Expression of octopaminergic receptor genes in 4 nonneural tissues in female Nicrophorus vespilloides beetles

Octopamine regulates the function of many tissues and physiological processes in invertebrates. The expression of octopamine receptor genes has been examined in multiple tissue types in several different insect orders. However, little work has addressed this issue in Coleoptera. Most studies characterize individual genes in different tissue types, but here we describe the expression of 6 octopamine receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of female Nicrophorus vespilloides beetles to characterize both different genes and different tissues within a single study. We then compare the gene expression profiles found in this beetle to other insects to examine the extent to which expression profiles are conserved across insects. We also examine the relative involvement of octopamine verses octopamine/tyramine receptors based on receptor gene expression in each tissue to help elucidate if tyramine plays a role in the regulation of these tissues. We find a high degree of overlap in the expression profile of the 6 genes examined in the thoracic musculature, a moderate amount for the oviducts, and divergent profiles for Malpighian tubules and fat body. Based on expression difference in receptor subtypes, our results also support the suggestion that tyramine is a biogenic amine with physiological actions separate from octopamine.


Introduction
The octopaminergic system, comprising six receptors and two biogenic amines, plays a fundamental role in the regulation of behavioral and physiological processes in invertebrates (Blenau & Baumann, 2001;Roder, 2005;Verlinden et al., 2010a). Although perhaps best known for its role in behavior, octopamine receptors are expressed in both neural and peripheral tissues and the effect of the octopaminergic system on peripheral tissues may be extensive (Table 1). For example, this system helps regulate energy metabolism, stress responses, immune responses, and ovulation in insects (Roder, 2005; Correspondence: Allen J. Moore, Department of Genetics, University of Georgia, 120 Green Street Davison Life Sciences Bldg Athens, GA 30602, USA. Tel: 706 542 4423; fax: 706 542 3910; email: ajmoore@uga.edu Verlinden et al., 2010a). The octopaminergic system has a well-established direct role in the regulation of muscles (Candy, 1978;Evans & Siegler, 1982), oviducts (Nykamp & Lange, 2000;Lee et al., 2003;Donini & Lange, 2004), Malpighian tubules (Blumenthal, 2003(Blumenthal, , 2009, and fat body (Wang et al., 1990). Octopamine also acts as a neurotransmitter, neurohormone, and neuromodulator (Roder, 2005;Verlinden et al., 2010a;Farooqui, 2013). The octopaminergic system shares many similarities with the vertebrate adrenergic system by regulating many similar physiological processes, sharing many functional characteristics (Roder, 2005;Verlinden et al., 2010a), and possibly having the same evolutionary origin (Pflüger & Stevenson, 2005;Caveney et al., 2006). Despite the extensive and central role of the octopamine system in behavior and physiology, gene expression in the octopaminergic system is surprisingly poorly characterized outside of a handful of insects (Table 1). In this study we characterized Table 1 Presence, absence, or nonavailability of information on octopaminergic receptor genes in musculature, oviducts, Malpighian tubules, and fat body. Only studies reporting on multiple genes or tissues were included.
Here, we investigated the gene expression profiles of receptors in the octopaminergic system in four nonneural tissue types in the beetle Nicrophorus vespilloides (Herbst) (Coleoptera: Silphidae), to characterize how genes in this system might influence physiology in a beetle. We have previously characterized these genes, pro-vided a phylogenetic comparison, and examined their expression in neural tissue across different social contexts (Cunningham et al., 2014). The tissue types we investigated here were thoracic musculature (with physiological functions associated with locomotion, flight, thermoregulation), oviducts (protein secretion, egg laying), Malpighian tubules (urine production and ion exchange), and fat body (energy storage and metabolism). We selected these tissues because of their roles in insect physiology and because the octopaminergic system is known to help control their function in at least some insects (Candy, 1978;Evans & Siegler, 1982;Wang et al., 1990;Kutsukake et al., 2000;Nykamp & Lange, 2000;Blumenthal, 2003;Lee et al., 2003;Donini & Lange, 2004;Roder, 2005;Blumenthal, 2009). We had two goals with this study. First, we provide basic information about the octopaminergic system from a beetle in these four tissue types and then compare the gene expression profiles to other insects to elucidate how conserved each tissue-specific gene expression profile is across insects. N. vespilloides is a subsocial beetle that has very complex social interactions including extensive parental care. This beetle is therefore potentially very different from the only other well-characterized beetle, the model species, Tribolium castaneum. Second, by simultaneously characterizing expression patterns of six receptors in multiple tissues, we predicted we would uncover functional specialization. Other studies often focus on a single tissue or single receptor. As predicted, the most abundant receptor depended on the tissue examined. However, in comparison with other insects, we found that expression patterns are conserved for some tissues but variable among taxa for others. Our results also reinforce the view that tyramine has an independent effect from octopamine.

