The association of crustacean cardioactive peptide with the spermatheca of the African migratory locust, Locusta migratoria

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Abstract

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons in the nerves that project to the spermatheca of 3–4 week old adult female locusts. In addition, lightly stained CCAP-like immunoreactive processes were localized over the spermathecae. The amount of CCAP in the spermathecal tissue was quantified using an enzyme-linked immunosorbent assay (ELISA) performed on extracts of the whole spermatheca, and on its constituent parts, namely the sperm sac, coiled duct and straight duct. The spermatheca contains 920±273 fmol (mean±SE) of CCAP equivalents, with the majority localized in the coiled duct. There are age-related differences in the amount of CCAP present in the spermathecae with less content in spermathecae from 1 to 5 day old and greater content in spermathecae from 3 to 4 week old adults. There was also no difference in CCAP content of spermathecae in mated and virgin 3 to 4 week old adults. Reversed phase-high performance liquid chromatography (RP-HPLC) followed by ELISA further confirmed the presence of CCAP-like material in extracts of locust spermathecae. Physiological assays demonstrated that CCAP increased the basal tonus and frequency of spontaneous contractions of the spermatheca, with thresholds between 10−10 and 10−9 M and maxima at 10−7 M CCAP. CCAP also increases the amplitude of neurally evoked contractions with a threshold less than 10−11 M and a maximum of 10−7 M CCAP. The present study suggests that CCAP acts as a neuromodulator/neurotransmitter at the spermathecal visceral tissue of female Locusta migratoria.

Introduction

Insect reproduction can be influenced by various factors affecting the physiological functioning of the visceral organs of the reproductive system. The female reproductive system of the African migratory locust, Locusta migratoria, can be used as a model system to examine physiological events associated with the ovaries, oviducts and the spermatheca. The walls of these reproductive tissues are covered by a muscular sheath consisting of circular and longitudinal muscle fibres arranged in layers (Lange, 1992; Lay et al., 1999). Upon mating, a male deposits sperm in the spermatheca of the female, which serves as a repository for spermatozoa. The spermatheca consists of an anteriorly localized sperm sac, followed by a coiled duct that wraps around the sperm sac and connects to the anterior and posterior straight duct (Clark and Lange, 2000). Within the ovaries, the eggs mature and during egg-laying they pass down the lateral and common oviducts on the way to the genital chamber where fertilization occurs prior to oviposition, via release of spermatozoa from the spermatheca (Lange, 1993). Previous studies in the desert locust, Schistocerca vaga (Okelo, 1979) and L. migratoria (Clark and Lange, 2000) have shown that the spermatheca is innervated bilaterally by motor neurons with axons in the ventral ovipositor nerve (VON). This nerve branches from the sternal nerve of the VIIIth abdominal ganglion. Dorsal unpaired median (DUM) neurons also have axons that project to the spermatheca in the VIIth and VIIIth abdominal ganglia (Clark and Lange, 2000).

There has been evidence indicating that the passage of an egg can influence the spermatheca to contract via a neural loop in L. migratoria (Clark and Lange, 2001). This evidence follows on the postulation that the spermatozoa stored in the sperm sac are forced to the most posterior portion of the straight duct through contractions of the anterior portion of the spermatheca. Once released from the sperm sac, the spermatozoa are held in the straight duct until the passage of an egg through the genital chamber leads to the contractions of the posterior region of the spermatheca and subsequent deposition of spermatozoa onto the micropyle of the egg (Okelo, 1979; Clark and Lange, 2000).

The reproductive tissues of L. migratoria continue to contract in slow and rhythmic peristaltic waves when isolated from the central nervous system (Davey, 1964; Lange et al., 1984). These contractions, however, can be influenced by both nervous and/or hormonal input. It has been shown in other insect species such as in the leaf beetle, Chelymorpha alternans (Rodriguez, 1994) and in the Australian field cricket, Teleogryllus commodus (Sugawara, 1989), that contractions of the spermathecal muscle tissue are essential for spermatozoa to move along the spermatheca. A variety of neurochemicals modulate the contractions of the lateral and common oviducts. These neurochemicals include the neuropeptides proctolin (Lange and Orchard, 1986) and FMRF-amide related peptides (Lange et al., 1991), the amines serotonin (Orchard and Lange, 1985), octopamine (Orchard and Lange, 1985) and tyramine (Donini and Lange, 2004) and more recently the cardioacceleratory peptide, crustacean cardioactive peptide (CCAP) (Donini et al., 2001). Similarly, contractions of the spermatheca of L. migratoria have also been found to be modulated by the neuropeptides proctolin (Lange, 1993) and FMRFamide-related peptides (Clark and Lange, 2002a) and the amines octopamine (Clark and Lange, 2003) and serotonin (Clark and Lange, 2002b).

