at the 5% level is indicated by asterisks (n = 9–10).

3.2. The effect of venom on Pyrap-Akh and Peram-Cah-II gene expressions, and on AKH level in CNS and haemolymph Venom injection is expected to cause intensive stress in P. apterus body, leading to the activation of the nervous and endocrine systems and corresponding biochemical- and physiological-defence reactions. This assumption was monitored by assessing Pyrap-Akh and PeramCah-II gene expressions in the firebug's CNS (Fig. 3), and by determining AKH levels in the CNS (Fig. 4) and haemolymph (Fig. 5). Twenty-four hours after the venom treatment, significant 5.4-fold and 3.6-fold increases were detected in Pyrap-Akh and Peram-Cah-II gene expressions, respectively (Fig. 3). The response of AKH levels in CNS and haemolymph was similar to the trend observed in genes' expression, although slightly less intensively, i.e., there was a significant increase in the CNS (2.5-fold; Fig. 4) and haemolymph (3.0-fold; Fig. 5). However, AKH levels in haemolymph should be regarded as semi-quantitative only, as they were estimated from the peak areas of the AKHs in haemolymph samples fractionated in the HPLC column (Fig. 5A and B). Peak areas calculated in the Clarity software were 64.275 units in controls and 195.056 units in venom treated bugs, indicating a 3.0fold increase of the AKH level in the haemolymph of venom treated bugs. 3.3. The effect of venom on general metabolic intensity Using the production of carbon dioxide by experimental firebugs 24 and 48 h after the venom treatment as a marker of metabolic intensity (Fig. 6), revealed that venom application significantly decreased metabolic intensity. Carbon dioxide production at 24 and 48 h decreased 1.4-fold and 1.9-fold, respectively, compared to Ringer saline-injected controls.

Fig. 5. The RP HPLC elution profiles of pre-purified extracts of 100 μL haemolymph from 3day old P. apterus adult females 24 h after the Ringer saline injection (control; A) and after the venom injection (B). (C) The relative level of AKHs in HPLC pre-purified haemolymph (in the peaks marked by the arrows in A and B) from 3-day old P. apterus adult females. The number above the column represents fold-difference of AKH level in haemolymph of the venom treated bugs as compared with untreated controls.

venom is also active in some Hemiptera (Drenth, 1974), but only when high doses are injected. The present study showed that H. hebetor venom induced 100% paralysis in the hemipteran P. apterus, when 0.2 venom gland-equivalent was used. Additionally, it evidenced that the

3.4. The effect of venom on lipid and carbohydrate levels in the haemolymph Venom injection itself and the subsequent releasing of natural AKHs from the corpora cardiaca into the haemolymph might primarily mobilize available nutrients from the fat body. Indeed, venom injection elicited a significant 1.9-fold increase of total lipids level in the haemolymph, but had no effect on carbohydrate level (Fig. 7). 4. Discussion Although most reports on the paralysis-inducing effect of H. hebetor venom toxins comprise holometabolous insect species from the orders Lepidoptera and Coleoptera (Pennacchio and Strand, 2006), the

Fig. 6. The carbon dioxide production in 3-day old P. apterus adult females 24 and 48 h after the venom (0.2 gland equiv.) treatments. The numbers above the venom columns represent fold-difference of carbon dioxide production as compared with corresponding controls. Statistically significant differences between the experimental and control groups evaluated using the Student's t-test at the 5% level are indicated by asterisks (n = 7–14).

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Fig. 7. The lipid and carbohydrate levels in haemolymph of 3-day old P. apterus adult females 24 h after the venom (0.2 gland equiv.) treatments. The number above the lipid venom column represents fold-difference of lipid level as compared with control. Statistically significant difference between the experimental and control groups evaluated using the Student's t-test at the 5% level is indicated by asterisk (n = 6–8).