Insects
We maintain N. vespilloides as an actively outbred colony at the University of Georgia. The founders of this colony were collected from the wild near the University of Exeter, Cornwall in the United Kingdom. See Head et al. (2012) for a description of the population and collecting methods. From dispersal until experimentation, beetles were kept in a common colony room set at 22 ± 1°C, under a 15 : 9 light : dark cycle, and fed decapitated mealworms (Tenebrio) ad libitum once a week after adult eclosion. Beetles were housed individually at dispersal in 5 oz circular deli containers (Eco products, Boulder, CO, USA) filled with 2.5 cm of moist soil.
We submerged whole bodies into ice-cold 1× PBS (National Diagnostics, Atlanta, GA, USA) for tissue collection. After dissection, we submerged tissues into at least 10× their volume of RNAlater (Ambion, Grand Island, NY, USA) on ice and processed the tissues according to the manufacturer's instructions for storage at −20°C until RNA extraction.
We used a Qiagen RNeasy Micro kit following the manufacturer's instructions to extract total RNA with Qiagen QIAzol (700 μL) as the lysis buffer and an addition of 150 μL of chloroform (J.T. Baker, Center Valley, PA, USA) following homogenization. We treated samples with DNase I (Qiagen) to minimize genomic DNA contamination according to manufacturer's instructions. We quantified RNA using a Qubit 2.0 flourometer (Invitrogen Corporation, Carlsbad, CA, USA) and synthesized cDNA with Quanta Biosciences qScript reverse transcriptase master mix (Quanta Biosciences, Gaithersburg, MD, USA) using 500 ng of RNA. We stored RNA at −80°C and cDNA at −20°C. We made multiple no template controls for each tissue type using the same protocol, except RNase-free water replaced RNA in the cDNA synthesis reaction.
We designed qRT-PCR primers for each gene of interest (GOI) using Primer3 (v4.0.0; Untergrasser et al., 2012) to flank exon boundaries using the draft genome of N. vespilloides as a reference and to produce ß100 bp amplicons. Primer information can be found in Appendix 1.
We used Roche LightCycler 480 SYBR I Green Master Mix with a Roche LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA) for qRT-PCR. We ran all samples with 3 technical replicates using 10 μL reactions. Each reaction contained 5 μL of SYBR mix, 2 μL of cDNA diluted 1 : 10 with qRT-PCR grade water, and 3 μL of a 1.33 μmol/L primer stock of both the sense and anti-sense primers. We used the manufacturer's recommended protocol with an annealing temperature of 60°C for 45 cycles of amplification. We ran tata-box binding protein (tbp) as an endogenous control gene with each tissue type. We established the stability of tbp in a previous study (Cunningham et al., 2014) using the same primer pair and the same experimental methods as in this study. To check for genomic contamination, we ran multiple no template controls of each tissue type with only 2 of 8 samples showing any, and minimal, amplification (1 thoracic musculature sample; 1 Malpighian tubule sample). See Appendix for details.
