In vivo effect of Neuropeptide F on ecdysteroidogenesis in adult female desert locusts (Schistocerca gregaria)

Journal of Insect Physiology 59 (2013) 624–630

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In vivo effect of Neuropeptide F on ecdysteroidogenesis in adult female desert locusts (Schistocerca gregaria) Pieter Van Wielendaele ⇑, Niels Wynant, Senne Dillen, Liesbeth Badisco, Elisabeth Marchal 1, Jozef Vanden Broeck Molecular Developmental Physiology and Signal Transduction, Department of Animal Physiology and Neurobiology, Zoological Institute, K.U. Leuven, Naamsestraat 59, B-3000 Leuven, Belgium

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Article history: Received 20 January 2013 Received in revised form 11 March 2013 Accepted 12 March 2013 Available online 21 March 2013 Keywords: Ecdysone Insect Oocyte maturation Peptide Reproduction

a b s t r a c t Neuropeptides are important regulatory factors that mediate key life processes, both in vertebrates and invertebrates. Many insect neuropeptides display pleiotropic activities, which means that they can influence multiple aspects of insect physiology. In the fruit fly, Drosophila melanogaster, Neuropeptide F (NPF) mediates diverse physiological processes, such as learning, stress responses, feeding and male courtship behavior. In locusts, only a truncated form of the predicted ‘‘full-length’’ NPF, the nonapeptide ‘‘trNPF’’, has been isolated. This nonapeptide previously proved to be biologically active, since it was shown to influence food intake and weight increase, as well as oocyte growth in adult female desert locusts (Schistocerca gregaria [Forskål]). In the present study, we have further analyzed the effect of trNPF on female reproductive physiology in S. gregaria. We confirmed that daily trNPF injections in adult females elicit an increase of oocyte size. In addition, an RNAi-mediated knockdown of the Schgr-NPF precursor transcript in adult female locusts resulted in the opposite effect, i.e. significantly smaller oocytes. Moreover, we discovered that daily injections of trNPF in adult female S. gregaria, caused higher ecdysteroid titers in the ovaries and accelerated the appearance of ecdysteroid peaks in the hemolymph of these animals. The RNAi-based knockdown of the Schgr-NPF precursor transcript clearly resulted in reduction of both hemolymph and ovarian ecdysteroid concentrations, confirming the stimulatory effects of trNPF injections on adult female ecdysteroid levels. The observed results are discussed in relation to previous reports on NPF activities in locusts and other insects. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Both in vertebrates and invertebrates, neuropeptides constitute a highly versatile class of extracellular messenger substances that play important roles in the regulation and coordination of internal physiology (Grimmelikhuijzen and Hauser, 2012; Kastin, 2006; Vanden Broeck, 2001). In insects, they are involved in the control of a wide variety of physiological processes, such as molting, growth, metabolism and reproduction (reviewed by Altstein and Nässel, 2010). One of these insect neuropeptides is ‘‘Neuropeptide F’’ (NPF), the invertebrate counterpart of ‘‘Neuropeptide Y’’ (NPY). Studies performed in the fruit fly, Drosophila melanogaster, demonstrated that NPF is a pleiotropic factor, since this peptide can influence multiple physiological processes in this species (Nässel and ⇑ Corresponding author. Address: Animal Physiology and Neurobiology, Naamsestraat 59, P.O. Box 02465, B-3000 Leuven, Belgium. Tel.: +32 16 32 42 60; fax: +32 16 32 39 02. E-mail address: [email protected] (P. Van Wielendaele). 1 Current address: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Canada. 0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.03.005