application of AKH delayed paralysis. The mechanism underlying AKH activity is still unknown, because the mechanism by which venom toxins elicit neuromuscular paralysis is also not known in detail. Still, it is known that the venom blocks receptors on the presynaptic membranes of neuromuscular junctions and inhibits exocytosis in presynaptic vesicles (Slavnova et al., 1987; Pennacchio and Strand, 2006). This finding was recently confirmed by Sláma (2012), who proved that the venom does not affect the neuron systems regulated by junction potential or interneuron communication, only disabling the transmission of the neuromuscular action potential. Further, it is generally accepted that AKHs play a role in neuronal signalling. This was first described by Milde et al. (1995), who showed that the injection of AKH into the mesothoracic ganglion of Manduca sexta increased the electrical activity of relevant nerves in the corresponding mesothoracic muscles. Recently, it has been proven that, at least in P. apterus brain, AKHs are localized in neuronal bodies and axons, where their secretion might play a role in neuronal signalling (Kodrík et al., 2015a). Unfortunately, there is no data on the role played by AKHs in neuromuscular junctions, but this cannot be completely excluded, as AKHs increase muscle tonus (O'Shea et al., 1984) and stimulate general locomotion (Socha et al., 1999; Kodrík et al., 2000). Although the mechanism by which AKHs affect insect's muscular system is not known, a possible control via nervous system and neuromuscular junctions has already been considered by Socha et al. (1999). Thus, interactions between H. hebetor venom toxins and AKHs in neuromuscular junctions cannot be completely excluded, despite the need for direct evidence. Further, one can speculate whether the assumed immune-response elicited by the venom application (Kryukova et al., 2011) could also participate in the induction of the neuromuscular paralysis. It is known that the neural and immune systems operate in mutual co-operation (Kawli et al., 2010); accordingly, it has been proven recently that overactivation of immune-response in Drosophila brain induces neurodegeneration, and that the NF-κB transcription factor is also involved (Cao et al., 2013). It is also known that the venom from the wasp N. vitripennis affects the NF-κB activity (Cao et al., 2013; Danneels et al., 2015). Thus, taken together all these facts, application of a parasitic wasp venom might affect neuronal/neuromuscular functions including those in the central nervous systems via activation of the innate immune system. However, without any supporting data this remains just a speculation. Stress usually increases the level of AKH in the insect body. Indeed, the stress induced by the application of H. hebetor venom significantly increased the expression of both P. apterus Akh genes and AKH levels in the CNS and haemolymph. Similar reactions were recorded after the application of various stressors (e.g., insecticides, bacterial toxins, oxidative stressors - see Kodrík et al., 2015b, 2015c). However, the AKH increase in the CNS varied depending on insect species and stressor type. It is generally known that the coupling between AKHs biosynthesis

in the CNS and their release into the haemolymph is very week or even non-existing in some insect species (Diederen et al., 2002). Naturally, AKH content is several hundred times higher in the CNS than in the haemolymph (Goldsworthy et al., 2002b; Candy, 2002), and the increased demands of AKH in insect target tissues are usually covered by stored AKH not affecting the rate of its synthesis. Accordingly, after stressor application, elevation of AKH levels in the haemolymph was always recorded in tested species, including the locust Schistocerca gregaria (Candy, 2002), the Colorado potato beetle Leptinotarsa decemlineata (Kodrík et al., 2007), and the firebug P. apterus (Kodrík and Socha, 2005; Kodrík et al., 2010; Velki et al., 2011), among others. In the present study, a venom-induced increase in AKH levels was recorded in both the CNS and haemolymph. Data on chemical stressors influencing Akh gene expression are essentially unavailable, and it has recently been shown that the application of the oxidative stressor paraquat onto Drosophila melanogaster body had no effect on Akh expression (Zemanová et al., 2016). Contrarily, in the present study, H. hebetor venom significantly stimulated the expression of both Akh genes in P. apterus suggesting that this stimulation might be species- or stressor-specific. Thus, a weak relationship between Akh gene expression and AKH levels in the CNS, similar to that suggested by Diederen et al. (2002) for AKH levels in the CNS and haemolymph, cannot be completely excluded. Application of the venom significantly reduced general metabolism, as indicated by the carbon dioxide production (see Fig. 6). This result was expected and is in accordance with the decreased rate of oxygen consumption in G. mellonella larvae paralysed by H. hebetor venom as reported in previous studies (Waller, 1965; Edwards and Sernka, 1969). However, it was not clear whether the metabolic decline resulted from muscle inhibition and starvation, which lower oxygen consumption, or from the direct action of the venom. Nevertheless, Slama's experiments (2012) with ligatured and venom-poisoned G. mellonella larvae unequivocally demonstrated that paralysis does not disintegrate a metabolically active state. This explains why H. hebetor venom application into P. apterus body significantly increased total lipids level in the haemolymph (see Fig. 7). Still, this might be a secondary effect, mediated by the increased level of AKH in the haemolymph. This consideration is supported by the fact that venom application had no effect on haemolymph carbohydrates. It is known that P. apterus metabolism depends mostly on lipids, and thus their level in the haemolymph is about 10 times higher than that of free carbohydrates (this study; Socha et al., 2005); in the fat body the level of lipids is about 100 times higher than that of glycogen (Socha et al., 2005). Additionally, the application of AKH into P. apterus body significantly increased the mobilization of lipids (Kodrík et al., 2000; Socha et al., 2004) but not that of carbohydrates (Socha et al., 2004). In summary, the present study demonstrated that the co-application of H. hebetor venom with Pyrap-AKH into P. apterus body significantly reduced total neuromuscular paralysis compared to the application of venom only. Although the mechanism of their action is still not known, both agents might affect neuromuscular junctions. Venom application significantly increased Akh gene expression, as well as the levels of AKH in the CNS and haemolymph. This increased level of AKH in the haemolymph probably led to the mobilization of the lipid stores into the haemolymph, while the level of carbohydrates remained unchanged. Total metabolism in venom-paralysed P. apterus individuals declined, probably due a direct inhibition of the muscular system. These findings are academically interesting but also contribute data that might be applicable in human or veterinary medicine in future. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpc.2017.02.011. Acknowledgements This study was supported by grant No. 17-03253S (DK) from the Czech Science Foundation, and by projects RVO 60077344 of the