We used the C T method (Livak & Schmittgen, 2001) to examine differences in expression, with tbp as our endogenous control gene (Cunningham et al., 2014). We calculated C T by taking individual C T values for each gene of interest and subtracting the mean C T value of the most highly expressed gene of interest within a tissue. All C T values approached normality. These values were then converted to relative expression by the 2 − C T method (Livak & Schmittgen, 2001). We visually inspected the data for outliers within technical replicates and removed those that were more than a cycle different from the other values. We tested for the difference in receptor gene expression using an ANOVA. We then used a post hoc Tukey honestly significant difference (HSD) test to assess pairwise significant difference between Fig. 1 Expression of receptor genes of the octopaminergic system, standardized to the mean C T expression of octβr2, in thoracic musculature from Nicrophorus vespilloides adult females. Gene expression was measured using qRT-PCR. Bars are mean ± SEM (n = 7). Letters above bars represent statistically significantly differences using a Tukey HSD post hoc comparison multiple-comparison test. Bars with different letters are statistically significantly different from each other at P < 0.05. the genes. We used JMP Pro (v11.0.0) for all statistical analyses.

Results
In the thoracic musculature, we found statistically significant differences in the expression of the six different receptor genes (F 5,34 = 34.820, P = 0.0001). octβr2 was the most abundant transcript in thoracic musculature followed by tyrr1, octαr, octβr1, tyrr2, and octβr3 (Fig. 1). A post hoc Tukey HSD test showed octβr2 was expressed at significantly greater levels than the other genes (P < 0.0001). tyrr1 was expessed at significantly higher levels than tyrr2, octβr1, and octβr3.
We also found statistically significant differences in gene expression for the six receptors in oviducts (F 4,30 = 14.035, P < 0.0001). octβr2 was again the most abundant transcript in oviducts, with a statistically significantly greater expression than all other receptor genes (Tukey HSD, P < 0.009; Fig. 2). The pattern of the expression of the other genes was different than in the thoracic Fig. 2 Expression of receptor genes of the octopaminergic system, standardized to the mean C T expression of octβr2, in oviducts from Nicrophorus vespilloides adult females. Gene expression was measured using qRT-PCR. Bars are mean ± SEM (n = 8). Letters above bars represent significantly different means using a Tukey HSD test. Bars with different letters are significantly different from each other at P < 0.05. musculature. In the oviduct, there was no statistically significant difference in the expression of octαr, tyrr2, tyrr1, and octβr1 and octβr3 was not consistently detectable among the biological and technical replicates (Fig. 2).
In Malpighian tubules, there were statistically significant differences in expression (F 3,28 = 26.274, P < 0.0001), but this was dominated by the expression of tyrr2 and octβr1 and octβr3 were not consistently detectable among the biological or technical replicates (Fig. 3). A post hoc Tukey HSD test showed tyrr2 was significantly different from the other genes (P < 0.0001), but no other pairwise comparison was significantly different.
In fat body, we again found statistically significant differences in the expression of the six receptor gene (F 5,38 = 3.754, P = 0.0074). octαr was the most abundant octopaminergic receptor transcript followed by tyrr2 and then octβr2, tyrr1, octβr3, and octβr1 (Fig. 4). A post hoc Tukey HSD test showed that only tyrr2 was significantly different from octβr1 and octβr3 (P < 0.04).

Fig. 3
Expression of receptor genes of the octopaminergic system, standardized to the mean C T expression of tyrr2, in Malpighian tubules in Nicrophorus vespilloides adult females. Gene expression was measured using qRT-PCR. Bars are mean ± SEM (n = 8). Letters above bars represent statistically significantly differences using a Tukey HSD post hoc comparison multiple-comparison test. Bars with different letters are statistically significantly different from each other at P < 0.05.