Wegener, 2011). In addition to its well-established regulatory role in feeding and foraging behavior (Lingo et al., 2007; Shen and Cai, 2001; Wu et al., 2003, 2005a,b), fruit fly NPF was also found to mediate learning and memory (Krashes et al., 2009), stress responses (Xu et al., 2010), alcohol sensitivity (Wen et al., 2005), reward-seeking behavior (Shohat-Ophir et al., 2012), circadian activity and male courtship behavior (Lee et al., 2006; Hermann et al., 2012). However, in other insect species, much less information is available about the physiological activities of NPF. While in several insects a ‘‘full-length’’ NPF (30–35 AA) was demonstrated (Brown et al., 1999; Nuss et al., 2010; Stanek et al., 2002), only a nonapeptide, consisting of the C-terminal region of the predicted ‘‘full-length’’ NPF, was purified and identified from the locusts, Locusta migratoria and Schistocerca gregaria (Schoofs et al., 2001; Clynen et al., 2009). This locust nonapeptide was designated as ‘‘truncated NPF’’ or ‘‘trNPF’’ to make the distinction with both ‘‘short NPF’’ (sNPF) and the longer, non-truncated version of NPF, which has not been demonstrated in these species (Van Wielendaele et al., 2013a). Recent studies on trNPF indicated that this peptide influences food intake and weight gain, as well as several aspects of male

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reproductive physiology in the desert locust, S. gregaria (Van Wielendaele et al., 2013a,b). Moreover, biological activity of trNPF had already been demonstrated in locusts by Schoofs and coworkers (2001), who found that this peptide can stimulate oocyte growth in adult female S. gregaria. Also other endocrine factors were previously reported to play a role in the regulation of various aspects of female reproductive physiology in locusts. In addition to neuropeptides, such as neuroparsins, ovary maturating parsin and insulin-related peptide, the lipophilic insect hormones, ecdysteroids and juvenile hormone, also take part in this regulation (reviewed by Verlinden et al., 2009). In adult female insects, ecdysteroid hormones are mainly produced by the ovaries (Brown et al., 2009). Ecdysteroids of locust females were shown to mediate oocyte maturation (Lanot et al., 1987), but are also stored within the oocytes to supply a source of hormone during embryonic development (Dinan and Rees, 1981; Isaac and Rees, 1985; Lagueux et al., 1977; Tawfik et al., 1999). Additional functions of ecdysteroids in the regulation of various aspects of female reproductive physiology have been reported in several other insect species (e.g. regulation of oocyte resorption, previtellogenic oocyte maturation and follicle cell development; Bellés et al., 1993; Buszczak et al., 1999; Parthasarathy et al., 2010; Soller et al., 1999). In the present study, we further analyzed the in vivo effects of trNPF on female locust reproductive physiology. Therefore, we performed trNPF injections and induced an RNA interference-mediated knockdown of the Schgr-NPF precursor transcript. We hereby focused on the effects of these treatments on the size of the growing terminal oocytes and on ecdysteroid levels in the hemolymph and ovaries of adult female S. gregaria. 2. Materials and methods 2.1. Rearing of desert locusts Gregarious desert locusts [S. gregaria (Forskål)], were reared under crowded conditions at constant temperature (32 ± 1 °C) and constant day/night cycle (14 h photoperiod). Each day, they were fed ad libitum with fresh cabbage leaves, while also oat flakes were supplied. The female locusts that were used in the experiments, were synchronized on the day of adult emergence. During each experiment, the different experimental groups of females were kept together in different compartments of the same cage which also contained a number of males (in order to ensure a normal reproductive process and to prevent phase shifts; Uvarov, 1966).

