Downregulation of the dopamine D2-like receptor in corpus allatum affects juvenile hormone synthesis in Drosophila melanogaster females

https://doi.org/10.1016/j.jinsphys.2011.12.006Get rights and content

Abstract

In Drosophila, juvenile hormone (JH) is synthesized de novo in the specialized endocrine gland, corpus allatum (CA). Dopamine (DA) controls JH levels by either stimulating or inhibiting its synthesis and degradation depending on the developmental stage. The present study focuses on the role of D2-like receptors in the regulation of JH synthesis by dopamine. We show that D2-like receptors (DD2R) are expressed in CA cells of Drosophila melanogaster females. In addition, the level of JH production was analyzed in D. melanogaster females with decreased DD2R expression in CA (vs. corresponding control flies) by assessing multiple indices of JH synthesis (JH-hydrolyzing activity and stress reactivity of the system of JH metabolism, activity and stress reactivity of the alkaline phosphatase, activity and stress reactivity of the tyrosine decarboxylase). The differential value obtained for each index suggests increased JH production in female flies that downregulate DD2R. Based on these findings, we postulate that the DA inhibiting effect on the JH synthesis in D. melanogaster is mediated at least in part via D2-like receptors.

Highlights

► D2-like receptor (DD2R) is expressed in corpus allatum (CA) of Drosophila adults. ► Downregulation of DD2R in CA decreases juvenile hormone (JH) degradation. ► Downregulation of DD2R in CA decreases tyrosine decarboxylase activity. ► Downregulation of DD2R in CA increases alkaline phosphatase activity.

Introduction

Juvenile hormones (JHs) comprise a family of acyclic sesquiterpenoid hormones that regulate a wide range of biological processes in insects (Goodman and Granger, 2005). In larval development JH plays a “status quo” role, which involves suppression of premature metamorphosis and determination of the ecdysteroid-induced molt as larval or pupal (Truman and Riddiford, 2007). Besides the “status quo” action, JH plays a gonadotropic role in adult insects together with the molting hormone 20-hydroxyecdysone (20E). According to the model generally accepted for Drosophila (Bownes, 1989, Gilbert et al., 2000, Raikhel et al., 2004), JH, synthesized de novo in a specialized endocrine gland, the corpus allatum (CA), stimulates ecdysone biosynthesis by the ovarian follicular cells. Ecdysone is converted to 20E in the hemolymph and in other tissues. 20E stimulates vitellogenin biosynthesis in the fat body, which is in turn taken up from the hemolymph by the ovaries. It was also shown that in insects, including Drosophila, JH is a part of the neuroendocrine stress response (Rauschenbach et al., 1995, Cymborowski, 1991) and plays a key role in insect adaptation to stressors (Gruntenko and Rauschenbach, 2008). Therefore, the strict regulation of JH titer in insect body is crucial throughout their development as well as adult life.

It is well known that dopamine (DA) acts as a neurotransmitter, neuromodulator and neurohormone in various insects, including Drosophila (Blenau and Baumann, 2001, Neckameyer and Leal, 2002, Waddel, 2010, Van Swinderen and Andretic, 2011). It has been established that dopamine (DA), as a neurohormone, is involved in the regulation of JH titer (Pastor et al., 1991, Woodring and Hoffmann, 1994, Granger et al., 1996, Gruntenko et al., 2000, Gruntenko et al., 2005). In Blattella germanica females, DA stimulates JH production at days 1 and 2 of the first ovarian cycle, and induces the opposite effect on days 6 and 7 (Pastor et al., 1991). Ontogenetic differences in the control of JH synthesis by DA have been established for larvae of Manduca sexta: DA stimulates hormone biosynthesis in CA in the first 2 days of the last larval stage, but inhibits CA on days 3–6, at the beginning of the prepupal stage (Granger et al., 1996). The existence of ontogenetic differences in the control of JH by DA has also been demonstrated for Drosophila females: DA increases JH levels (inhibiting JH degradation) in young females and decreases JH levels (stimulating JH degradation) in sexually mature flies (Gruntenko et al., 2000, Gruntenko et al., 2005, Gruntenko and Rauschenbach, 2008). There is a negative feedback in this regulation – in young females a rise in JH titer leads to decreased DA levels, while in mature females it results in an increase in DA levels (Gruntenko et al., 2003b, Rauschenbach et al., 2004). It should be emphasized, that a change in DA content had no effect on JH metabolism in Drosophila males (Gruntenko and Rauschenbach, 2008).

