Comparison of proctolin and FMRFamide actions on the motility of ...

Invertebrate Reproduction and Development, 54:1 (2010) 1–6 Balaban, Philadelphia/Rehovot 0168-8170/10/$05.00 © 2010 Balaban

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Comparison of proctolin and FMRFamide actions on the motility of male and female beetle reproductive tracts PAWE£ MARCINIAK* and GRZEGORZ ROSIÑSKI Department of Animal Physiology and Development, Adam Mickiewicz University, Umultowska 89 Str., 61-614 Poznan, Poland email: [email protected] Received 13 November 2009; Accepted 22 January 2010

Abstract Two myotropic peptides, proctolin and FMRFamide, were tested for their action on the frequency of contractions of the the male ejaculatory duct and female oviduct of two beetle species: Tenebrio molitor and Zophobas atratus. The tested peptides caused changes in endogenous contraction frequencies of the reproductive tracts in both species. Proctolin exerted a strong stimulatory effect on spontaneous muscular contractions of the ejaculatory ducts and oviducts of T. molitor and Z. atratus. FMRFamide action, however, differed between species and organ tested. At high doses (10 !7–10 !5 M) it stimulated oviduct contractions in Z. atratus but inhibited the T. molitor oviduct contractility. In T. molitor, the peptide had a stimulatory effect on oviduct contractions at low doses (10 !11–10 !9 M). In contrast to proctolin, FMRFamide is a weak inhibitor of the ejaculatory ducts of both beetles at high concentrations (10 !6–10 !5 M). Differentiated responses of the ejaculatory ducts and oviducts of T. molitor and Z. atratus suggest that these peptides are functional and specific regulators of both reproductive tracts. Key words: Proctolin, FMRFamide, ejaculatory duct, oviduct, Tenebrio molitor, Zophobas atratus

Introduction The reproductive system of insects, as in other animals, is under hormonal and neural control. Major hormones that regulate various reproductive processes are mainly juvenile hormone (JH), ecdysteroids and neuropeptides (Gäde & Hoffman, 2005). They stimulate accessory gland development in males, oocyte and egg growth and maturation in females and other reproductive events in insects (Simonet et al., 2004). A very important part of reproductive physiology is contractions and movements of reproductive tracts, such as the oviduct in females and ejaculatory duct in males, which play significant roles during fertilization and oviposition

*

Corresponding author.

(Lange & da Silva, 2007). Neurohormonal regulation of oviduct contractions is well studied only in the locust Locusta migratoria (Lange & da Silva, 2007). To date, no studies have been undertaken on beetles. Recently, Rankin et al. (2009) studied the ejaculatory duct of earwigs, examining two peptides, proctolin and cockroach allatostatin (FGLa AST). The neuropeptide proctolin was firstly identified in the cockroach Periplaneta americana (Brown & Starrat, 1975). Thus far, this pentapeptide has been found to be a co-transmitter at various insect neuromuscular junctions and to be widely distributed and active on insect and arthropod muscles (Orchard et al., 1989; Konopiñska &

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Rosiñski, 1999). In reproductive organs, proctolin increases the frequency and amplitude of spontaneous muscle contractions and also increases the amplitude of neurally-evoked contractions of oviducts in L. migratoria, P. americana and Rhodinus prolixus (Lange et al., 1991; Lange, 2002). Moreover, proctolin has been associated with the spermatheca of Gryllus bimaculatus, L. migratoria and R. prolixus (Lange & da Silva, 2007). Furthermore, it stimulates the rate of reproductive tract contractions in earwigs (Rankin et al., 2009) and accessory gland movements in crickets (Kimura et al., 1989). These studies suggest that proc-tolin might be an important factor in regulating the movement of insect reproductive organs. FMRF-amide-related peptides (FaRPs) are one of the most diverse group of neuropeptides in insects (Hewes & Taghert, 2001; Nässel, 2002; De Loof, 2008). Since 1977, when the neuropeptide FMRFamide was first discovered in the neural ganglia of the calm Macrocallista nimbosa (Price & Greenberg, 1977), the physiological role of this peptide and other related peptides has been investigated. Despite the fact that FMRFamide does not occur in insects, FaRPs have been shown to affect muscles (Cuthbert & Evans, 1989), neurons and gland activities (Neupert & Gundel, 2007). Peeff et al. (1993) showed that FMRFamide stimulates contractions of the oviduct of L. migratoria in a dose-dependent manner. Moreover, another bioanalog of this family, FLRFamide, also induces contractions of isolated oviducts (Peeff et al., 1993). The same study revealed that N-extended analogs of FLRFamide, Schisto-FLRFamide (PDVDHVFLRFa) and ADVGHVFLRF-amide, has been found to decrease spontaneous and neurogenic contractions in the locust oviduct (Lange et al., 1991; Peeff et al., 1993, 1994). In addition, another extended bioanalog of FLRFamide, leucomyosup-pressin (LemMS; QDVDHVFLRFamide), decreased oviduct contraction frequency in L. migratoria (Peeff et al., 1993) and Leucophaea maderae (Cook & Wagner, 1991). These data suggest that FaRPs are widely involved in control of the motility of reproductive organs. The aim of this study was to evaluate the role of the neuropeptides proctolin and FMRFamide in the regulation of spontaneous contraction activity of the oviduct and ejaculatory duct of two beetle species: Tenebrio molitor and Zophobas atratus. Thus far, only one peptide Neb-colloostatin, which is known as an oostatic factor, was found to change the beetle oviduct contraction frequency (Kuczer et al., 2005). No comparative studies have been undertaken on the motility and hormonal regulation of the reproductive tracts of male beetles.