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The administered dose of 20 pmol trNPF applied in both experiments was chosen based on a series of preliminary in vivo dose–response experiments analyzing the effective trNPF dose needed in these injection experiments. 2.3. RNAi experiments For determining the effects of RNAi-mediated knockdown of the Schgr-NPF precursor transcript (Genbank: JQ236862), this mRNA was down-regulated according to a previously described method, that has been proven to result in a strong reduction of this transcript and subsequent significant physiological effects (Van Wielendaele et al., 2013a,b). Double stranded (ds)RNA directed against a part of the Schgr-NPF precursor transcript (npf dsRNA1, 370 bp) or control dsRNA (gfp dsRNA, 509 bp) was produced by means of the MEGAscriptÒ RNAi Kit (Ambion, Austin, TX, USA). The dsRNA was purified out of the reaction mixture with a phenol/chloroform extraction followed by an ethanol precipitation. The dsRNA pellet was redissolved in the elution buffer provided in the MEGAscriptÒ RNAi Kit. The quality and concentration of the produced dsRNA was determined by means of spectrophotometry (Nanodrop ND-1000). To check the integrity of the dsRNA, a small amount of the reaction product was analyzed by agarose gel electrophoresis. The produced dsRNA was directly injected into the females’ hemocoel between the first and second abdominal segments in the direction of the head, using a 710RN 100 ll Syringe (Hamilton, Bonaduz, Switzerland). In order to verify the knockdown effect of npf dsRNA injection, females were injected at day 5 of the adult stage and knockdown efficiency was determined in the brains, optical lobes and suboesophageal ganglion using qRT-PCR (with the genes for elongation factor 1 alpha and ribosomal protein 49 as reference genes, as indicated by reference gene analysis using geNorm; Vandesompele et al., 2002; Van Hiel et al., 2009). For each tissue, a significant reduction (>90% down regulation) in npf mRNA levels was observed at 7 days after dsRNA injection, on day 12 of the adult stage (Fig. 1). For determining the effect of dsRNA injection on oocyte size and ecdysteroid levels, females were injected at day 5 and 10 of the adult stage. At several time points during the experiment, hemolymph samples were taken for measurement of ecdysteroid titers using EIA. At day 15 of the adult stage, ovaries were dissected and weighed and oocyte size was determined. Next, ovary samples were prepared for EIA ecdysteroid measurements. In order to further confirm the effects of knockdown of the Schgr-NPF precursor transcript, an additional npf dsRNA (npf dsRNA2, 407 bp) was produced that did not overlap

2.2. Peptide injection experiments Female adults were injected daily from day 1 until day 12 of the adult stage using a 710RN 100 ll Syringe (Hamilton, Bonaduz, Switzerland). Injections were performed directly in the hemocoel between the first and second abdominal segments in the direction of the head. Each day, experimental animals were injected with 20 pmol trNPF (dissolved in 4 ll Milli-QÒ water), while control animals were injected with 4 ll Milli-QÒ water. Initially, this injection procedure was applied for verifying the effect of trNPF on oocyte maturation, by dissecting the ovaries and measuring oocyte size on day 12 (see Section 2.4). Later on, this experimental scheme was also applied for assessing effects of trNPF on ecdysteroid levels in hemolymph and ovaries. For this, hemolymph samples were taken at several time points during the experiments, for determination of ecdysteroid levels by means of enzyme immunoassay (EIA, see Section 2.5). On the last day of the experiment (day 12 of the adult stage), hemolymph samples were collected and ovaries were dissected. After determination of ovary weight, the ovary samples were prepared for determination of ecdysteroid levels using EIA.

Fig. 1. Effect of npf dsRNA injection on the Schgr-NPF precursor transcript levels in adult females. Adult females were injected with gfp dsRNA or npf dsRNA. Transcript levels were determined in different tissues at 7 days after dsRNA injection. Results were obtained by analyzing three independent groups of seven individuals per condition and are represented as means ± SD. Abbreviations used on the X-axis: Br: brain, Op: optic lobes, Sog: suboesophageal ganglion. ⁄Significant difference in the respective tissue between the two treatments (linear regression analysis, P < 0.01, n = 21).