Institute of Entomology. The authors thank Dr. Z. Svobodová (Institute of Entomology) for help with statistical analysis, and Mrs. Štěrbová for technical assistance. The authors are also thankful to the insects whose sacrifice made this work possible. The English grammar and stylistics were checked by the Editage Author Services. References Alquicer, G., Kodrík, D., Krishnan, N., Večeřa, J., Socha, R., 2009. Activation of insect antioxidative mechanisms by mammalian glucagon. Comp. Biochem. Physiol. B 152, 226–233. Altuntas, H., Kilic, A.Y., Sivas-Zeytinoglu, H., 2010. The effects of parasitism by the ectoparasitoid Bracon hebetor Say (Hymenoptera: Braconidae) on host hemolymph proteins in the Mediterranean flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Turk. J. Zool. 34, 409–416. Andrew, N., Basio, M., Kim, Y., 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31, 341–347. Baker, J.E., Fabrick, J.A., 2000. Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: Braconidae). Insect Biochem. Mol. Biol. 30, 937–946. Beckage, N.E., Gelman, D.B., 2004. Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. Annu. Rev. Entomol. 49, 299–330. Bednářová, A., Kodrík, D., Krishnan, N., 2013. Unique roles of glucagon and glucagon-like peptides: parallels in understanding the functions of adipokinetic hormones in stress responses in insects. Comp. Biochem. Physiol. A 164, 91–100. Benson, J.F., 1974. Population dynamics of Bracon hebetor Say (Hymenoptera, Braconidae) and Ephestia cautella (Walker) (Lepidoptera, Phycitidae) in a laboratory ecosystem. J. Anim. Ecol. 24, 71–86. Bil, M., Broeckx, V., Landuyt, B., Huybrechts, R., 2014. Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis. Gen. Comp. Endocrinol. 208, 49–56. Bodláková, K., Jedlička, P., Kodrík, D., 2017. Adipokinetic hormones control amylase activity in the cockroach (Periplaneta americana) gut. Insect Sci. http://dx.doi.org/10.1111/ 1744-7917.12314. Candy, D.J., 2002. Adipokinetic hormones concentrations in the haemolymph of Schistocerca gregaria, measured by radioimmunoassay. Insect Biochem. Mol. Biol. 32, 1361–1367. Cao, Y., Chtarbanova, S., Petersen, A.J., Ganetzky, B., 2013. Dnr1 mutations cause neurodegeneration in Drosophila by activating the innate immune response in the brain. Proc. Natl. Acad. Sci. U. S. A. 110, E1752–E1760. Carroll, N.V., Longley, R.W., Roe, J.H., 1956. The determination of glycogen in liver and muscle by use of anthrone reagent. J. Biol. Chem. 220, 583–593. Danneels, E.L., Gerlo, S., Heyninck, K., van Craenenbroeck, C.K., de Bosscher, B.K., Haegeman, G., de Graaf, D.C., 2014. How the venom from the ectoparasitoid wasp Nasonia vitripennis exhibits anti-inflammatory properties on mammalian cell lines. PLoS One 9, e96825. Danneels, E.L., Formesyn, E.M., De Graaf, D.C., 2015. Exploring the potential of venom from Nasonia vitripennis as therapeutic agent with high-throughput screening tools. Toxins 7, 2051–2070. De Graaf, D.C., Aerts, M., Brunain, M., Desjardins, C.A., Jacobs, F.J., Werren, J.H., Devreese, B., 2010. Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies. Insect Mol. Biol. 19, 11–26. Diederen, J.H.B., Oudejans, R.C.H.M., Harthoorn, L.F., Van der Horst, D.J., 2002. Cell biology of the adipokinetic hormone-producing neurosecretory cells in the locust corpus cardiacum. Microsc. Res. Tech. 56, 227–236. Doležel, D., Šauman, I., Košt'ál, V., Hodková, M., 2007. Photoperiodic and food signals control expression pattern of the clock gene, period, in the linden bug, Pyrrhocoris apterus. J. Biol. Rhythm. 22, 335–342. Drenth, D., 1974. Susceptibility of different species of insects to an extract of the venom gland of the wasp Microbracon hebetor (Say). Toxicon 12, 189–192. Edwards, J.S., Sernka, T.J., 1969. On the action of Bracon venom. Toxicon 6, 303–305. Gäde, G., Auerswald, L., 2003. Mode of action of neuropeptides from the adipokinetic hormone family. Gen. Comp. Endocrinol. 132, 10–20. Gäde, G., Goldsworthy, G.J., 2003. Insect peptide hormones: a selective review of their physiology and potential application for pest control. Pest Manag. Sci. 59, 1063–1075. Gäde, G., Hoffmann, K.H., Spring, J.H., 1997. Hormonal regulation in insects: facts, gaps, and future directions. Physiol. Rev. 77, 963–1032. Goldsworthy, G.J., Opoku-Ware, K., Mullen, L.M., 2002a. Adipokinetic hormone enhances laminarin and bacterial lipopolysaccharide-induced activation of the prophenoloxidase cascade in the African migratory locust, Locusta migratoria. J. Insect Physiol. 48, 601–608. Goldsworthy, G.J., Kodrík, D., Comley, R., Lightfoot, M., 2002b. A quantitative study of the adipokinetic hormone of the firebug, Pyrrhocoris apterus. J. Insect Physiol. 48, 1103–1108. Goldsworthy, G.J., Chandrakant, S., Opoku-Ware, K., 2003. Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide. J. Insect Physiol. 49, 795–803. Hartzer, K.L., Zhu, K.Y., Baker, J.E., 2005. Phenoloxidase in larvae of Plodia interpunctella (Lepidoptera: Pyralidae): molecular cloning of the proenzyme cDNA and enzyme activity in larvae paralyzed and parasitized by Habrobracon hebetor (Hymenoptera: Braconidae). Arch. Insect Biochem. Physiol. 59, 67–79.