Material and Methods Insects Tenebrio molitor L. adults were obtained from a culture maintained as described previously (Rosinski et al. 1979) and Zophobas atratus Fab. was reared according to the Quennedy procedure (Quennedy et al., 1995). Peptides FMRFamide was purchased from Bachem (Switzerland) and proctolin was obtained from Sigma (Germany). Peptides were dissolved in saline (274 mM NaCl, 19 mM KCl, 9 mM CaCl, 5 mM glucose, 5 mM HEPES, pH 7.0) to yield a stock solution of 1 mM and were stored at !30ºC. Working dilutions were made from the stock solution in saline. Oviduct and ejaculatory duct preparation Adult males and females of T. molitor and Z. atratus were decapitated and their wings and legs removed. The central portion of the integument on the dorsal abdomen was cut out and the remainder of the body was placed in a small Sylgard-filled Petri dish, pinned through the ventral surface of the abdomen and flooded with saline. The alimentary tract, fat body fragments and Malpighian tubules were removed from the surface of the reproductive organs. The ovaries and the oviduct attached to the ventral sclerite were removed from the abdomen, the accessory glands and the spermatheca were cut off, and the oviduct with ovaries was used for the bioassay. The ejaculatory duct was isolated in a similar way to the oviduct, and the final preparation for bioassay consisted of the ejaculatory duct with the accessory glands. Each dissected preparation was transferred to an incubation chamber with a 50 µl perfusion well prepared in a 5 cm Petri dish filled with Sylgard. Reproductive tract videomicroscopy bioassay The system for registering contractile activity of both organs, data acquisition and analysis, was the same as described previously (Marciniak et al., 2008). In brief, the incubation chamber with the preparation in the perfusion well was installed horizontally on the stage of an Olympus SZX12 stereomicroscope equipped with a Pixeling 662 camera. An open perfusion system was used, with an injection port 70 mm above the superfusion well. The oviduct or ejaculatory duct was subjected to a constant perfusion with fresh saline at a rate of about 140 µl/min. All tested peptide concentrations were applied at the injection port with a Hamilton syringe.


Many pulse applications of samples (10 µl) could be sequentially assayed in a single preparation. The system was designed to enable samples to be added without causing a change in pressure. After the initial 15–20 min acclimatization, the activity of the isolated preparation was recorded for 2 min. Next the peptide was applied and the activity was recorded for further 2 min. This procedure was repeated with 5 min intervals for each concentration tested. We developed the edge tracing software (AnTracker; www.mikroskop.com.pl) to create a trace of the movement of the edge (proximal) of the common oviduct or ejaculatory duct. Myograms were constructed from the videoclips as follows: a point was overlaid on the binarized and thresholded video image to follow the black/white boundary at the proximal edge of the common oviduct or ejaculatory duct. A plot of the change in position of the boundary from frame to frame was plotted. Each peak on the obtained myograms corresponds to the single contraction manifested as a change in the position of the preparation boundary.