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with the first npf dsRNA (npf dsRNA1). The data presented in this paper were obtained using npf dsRNA1, but were confirmed in additional experiments with npf dsRNA2 (data not shown). 2.4. Measuring oocyte size For determining effects on oocyte size, the length of 10 terminal oocytes (i.e. oocytes at the base of the ovarioles and hence the largest oocytes) was measured for every female involved in this experiment. The size of the individual oocytes was measured using a piece of millimeter-squared paper and the average oocyte size was calculated for each individual. Experimental data of the different experimental conditions were analyzed by means of an unpaired t-test (GraphPad Prism 5 program; GraphPad Software Inc., San Diego, CA, USA), after checking the validity of the assumptions on which this test is based. 2.5. Ecdysteroid sampling, extraction and quantification For determining ovary and hemolymph ecdysteroid levels, sampling, extraction and quantitative ecdysteroid measurements using enzyme immune assay (EIA), were performed as previously described by Van Wielendaele et al. (2012). 2.5.1. Collection and extraction of hemolymph samples At different time points during the experiments, 5 ll hemolymph was collected from every female using capillary tubes. Each hemolymph sample was diluted in 100 ll ice-cold methanol and stored at 20 °C until further processing. Samples were extracted three times with 100% methanol by cyclic centrifugation and collection of the supernatants. All supernatants originating from the same sample were combined and dried in a vacuum centrifuge, until completely dry. The resulting pellets were dissolved in a sample buffer for Enzyme Immuno Assay (EIA) measurement (0.1 M phosphate buffer, pH 7.4) and stored at 20 °C. 2.5.2. Collection, extraction and preparation of ovary samples Complete ovaries were dissected and carefully rinsed in S. gregaria saline (1 L: 8.766 g NaCl; 0.188 g CaCl2; 0.746 g KCl; 0.407 g MgCl2; 0.336 g NaHCO3; pH 7.2). Next, they were placed in 2 ml ice-cold pure methanol and kept at 20 °C until further processing. Ecdysteroid extraction was performed as described by Tawfik et al. (1999). Ovaries were homogenized by means of a bar sonicator, while still being submerged in pure methanol. The resulting homogenates were heated to 60 °C for 10 min, followed by centrifugation at 10000 g (10 min). The supernatants were collected and the pellets were re-extracted twice by adding 1 ml 70% methanol. The different supernatants originating from the same sample were combined and dried by evaporation in a vacuum centrifuge, until completely dry. In order to obtain optimal EIA measurements, apolar lipids were removed from the samples. This was done by dissolving the pellets in 1 ml 70% methanol and 1 ml 100% hexane, followed by mixing, centrifuging and discarding the upper hexane phase. The remaining methanol phase of each sample was divided in two equal halves. Both halves were desiccated with the vacuum centrifuge, until completely dry. The pellet of one of both halves was directly dissolved in the sample buffer for EIA measurement. The pellet of the other half (of each sample) was dissolved in 2 ml sodium acetate buffer (50 mM, pH 5.1), containing 1 mg type H-1 b-glucuronidase/arylsulphatase l from Helix pomatia (Sigma– Aldrich, St. Louis, MO, USA) and 1 mg type II acid phosphatase from potatoes (Sigma–Aldrich). A large portion of the ecdysteroids present in locust eggs is conjugated. This conjugation hampers binding of the ecdysteroids to the antibody used in the EIA measurement. The enzymes present in the buffer, will convert the conjugated ecdysteroids into ‘‘free’’ ecdysteroids, which can be recognized by

the antibody used in the EIA. The reaction mixtures were incubated at 37 °C for 24 h. Reactions were terminated by adding pure methanol. Next, all mixtures were dried with the vacuum centrifuge. The desiccated samples were then dissolved in the sample buffer for EIA measurement. 2.5.3. Ecdysteroid quantification Ecdysteroid levels were evaluated using enzyme immunoassay (EIA), according to the method of Porcheron et al. (1989), modified by using a peroxidase conjugate of 20E as tracer and a rabbit polyclonal antiserum (L2) against ecdysteroids (Pascual et al., 1995). The EIA technique relies on the competition between the sample ecdysteroids and the ecdysteroid tracer for binding to the antiecdysteroid antibodies present in the L2 antiserum. After incubation to allow the immunological reaction to take place and subsequent washing steps, a coloration reaction was started by the addition of UHP (urea–hydrogen peroxide adduct, Sigma–Aldrich) and TMB (tetramethyl benzidine, Sigma–Aldrich). Absorbance was measured every 5 min at 370 nm for 1 h. In order to quantify the ecdysteroid levels, a serial dilution (ranging from 10 8 M till 10 12 M) of ecdysone (E) or 20-hydroxy-ecdysone (20E) was placed on each 96-well plate. Results were determined by comparison with dose–response curves obtained using these diluted standards and were calculated as E equivalents or 20E equivalents. For the ovary samples, ecdysone was used as standard since it was shown to be the main ecdysteroid in adult ovaries of S. gregaria (Tawfik et al., 1999). Although ecdysone and 20-hydroxy-ecdysone are similarly abundant in female adult hemolymph samples (Tawfik et al., 1997), 20-hydroxyecdysone was chosen as standard because of its general physiological importance. In order to test the statistical significance of the observed differences, unpaired t-tests were performed (using the GraphPad Prism 5 software), after checking the validity of the assumptions on which this test is based. 3. Results 3.1. Effects on oocyte size To investigate the possible role of trNPF in female reproductive physiology, we first verified the effect of trNPF injections on oocyte growth (as previously described by Schoofs et al., 2001) by applying daily injections of trNPF to adult female desert locusts (Fig. 2). Furthermore, the effect of a systemic RNAi-mediated knockdown