17

Kawli, T., He, F., Tan, M.W., 2010. It takes nerves to fight infections: insights on neuro-immune interactions from C. elegans. Dis. Model. Mech. 3, 721–731. Kodrík, D., 2008. Adipokinetic hormone functions that are not associated with insect flight. Physiol. Entomol. 33, 171–180. Kodrík, D., Socha, R., 2005. The effect of insecticide on adipokinetic hormone titre in insect body. Pest Manag. Sci. 61, 1077–1082. Kodrík, D., Socha, R., Šimek, P., Zemek, R., Goldsworthy, G.J., 2000. A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera). Insect Biochem. Mol. Biol. 30, 489–498. Kodrík, D., Socha, R., Zemek, R., 2002a. Topical application of Pya-AKH stimulates lipid mobilization and locomotion in the flightless bug, Pyrrhocoris apterus (L.) (Heteroptera). Physiol. Entomol. 27, 15–20. Kodrík, D., Šimek, P., Lepša, L., Socha, R., 2002b. Identification of the cockroach neuropeptide Pea-CAH-II as a second adipokinetic hormone in the firebug Pyrrhocoris apterus. Peptides 23, 585–587. Kodrík, D., Krishnan, N., Habuštová, O., 2007. Is the titer of adipokinetic peptides in Leptinotarsa decemlineata fed on genetically modified potatoes increased by oxidative stress? Peptides 28, 974–980. Kodrík, D., Bártů, I., Socha, R., 2010. Adipokinetic hormone (Pyrap-AKH) enhances the effect of a pyrethroid insecticide against the firebug Pyrrhocoris apterus. Pest Manag. Sci. 66, 425–431. Kodrík, D., Vinokurov, K., Tomčala, A., Socha, R., 2012. The effect of adipokinetic hormone on midgut characteristics in Pyrrhocoris apterus L. (Heteroptera). J. Insect Physiol. 58, 194–204. Kodrík, D., Stašková, T., Jedličková, V., Weyda, F., Závodská, R., Pflegerová, J., 2015a. Molecular characterization, tissue distribution, and ultrastructural localization of adipokinetic hormones in the CNS of the firebug Pyrrhocoris apterus (Heteroptera, Insecta). Gen. Comp. Endocrinol. 210, 1–11. Kodrík, D., Bednářová, A., Zemanová, M., Krishnan, N., 2015b. Hormonal regulation of response to oxidative stress in insects - an update. Int. J. Mol. Sci. 16, 25788–25816. Kodrík, D., Plavšin, I., Velki, M., Stašková, T., 2015c. Enhancement of Insecticide Efficacy by Adipokinetic Hormones. In: Montgomery, J. (Ed.), Insecticides: Occurrence, Global Threats and Ecological Impact. Nova Science Publishers, New York, pp. 77–91. Kovalenkov, V.G., 1984. The biomethod in integrated protection of cotton. J. Zas. Rast. 8, 12–14. Kryukova, N.A., Dubovskiy, I.M., Gryzanova, E.A., Naumkina, V.V., Glupov, V.V., 2007. Cell immunity response of the Galleria mellonella (L.) (Lepidoptera, Piralidae) during parasitization of Habrobracon hebetor (Say) (Hymenoptera, Braconidae). Euroasian Entomol. J. 6, 361–364. Kryukova, N.A., Dubovskiy, I.M., Chertkova, E.A., Vorontsova, Y.L., Slepneva, I.A., Glupov, V.V., 2011. The effect of Habrobracon hebetor venom on the activity of the prophenoloxidase system, the generation of reactive oxygen species and encapsulation in the haemolymph of Galleria mellonella larvae. J. Insect Physiol. 