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Results Effect of proctolin and FMRFamide on ejaculatory duct contractions Ejaculatory duct contraction frequencies in both beetles remained slightly irregular during superfusion with physiological saline and showed on average 7 ± 4 and 7 ± 2 contractions per minute for Z. atratus and T. molitor, respectively. The application of physiological saline induced no significant changes in contractile activity wheras proctolin and FMRFamide caused differentiated and reversible effects on the ejaculatory duct contractions (Fig. 1). Proctolin induced a strong stimulatory effect on the ejaculatory ducts of both beetles. The contraction activity was progressively stimulated while the peptide concentrations were increased from 10!11 to 10!5 M. Both species demonstrated similar sensitivity to this peptide. The threshold concentration for an observable effect was 10 !11 M in both beetles (Fig. 2).

Fig. 1. Typical responses on the spontaneous contraction activity of T. molitor and Z. atratus ejaculatory ducts to 1×10 !7 M proctolin (A) and 0.8×10 !7 FMRFamide (B). Peptide application is indicated by an arrow.

Fig. 2. Dose-response curves for the effect of proctolin (A) and FMRFamide (B) on the contraction frequency of the ejaculatory duct of Z. atratus (") and T. molitor (!). Mean ± SEM is given from 6–8 determinations. Statistically significant differences (p< 0.05) from controls (Ringer saline) are indicated by asterisks (Wilcoxon test).

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FMRFamide induced species specific and dosedependant bimodal effects on the beetle ejaculatory ducts. In T. molitor, the higher (10!5 M) concentration of FMRFamide showed a weak inhibition of the ejaculatory duct contractions, whereas in lower concentrations (10!7 M) it slightly stimulated contractions or caused no effect. The inhibition was also observed in a lower concentration range (10!11–10!8 M). Comparison of the dose-response curves (Fig. 2) indicates slightly different species specificity of the peptide action. The ejaculatory duct of Z. atratus was more sensitive to this peptide. In the concentration range 10!9–10!5 M FMRFamide caused inhibition of the ejaculatory duct contractions. Effect of peptides on oviduct contractions The contractions of oviducts remained regular during superfusion with physiological saline with ave-rage contraction rates of 9±7 and 7±4 per minute for Z. atratus and T. molitor respectively. Both examined peptides caused fast, dose-dependent and reversible effects on the oviduct contractions (Fig. 3). The appli-

cation of physiological saline caused no significant changes in contractile activity of oviducts in both species. Proctolin produced a strong stimulatory effect on the oviducts of both beetle species. The contraction activity was progressively stimulated as the peptide concentration increased (Fig. 4). The oviduct of Z. atratus was less sensitive to proctolin than that of T. molitor. The threshold peptide concentration for an observable effect on the oviduct of Z. atratus was 10!9 M, whereas for T. molitor it was about 10!11 M. However, at a higher concentration range (10!7–10!5 M), proctolin exerted a stronger stimulatory effect on the oviduct of Z. atratus in comparison to T. molitor (Fig. 4). FMRFamide caused a bimodal effect on the oviduct of both beetles, however, with a different range of peptide concentrations. The oviduct of T. molitor was more sensitive to this peptide. In low concentrations (10!11–10!8 M), FMRFamide increased the contraction frequency by 30–70% whereas at higher concentrations (10 !7–10!6 M) it caused no effect, or slightly decreased the contraction frequency by 20–30% (Fig. 4). In

Fig. 3. Typical responses on the spontaneous contraction activity of the oviduct of T. molitor and Z. atratus to 1×10 !7 M proctolin (A) and 0.8×10 !7 M FMRFamide (B). Peptide application is indicated by an arrow.

Fig. 4. Dose-response curves for the effect of proctolin (A) and FMRFamide (B) on the contraction frequency of the oviduct of Z. atratus (") and T. molitor (!). Mean ± SEM are given from 6–8 determinations. Statistically significant differences (p< 0.05) from controls (Ringer saline) are indicated by asterisks (Wilcoxon test).


contrast, in Z. atratus FMRFamide was inactive at lower concentration ranges (10!9–10!7 M) but at higher concentrations (10!5 M), it strongly increased the contraction frequency of the oviduct by 80% (Fig. 4).