Fig. 2. Effect of trNPF injection on oocyte size in adult females. Adult females were injected daily with 20 pmol trNPF (dissolved in 4 ll Milli-QÒ water) starting from day 1 until day 12 of the adult stage. Control animals were injected in the same way, but only with 4 ll Milli-QÒ water. The resulting average oocyte size (measured on day 12) is represented on the Y-axis. Results were obtained by analyzing 20 individuals per condition and are represented as means ± S.D. ⁄Significant difference compared to the control group (unpaired t-test; P < 0.05; n = 20).

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of the Schgr-NPF precursor transcript on oocyte size was analyzed in adult females by injecting dsRNA on day 5 and 10 and measuring oocyte size on day 15 of the adult stage. When we compare the average oocyte size of animals injected with npf dsRNA or with the gfp dsRNA control (Fig. 3), we can conclude that injections of npf dsRNA resulted in significantly smaller oocytes. The mean oocyte size for the npfRNAi condition was about twice as small as the mean value for the control condition. 3.2. Effect of trNPF injections on ecdysteroid concentrations In order to further analyze the effect of trNPF on female reproductive physiology, we determined the effect of daily trNPF injections on ecdysteroid levels in the hemolymph and ovaries of adult females. Adult females were injected each day with trNPF or MilliQÒ water, starting from the first day of the adult stage until day 12. At several time points during the experiment, hemolymph samples were taken and on day 12 ovaries were dissected for determination of ecdysteroid levels using EIA. The average hemolymph ecdysteroid concentrations for the different time points are represented in Fig. 4. On day 1, 3, 5, 7 and 11, ecdysteroid levels in both conditions were not statistically significant. However, on day 9 and 12, ecdysteroid concentrations were significantly higher in the animals injected with trNPF. The ovary ecdysteroid levels were also affected by the peptide injections. Both for free ecdysteroids (EIA measurement without enzymatic treatment) and total ecdysteroids (EIA measurement preceded by enzymatic treatment), ecdysteroid levels were significantly higher in the ovaries of animals treated with trNPF (Fig. 5).

Fig. 4. Effect of daily trNPF injection on hemolymph ecdysteroid levels in adult female S. gregaria. Adult females were injected daily with 20 pmol trNPF (dissolved in 4 ll Milli-QÒ water) from day 1 until day 12 of the adult stage. Control animals were injected in the same way, but only with 4 ll Milli-QÒ water. Hemolymph samples were taken at different time points during the experiment and the ecdysteroid content was measured with the EIA. The resulting average ecdysteroid titers for the different time points are represented on the Y-axis. Results were obtained by analyzing 25 individuals per condition and are represented as means ± SD. ⁄Significant differences compared to the respective control values (unpaired t-test; P < 0.05; n = 25).

3.3. Effect of npf RNAi on ecdysteroid concentrations Adult females were injected with npf dsRNA or gfp dsRNA on day 5 and 10 of the adult stage. At several time points during the experiment, hemolymph samples were taken. On day 15, ovaries were dissected. All samples were extracted and measured with EIA for determination of ecdysteroid contents. The average ecdysteroid contents for the hemolymph samples are presented in Fig. 6. Compared to the control group, ecdysteroid levels in the experimental condition are significantly lower at day 11 and 15, but are not statistically different from the control levels on the other days of the experiment. While the control levels display peak concentrations on day 11 and 15, the experimental levels slightly rise to their highest measured value on day 15. The ovary ecdyster-