57, 796–800. Kryukova, N.A., Chertkova, E.A., Semenova, A.D., Glazachev, Y.I., Slepneva, I.A., Glupov, V.V., 2015. Venom from the ectoparasitic wasp Habrobracon hebetor activates calcium-dependent degradation of Galleria mellonella larval hemocytes. Arch. Insect Biochem. Physiol. 90, 117–130. Lee, G., Park, J.H., 2004. Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167, 311–323. Lorenz, M.W., Zemek, R., Kodrík, D., Socha, R., 2004. Lipid mobilisation and locomotor stimulation in Gryllus bimaculatus (de Geer) (Ensifera, Gryllidae) by topically applied adipokinetic hormone. Physiol. Entomol. 29, 146–151. Milde, J.J., Ziegler, R., Wallstein, M., 1995. Adipokinetic hormone stimulates neurons in the insect central nervous system. J. Exp. Biol. 198, 1307–1311. Mullen, L.M., Goldsworthy, G.J., 2006. Immune responses of locusts to challenge with the pathogenic fungus Metarhizium or high doses of laminarin. J. Insect Physiol. 52, 389–398. Nay, J.E., Perring, T.M., 2005. Impact of ant predation and heat on carob moth (Lepidoptera: Pyralidae) mortality in California date gardens. J. Econ. Entomol. 98, 725–731. O'Shea, M., Witten, J., Schaffer, M., 1984. Isolation and characterization of two myoactive neuropetides: further evidence for an invertebrate peptide family. J. Neurosci. 4, 521–529. Pennacchio, F., Strand, M.R., 2006. Evolution of developmental strategies in parasitic Hymenoptera. Annu. Rev. Entomol. 51, 233–258. Pennacchio, F., Caccia, S., Digilio, M.C., 2014. Host regulation and nutritional exploitation by parasitic wasps. Curr. Opin. Insect Sci. 6, 74–79. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, e45. Plavšin, I., Stašková, T., Šerý, M., Smýkal, V., Hackenberger, H.K., Kodrík, D., 2015. Hormonal enhancement of insecticide efficacy in Tribolium castaneum: oxidative stress and metabolic aspects. Comp. Biochem. Physiol. C 170, 19–27. Quistad, G.B., Leisy, D.J., 1996. Insecticidal toxins from the parasitic wasp, Bracon hebetor US Patent No.5, 554 592. Quistad, G.B., Nguyen, Q., Bernasconi, P., Leisy, D.J., 1994. Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor. Insect Biochem. Mol. Biol. 24, 955–961. Shaik, H.A., Sehnal, F., 2009. Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration. J. Insect Physiol. 55, 781–787. Sláma, K., 2012. Neuromuscular Paralysis Induced in Insect Larvae by the Proteinic Venom of a Parasitic Wasp. In: Berhardt, L.V. (Ed.), Paralysis: Causes, Classification and Treatments, Vol. 43. Advances in Medicine and Biology. Nova Science Publishers, New York, pp. 195–212. Sláma, K., Lukáš, J., 2011. Myogenic nature of insect heartbeat and intestinal peristalsis, revealed by neuromuscular paralysis caused by the sting of a braconid wasp. J. Insect Physiol. 57, 251–259.