Discussion This comparative study has examined the myotropic action of two neuropeptides, proctolin and FMRFamide, on the contraction frequency of the male ejaculatory duct and female oviduct of two beetle species, T. molitor and Z. atratus. To date, few such studies have been undertaken on beetles (Kuczer et al., 2005). Hormonal regulation of the spontaneous contractions of the oviduct of L. migratoria (Lange & da Silva, 2007) and L. maderae (Cook & Wagner, 1991) was in a large range. Proctolin, the widely known insect myotropic pentapeptide (Konopiñska & Rosiñski, 1999), stimulated contractions of the oviducts of both beetle species in a concentration range of 10!11–10 !6 M. Starrat et al. (1997) and Lange (2002) showed that it stimulated contraction frequencies of the locust oviduct when the peptide concentration progressively increased from 10–11 M to 10!5 M. Our results are similar to data obtained on locusts. Proctolin stimulated contractions of the oviduct at 10 !11–10 !6 M in both beetles. It suggests that proctolin is a very important myotropic regulator in insect reproduction physiology, and in contrast to FMRFamide, its action is mainly myostimulatory. Proctolin exerts a myostimulatory effect on the oviduct in the same manner as crustacean cardioactive peptide (CCAP). CCAP at a concentration of 10!10–10 !7 M caused an increase in the contraction frequency of the oviduct of the locust (Donini et al., 2001). In studies conducted by Peef et al. (1993), FMRFamide was found to stimulate the contraction frequency of isolated locust oviducts in a dosedependent manner when the peptide concentration was increased from 10 !6 to 10!4 M. Our results are similar. FMRFamide also increased the contraction frequency but only in T. molitor; however, the threshold for any observable effect for T. molitor was lower (10!7 M) than in L. migratoria (10!6 M). Zophobas atratus showed the opposite effect. FMRFamide in high concentrations did not stimulate the oviduct, or only slightly increased the frequency of contraction, whereas at low concentrations it exerted a stimulatory effect by 30%. None of the thus far tested FaRPs exerted any effect in such low concentrations on locust oviducts (Peef et al., 1993). This suggest that beetles are more sensitive to FMRFamide than locusts. Another FaRP bioanalogue, leucomyosuppressin, decreased the amplitude and frequency of oviduct contractions at low concentrations (10!10–10!7 M) in the

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cockroach L. maderae (Cook & Wagner, 1991) but only at a concentration 10!7 M in the locust (Peef et al., 1993). However, core sequence-FLRFamid responsible for inhibitory activity of this peptide differs from FMRFamide due to the substitution of Leu/Met. This might be the possible reason of its higher activity in low concentrations. In beetles, leucomyosuppressin was tested also in a heart bioassay and exerted an inhibition with a range of 10!12–10 !7 M for Z. atratus and 10!8– 10!4 M for T. molitor (Skonieczna & Rosiñski, 2004). This might be the evidence that beetles are more sensitive to FaRPs than other insect species. However, the heart of beetles may be more sensitive to this peptide than the oviduct. The ejaculatory duct is a part of the male reproductive system which transfers sperm to the female. Substances which alter its contractile movements may influence reproduction. To date, only one study using the earwig, E. annulipes, as a model has been under-taken using neuropeptides as contractile modulators of the ejaculatory duct (Rankin et al., 2009). In this study, proctolin was shown to increase movements of the ejaculatory ducts at concentrations of 10!9 and 10 !7 M, but caused no effect at 10!5 M (Rankin et al., 2009). In our bioassay, proctolin and FMRFamide increased the ejaculatory duct contraction frequency in both species of beetle. It suggests that proctolin might be a very important peptide regulator in contraction modulations of insect male reproductive tracts. Other studies con-ducted by Kimura et al. (1989) associate proctolin with male reproductive tract of the cricket G. bimaculatus. They showed that 10!9 M of this peptide stimulated contractions of the male accessory glands and furthermore was present in these glands (Kimura et al., 1989). A low concentration range in which proctolin increases the ejaculatory duct contraction frequency shows that the male reproductive system of insects is very sensitive to this peptide. Another peptide tested on the ejaculatory duct of the earwig was cockroach allatostatin, which was first found in Diploptera punctata (Woodhead et al., 1989). This peptide caused no effect in low concentrations (10!9– 10 !7 M), whereas at 10!5 M it decreased duct motility (Rankin et al., 2009). Comparing together the effects of all three peptides on insect ejaculatory ducts indicated that they have different action modes. This suggests that neurohormonal regulation of the contraction frequency of the male reproductive tract is complex and needs further investigation. In conclusion, two tested peptides, proctolin and FMRFamide, have been shown to influence contractile activity of the oviducts and ejaculatory ducts of two beetle species. FMRFamide exerted species-specific and organ-specific bimodal effects on the oviducts of the

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tested species, whereas proctolin progressively stimulated the contractions of both reproductive tracts. The data suggest that hormonal regulation mechanisms of male and female reproductive tract contractions are complex and additional studies must be conducted.

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