Fig. 5. Effect of daily trNPF injection on ovary ecdysteroid levels in adult female S. gregaria. Adult females were injected daily with 20 pmol trNPF (dissolved in 4 ll Milli-QÒ water) from day 1 until day 12 of the adult stage. Control animals were injected in the same way, but only with 4 ll Milli-QÒ water. On day 12, ovaries were dissected and weighed. Ecdysteroids were extracted from the ovary samples and measured with EIA before (free ecdysteroids) and after enzyme treatment (‘‘total’’ ecdysteroids). The resulting average ecdysteroid content per mg tissue is represented on the Y-axis. Results were obtained by analyzing 25 individuals per condition and are represented as means ± SD. ⁄Significant differences compared to the respective control values (unpaired t-test; P < 0.05; n = 25).

oid levels are presented in Fig. 7. Both for free and total ecdysteroids, levels were significantly lower in the ovaries of animals treated with npf dsRNA (Fig. 7).

4. Discussion

Fig. 3. Effect of RNAi-mediated knockdown of the Schgr-NPF precursor transcript on oocyte size in adult females. Adult females were injected with 4 lg npf dsRNA on day 5 and 10 of the adult stage. Control animals were injected in the same way with 4 lg gfp dsRNA. On day 15, all animals were dissected and oocyte size was measured. The resulting average oocyte size is represented on the Y-axis. Results were obtained by analyzing 20 individuals per condition and are represented as means ± S.D. ⁄Significant difference compared to the control group (unpaired t-test; P < 0.05; n = 20).

In this paper, the effects of trNPF injections and RNAi-mediated knockdown of the Schgr-NPF precursor transcript are analyzed on female reproductive physiology during the first gonotrophic cycle of female S. gregaria. As mentioned above, the predicted ‘‘long’’ NPF of locusts has not been demonstrated, while its 9-AA C-terminal part, trNPF, has been identified both in S. gregaria and L. migratoria (Clynen et al., 2009; Schoofs et al., 2001). The experimental data presented in the current study confirm that locust trNPF is indeed biologically active, as previously demonstrated (Schoofs et al., 2001; Van Wielendaele et al., 2013a,b). The data presented in Fig. 2

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Fig. 6. Effect of RNAi-mediated knockdown of the Schgr-NPF precursor transcript on hemolymph ecdysteroid levels in adult females. Adult females were injected with 4 lg npf dsRNA or 4 lg gfp dsRNA on day 5 and 10 of the adult stage. Hemolymph samples were taken on several time points until day 15 and were measured with EIA. The resulting average ecdysteroid levels for each analyzed time point are represented on the Y-axis. Results were obtained by analyzing 25 individuals per condition and are represented as means ± S.D. ⁄Significant differences compared to the respective control values (unpaired t-test, P < 0.01; n = 25).

Fig. 7. Effect of RNAi-mediated knockdown of the Schgr-NPF precursor transcript on ovary ecdysteroid levels in adult females. Adult females were injected with 4 lg npf dsRNA or 4 lg gfp dsRNA on day 5 and 10 of the adult stage. On day 15 of the adult stage, ovaries were dissected. Ecdysteroids were extracted and measured with the enzyme immunoassay before (free ecdysteroids) and after enzyme treatment (‘‘total’’ ecdysteroids). The resulting ecdysteroid content per mg tissue is represented on the Y-axis. Results were obtained by analyzing 25 individuals per condition and are represented as means ± SD. ⁄Significant differences compared to the respective control values (unpaired t-test, P < 0.01; n = 25).

confirm the previously reported effect of trNPF injections on oocyte growth in adult female S. gregaria (Schoofs et al., 2001). Moreover, we demonstrate that treatment with npf dsRNA negatively affects oocyte growth (Fig. 3), providing an independent experimental confirmation for this effect of trNPF on oocyte growth. In addition, we also show that trNPF injections induce increased levels of ecdysteroids in the ovaries, as well as in circulation, while the RNAi knockdown condition generated opposite effects on ecdysteroid levels (Figs. 4–7). In locusts, the only other peptide that was hitherto known to increase in vivo ecdysteroidogenesis in adult females is the ‘Ovary Maturating Parsin’ (Girardie et al., 1991, 1998). This paper is the first report on NPF influencing ecdysteroid levels in insects. The dose of 20 pmol trNPF applied in the injection experiments described in this paper, was chosen based on preliminary experiments. In their 2001 paper, Schoofs and coworkers also analyzed the effect of daily application of various doses of trNPF on oocyte size, after which only the effect of the highest daily dose (1 lg or 890 pmol trNPF) was investigated more profoundly. Comparison with the dose–response data of Schoofs and coworkers (2001),