DA mediates its physiological effects through the activity of either D1-like (D1 and D5) or D2-like (D2, D3 and D4) G-protein-coupled receptors (Missale et al., 1998, Vallone et al., 2000). D1-like receptors have been shown to couple to stimulatory G proteins (Gαs) thus activating adenylate cyclase and triggering a cascade that leads to up-regulation of the target genes (Missale et al., 1998, Vallone et al., 2000). D2-like receptors signal via inhibitory G proteins (Gαi/o), which results in the inhibition of adenylate cyclase and leads to down-regulation of the target genes (Missale et al., 1998, Vallone et al., 2000). The Drosophila D2-like receptor (DD2R) was previously cloned and characterized (Hearn et al., 2002). Draper and co-authors (2007) determined the spatial expression pattern of DD2R in Drosophila melanogaster, as well as functionally characterized flies with reduced DD2R levels.

In this research, to study whether the inhibiting effect of DA on JH synthesis in СА is mediated through D2-like receptors, we have utilized transgenic D. melanogaster that downregulate DD2R specifically in CA. These flies were obtained by crossing UAS-ds-DD2R RNAi flies (carrying an inverted repeat construct of the DD2R cDNA sequence [Draper et al., 2007]) to the Aug21-Gal4 ‘driver’ line (that expresses the Gal4 transcription factor specifically in CA [Mirth et al., 2005, Liu et al., 2009, Gruntenko et al., 2010]). Six parameters of JH synthesis, previously established by our group to be reliable indicators of either an increase, or a decrease in JH synthesis in Drosophila female (Rauschenbach et al., 2004, Rauschenbach et al., 2007, Rauschenbach et al., 2011, Bogomolova et al., 2009, Gruntenko et al., 2010), were subsequently assessed in the Aug21>;UAS-ds-DD2R RNAi, (vs. control) flies. These include levels of JH degradation and stress reactivity of the system of JH metabolism, activity and stress reactivity of the alkaline phosphatase (ALP, the enzyme that controls the pool of DA precursor, tyrosine [Wright, 1987]), and activity and stress reactivity of tyrosine decarboxylase (TDC, the enzyme that limits the rate of octopamine synthesis [Wright, 1987]).

Section snippets

Drosophila strains and genetic experiments

The following three Drosophila strains were utilized for the current studies: (1) the Aug21-Gal4/Cyo::arm-GFP strain carrying the driver Aug21-Gal4 (Aug21>[Mirth et al., 2005]) over the Cyo::arm-GFP balancer chromosome (strain was kindly provided by Prof. S. Li, Institute of Plant Physiology and Ecology, Shanghai Institute of Biological Sciences of the Chinese Academy of Sciences); (2) the homozygous UAS-ds-DD2R strain, carrying the pUAS-ds-DD2R interference transgene (Draper et al., 2007), and

DD2R is expressed in the corpus allatum of D. melanogaster

The specific expression of the driver Aug21> has been found in the СА and the salivary glands of Drosophila larvae (Mirth et al., 2005). In Drosophila adult the corpus allatum (CA)/corpus cardiacum (CC, the source of adipokinetic hormone) complex is located above the junction between the crop and the midgut (Fig. 1a, schematic representation reproduced from Toivonen and Partridge (2009)). Here we show (Fig. 1b) that in the Drosophila adults (Aug21>;w1118;UAS-RFP females) the driver Aug21> is

Discussion

Reports in the literature have established that a proper balance between the JH and 20E is of a paramount importance for a normal oogenesis in Drosophila (Soller et al., 1999). Similarly, we have shown that an imbalance of these hormones (shifting the balance in favor of either JH or 20E) leads to reproductive defects in Drosophila (Gruntenko et al., 2003a, Rauschenbach et al., 2004). At the same time, we have found that (i) a mutation- stress- or pharmacologically-induced change in one of

Acknowledgements

We are grateful to Dr. Isabelle Draper (Molecular Cardiology Research Institute, Tufts Medical Center, Boston, Massachusetts) for D. melanogaster strains, DD2R antibodies, manuscript reading and valuable suggestions. We are also grateful to Prof. Sheng Li (Institute of Plant Physiology and Ecology, Shanghai Institute of Biological Sciences of the Chinese Academy of Sciences) for D. melanogaster strain Aug21-Gal4/Cyo::arm-GFP. The study was supported by the Grants of RFBR ## 09-04-00441,

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