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Slavnova, T.I., Antonov, S.M., Magazanik, L.G., Tonkikh, A.K., Kosovskii, A.V., Sadykov, A.A., Abduvakhabov, A.A., 1987. Effect of toxin from the venom of the ichneumon Habrobracon hebetor (Say) on neuromuscular transmission in insects. Dokl. Akad. Nauk SSSR 297, 492–494. Socha, R., Kodrík, D., 1999. Differences in adipokinetic response of Pyrrhocoris apterus (Heteroptera) in relation to wing dimorphism and diapause. Physiol. Entomol. 24, 278–284. Socha, R., Kodrík, D., Zemek, R., 1999. Adipokinetic hormone stimulates insect locomotor activity. Naturwissenschaften 88, 85–86. Socha, R., Kodrík, D., Šimek, P., Patočková, M., 2004. The kind of AKH-mobilized energy substrates in insects can be predicted without a knowledge of the hormone structure. Eur. J. Entomol. 101, 29–35. Socha, R., Kodrík, D., Šula, J., 2005. Wing morph specific differences in the metabolism and endocrine control of reserve mobilization in adult males of a flightless bug, Pyrrhocoris apterus (L.) (Heteroptera). J. Comp. Physiol. B. 175, 557–565. Velki, M., Kodrík, D., Večeřa, J., Hackenberger, B.K., Socha, R., 2011. Oxidative stress elicited by insecticides: a role for the adipokinetic hormone. Gen. Comp. Endocrinol. 172, 77–84.

Visser, B.J., Labruyere, W.T., Spanjer, W., Piek, T., 1983. Characterization of two paralysing protein toxin (A-MTX and B-MTX) isolated from a homogenate of the wasp Microbracon hebetor (Say). Comp. Biochem. Physiol. B 75, 523–530. Waller, J.B., 1965. The effect of the venom of Bracon hebetor on the respiration of the wax moth Galleria mellonella. J. Insect Physiol. 11, 1595–1965. Withers, P.C., 1977. Measurement of VO2, VCO2 and evaporative water loss with a flowthrough mask. J. Appl. Physiol. 42, 120–123. Zemanová, M., Stašková, T., Kodrík, D., 2016. Role of adipokinetic hormone and adenosine in the anti-stress response in Drosophila melanogaster. J. Insect Physiol. 91-92, 39–47. Zöllner, N., Kirsch, K., 1962. Uber die quantitative Bestimmung von lipoide (Mikromethode) mittels der vielen naturlichen Lipoiden (Allen dekannten Plasmalipoiden) gemeinsamen sulfo-phospho-vanillin-Reaktion. Z. Gesamte Exp. Med. 135, 545–561.