shows that the dose of 20 pmol, applied in our experiments, is within the range of effective doses reported in their study, i.e. between 8.9 pmol (0.01 lg) and 890 pmol (1 lg). Furthermore, application of this dose resulted in effects on oocyte size (Fig. 2) that are in line with these previously reported dose–response data (Schoofs et al., 2001). Considering that female locusts have a few hundred microliters of hemolymph, injection of 20 pmol in the hemocoel would generate an initial concentration of approximately 50 nM, immediately post-injection. Comparison with studies on NPF and NPF receptors in other insect species suggests that this concentration is within the physiological activity range. Prior to the actual RNAi-experiments, the knockdown effect of npf dsRNA injection was verified in adult female S. gregaria in the same period as the actual RNAi-experiments were scheduled (from day 5 until day 15 of the adult stage, the period of female sexual maturation). A previous transcript profiling study indicated that the NPF-precursor encoding mRNA predominantly occurs in the brain, optic lobes, midgut and suboesophageal ganglion of S. gregaria (Van Wielendaele et al., 2013a). Therefore, transcript levels were determined in these tissues for assessing the transcript knockdown efficiency upon injection of npf dsRNA. In all analyzed tissues, a very robust down regulation (>90% reduction of the npf mRNA levels) was observed in the npf dsRNA treated locusts, when compared to control animals treated with gfp dsRNA (Fig. 1). The knockdown effect was found to be strong enough to generate measurable physiological effects in the experiments described in this study (Figs. 3, 6 and 7). The occurrence of a very sensitive and robust RNA interference response in locusts has recently been reported in several other studies (e.g.Badisco et al., 2011; Marchal et al., 2012; Van Hoef et al., 2011; Ott et al., 2012; Wynant et al., 2012). As mentioned above, we found that our experimental treatments influenced ecdysteroid levels in the ovaries and hemolymph of female adult locusts. In adult female insects, the ovaries are the main production site for ecdysteroids (Brown et al., 2009). A large fraction of the ecdysteroids produced by the female gonads is stored in the oocytes as conjugates, that will be utilized as a source of ecdysteroids during embryonic development (Bownes et al., 1988; Kozlova and Thummel, 2003; Yamada et al., 2005). In addition, ecdysteroids can play important paracrine and autocrine roles in insect ovaries during oogenesis (e.g.Bellés et al., 1993; Buszczak et al., 1999; Lanot et al., 1987; Parthasarathy et al., 2010; Soller et al., 1999), while some fraction is also leaked into the hemolymph. In adult locusts, the exact physiological role of these hemolymph ecdysteroids remains to be determined. In any case, ecdysteroids are synthesized by the ovaries (follicle cells) and incorporated in the growing oocytes during the vitellogenic period, explaining temporal correlations of their synthesis (and appearance) with oocyte growth during the gonotrophic cycle of the locust (Dinan and Rees, 1981; Tawfik et al., 1997, 1999). Solely based on the observations reported in this paper, we cannot determine the exact mode of action of trNPF (e.g. direct or indirect action) in causing the described effects. Nevertheless, several hypotheses regarding the possible mode of action of (tr)NPF can be proposed. First of all, the existence of direct effects of trNPF on ovarian ecdysteroidogenesis, vitellogenesis or oocyte growth (through direct action on the fat body or ovaries) cannot be readily excluded. However, there are currently no reports on the occurrence of NPF receptors in insect ovaries or fat body. Since sequence information concerning the locust trNPF receptor is still lacking, we are currently unable to determine whether this receptor is present in the ovaries or fat body of female locusts. It is also possible that trNPF indirectly exerted its in vivo effects on female reproductive physiology. As in other invertebrate species (and as NPY in vertebrates), NPF promotes food intake in adult S. gregaria (Van Wielendaele et al., 2013a). This feeding-regulating