The neurochemical CCAP is a cyclic nonapeptide (Pro–Phe–Cys–Asn–Ala–Phe–Thr–Gly–Cys–NH2) that was originally isolated and sequenced from the neurohaemal pericardial organs of the shorecrab, Carcinus maenas (Stangier et al., 1987). Since then, CCAP has been isolated from a variety of insects including, L. migratoria (Stangier et al., 1989), the desert locust Schistocerca gregaria (Veelaert et al., 1997), the tobacco hornworm Manduca sexta (Cheung et al., 1992) and the cockroach Periplaneta americana (Sakai et al., 2004). The CCAP gene has also been recently identified and sequenced in M. sexta (Loi et al., 2001), and the receptor cloned in Drosophila melanogaster (Park et al., 2002; Cazzamali et al., 2003). These findings suggest that CCAP has been highly conserved during evolution.

Previous studies by Dircksen and Homberg (1995) have suggested that CCAP may act as a neurohormone in L. migratoria. In addition, the peptide has also been found to act as a neurotransmitter since CCAP-like immunoreactivity has been found within the central nervous systems of crustaceans (Dircksen and Keller, 1988) and insects. Studies have shown cardioacceleratory actions on M. sexta (Lehman et al., 1993), C. maenas (Stangier et al., 1987), and D. melanogaster (Nichols et al., 1999). In addition, CCAP has been shown to induce cardiac reversal in M. sexta and D. melanogaster (Dulcis et al., 2001, Dulcis et al., 2005; Dulcis and Levine, 2004). Other actions include involvement with the onset of ecdysis (Gammie and Truman, 1997; Philippen et al., 2000; Park et al., 2003), release of adipokinetic hormone (Veelaert et al., 1997), and stimulation of hindgut contractions (Stangier et al., 1989; Donini et al., 2002; Lange and Patel, 2005). Recently, the effects of CCAP on reproductive tissues of L. migratoria have been investigated. Donini et al. (2001) found that CCAP increases the basal tonus, frequency and amplitude of phasic and neurally evoked oviduct contractions in a dose-dependent manner. However, no CCAP-like immunoreactivity has been identified on the oviduct or in the nerves innervating the oviducts of L. migratoria, suggesting its role might be as a neurohormone (Dircksen et al., 1991, Donini et al., 2001). There has been no identified role of CCAP on the spermatheca in insects.

Since the spermatheca and oviducts of L. migratoria are closely associated, it is of interest to determine whether CCAP acts in a similar way in both of these reproductive visceral tissues. The present study examines the presence of, quantification and physiological effects of CCAP associated with the spermatheca of L. migratoria. This study provides evidence that CCAP acts as a possible neurotransmitter/neuromodulator of the locust spermatheca.

Section snippets

Animals

Experiments were conducted on 3–4 week old adult females of L. migratoria obtained from a colony at the University of Toronto at Mississauga, Ontario, Canada. Animal colonies were fed fresh wheat seedlings supplemented with bran and carrots and kept on a 12:12 h light-dark regime at a temperature range of 30–34 °C.

Chemicals

CCAP was purchased from Peninsula / BACHEM Laboratories (San Carlos, CA, USA), and made up to yield a stock solution of 10−3 M, using double distilled water. The stock solution was

Immunohistochemistry

Immunohistochemical analysis provided evidence for the association of CCAP with the VIIIth abdominal ganglion (not shown, n=40) as previously identified by Dircksen et al. (1991), the VIII sternal nerve, the VON of the VIIIth abdominal ganglion (n=12) and in processes on the spermatheca of L. migratoria (n=10). A CCAP-like immunoreactive axon is evident throughout the VON (Figs. 1A and B) and in the branch projecting towards the spermatheca and body wall (Fig. 1B). This CCAP-like

Discussion

Clark and Lange (2000) have previously identified the extensive innervation of the spermatheca originating from the VON and branching extensively over all regions of the spermatheca with more extensive projections over the coiled duct and sperm sac. CCAP-like immunoreactive axons are present in the VIIIth sternal nerve and VON of the VIIIth abdominal ganglion that project to the spermatheca. In addition, lightly stained CCAP-like immunoreactive processes and varicosities are seen over the

Acknowledgements

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to A.B. Lange. The authors wish to thank Dr. Ian Orchard for his revisions of the manuscript and valuable input and Dr. Ken Dawson-Scully for his aid with the digital imaging.

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