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activity of trNPF might be a possible explanation for our observations, although also other biological activities of trNPF might have contributed to, or have resulted in, the observed effects. By positively influencing food intake, trNPF injections might have increased the acquisition of energy and nutrients, in support of several anabolic processes, speeding up female reproductive maturation and vitellogenesis. This might then have resulted in the larger oocyte size of females injected with trNPF. Also other anabolic processes related to reproduction, such as ecdysteroidogenesis, might have been positively influenced in this way. In this respect, it is important to note that the ecdysteroid levels and contents of the experimental animals in our study can indeed be interpreted as precocious (in trNPF-injected females) or delayed (in the RNAi-mediated npf knockdown condition) manifestations of the naturally occurring ecdysteroid dynamics in hemolymph and ovaries (Figs. 4–7), when comparing our current data with literature reports on hemolymph and ovarian ecdysteroid levels in relation to oocyte growth during female sexual maturation (Tawfik et al., 1997, 1999; Van Wielendaele et al., 2012). Furthermore, several reports confirm that diet and food quality indeed can influence sexual maturation in female locusts (Hatle et al., 1979; Maeno and Tanaka, 2011; Van Huis et al., 2008). Also in other insect species, similar observations have been made (e.g. for cockroaches: Cooper and Schal, 1992; for butterflies: Shobana et al., 2010; and for bugs: Bonte and De Clercq, 2008). Unfortunately, it is currently not clear whether the stimulatory influence of trNPF on food intake (as demonstrated by Van Wielendaele et al., 2013a) would indeed be sufficient for generating the effects described in this study. Prior to the actual usage of nutrients in a specific anabolic process, food not only needs to be ingested, it also needs to be digested and the resulting nutrients need to be absorbed, transported, metabolized and differentially allocated to the organs involved in this specific process. It is possible that trNPF (directly or indirectly) influenced some of these intermediate processing steps, when causing the effects described in this manuscript. A previous study on locusts describes that injections of other peptides with effects on food intake (as demonstrated in locusts or other insect species; Audsley and Weaver, 2009; Spit et al., 2012) do not affect locust oocyte size (Cerstiaens et al., 1999). It might therefore be suggested that modulation of food intake alone is indeed not sufficient to generate effects on oocyte size in locusts, and this may also be the case for effects on ecdysteroidogenesis. Insect NPF has been suggested to be implicated in the regulation of a wide diversity of physiological processes and it might exert biological activities that still remain to be elucidated (Nässel and Wegener, 2011). Therefore, our current hypothesis is that trNPF probably elicits (directly and indirectly) a sequence of effects, which are functionally interconnected, cooperatively driving the animal’s physiology in a given direction (i.e. reproduction). In this view, both direct and indirect effects of the peptide might possibly contribute to the observed endpoints. Because of the complex functional interactions between several of the physiological processes underlying nutrient acquisition, metabolism and usage, it seems difficult to readily elucidate the actual contributions of the distinct biological activities that might be involved. As mentioned in the introduction, several other neuropeptides were previously described to play a role in female reproductive physiology in locusts. While some of these peptides were reported to influence oocyte growth (e.g. insulin-related peptide and neuroparsins; Badisco et al., 2011; Girardie et al., 1987), others also appear to affect the ecdysteroid levels in adult female locusts (ovary maturating parsin and CRF-related diuretic hormone: Girardie et al., 1991, 1998; Van Wielendaele et al., 2012; trNPF: this study). Further investigations will be needed to clarify the exact regulatory hierarchy causing the observed in vivo effects.

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Acknowledgements The authors thank Roger Jonckers for taking care of the locust colony and Joost Van Duppen for technical support. The antiserum and tracer for the EIA were a kind gift from Dr. De Reggi (Marseille, France) and Dr. Delbecque (Bordeaux, France). The authors also gratefully acknowledge the Interuniversity Attraction Poles program (Belgian Science Policy, Grant IAP P6/14), the K.U. Leuven Research Foundation (GOA/11/02), the Research Foundation of Flanders (FWO) and the Agency for Innovation by Science and Technology (IWT) for financial support.

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