Discussion
We characterized the expression of six octopaminergic receptor genes in thoracic musculature, oviducts, Malpighian tubules, and fat body of the beetle N. vespilloides. We chose these tissues because they have very different physiological functions and because of the important role the octopaminergic system in regulating their function in other insects (Candy, 1978;Evans & Siegler, 1982;Wang et al., 1990;Kutsukake et al., 2000;Nykamp & Lange, 2000;Blumenthal, 2003;Lee et al., 2003;Donini & Lange, 2004;Blumenthal, 2009). This allows us to make an informal comparison of our results to the expression profiles reported from other insect orders and help address a gap in our understanding about the octopaminergic system across different taxa. Overall, we find similar gene expression profiles for species in octopaminergic receptors in thoracic musculature (flight, locomotion, and thermoregulation) and oviduct (reproduction), while we see more variable gene expression profiles across species in the Malpighian tubules (ion exchange) Fig. 4 Expression of receptor genes of the octopaminergic system, standardized to the mean C T expression of octαr, in fat body from Nicrophorus vespilloides adult females. Gene expression was measured using qRT-PCR. Bars are mean ± SEM (n = 8). Letters above bars represent statistically significantly differences using a Tukey HSD post hoc comparison multiple-comparison test. Bars with different letters are statistically significantly different from each other at P < 0.05. and fat body (metabolism). We also find that receptor subtypes that have high affinity for tyramine compared to octopamine are more highly expressed in thoracic musculature, Malpighian tubules, and fat body than previously appreciated, along with the moderate role in regulating the oviducts as seen in other species. Our study is also among the first to examine the expression of tyrr2 where this pattern is especially evident. These results provide additional support for the emerging consensus of tyramine as a standalone neurohormone.
We found detectable levels of transcripts for all 6 octopaminergic receptors in thoracic musculature of N. vespilloides. Overall, there is a high level of consistency in the expression profiles of octopaminergic genes across several species of insects for this tissue. We found octβr2 was the most highly expressed receptor in N. vespilloides, as it is in the lepidopteran Trichoplusia ni (Lam et al., 2013). The orthopteran Schistocerca gregaria has an octβr (likely octβr2) expressed higher than octαr in muscle (Verlinden et al., 2010b), also seen in our results. We found that tyrr1 was the second highest expressed receptor gene in muscle in N. vespilloides. Functioning tyrr1 is necessary for proper body wall muscle function in Drosophila larvae (Kutsukake et al., 2000), so its expression in muscle is not surprising. A tyrr (likely tyrr1) is also present in the cockroach Periplaneta americana muscle (Rotte et al., 2009); however, very little tyrr (likely tyrr1) expression was found in T. ni muscle (Lam et al., 2013). octαr was found at moderate levels, consistent with its detectable but not abundant presence in T. ni (Lam et al., 2013) and S. gregaria (Verlinden et al., 2010b). octβr1 and octβr3 were detected at very low levels in this tissue, consistent with expression levels seen in T. ni (Lam et al., 2013). We also found low-level expression of tyrr2, a receptor type that shows a much greater sensitivity to tyramine compared to octopamine (Cazzamali et al., 2005;Huang et al., 2009), which might suggest a reduced role for tyramine in regulating this tissue compared to octopamine.
Five of the 6 octopaminergic system receptor genes were detectable in the oviducts. octβr3 was not detectable in N. vespilloides, but is detectable in 3 species of Lepidoptera (Lam et al., 2013). As with thoracic musculature, octβr2 was the most highly expressed receptor gene followed by octαr. octβr2 is also the most highly expressed gene in oviducts in T. ni (Lam et al., 2013). octαr is necessary for ovulation in Drosophila melanogaster (Lee et al., 2003), so its presence in this tissue is expected. We also note moderate expression of tyrr2. Tyramine is suggested to be a cotransmitter with octopamine during ovulation of a locust and tyrr2 presence in oviduct tissue supports this potential role (Donini & Lange, 2004). octβr1 and tyrr1 were found at low levels in oviduct tissue, a pattern also seen with 3 species of lepidopterans (Lam et al., 2013).
Four of the 6 octopaminergic receptors were detectable in N. vespilloides Malpighian tubules, with no detectable expression of octβr1 or octβr3. octβr1 is the most highly expressed octopaminergic receptor gene in the Malpighian tubules of Trichoplusia ni and is very highly expressed in two other lepidopteran species (Lam et al., 2013), but had no detectable expression in N. vespilloides. T. ni has marginally detectable expression of octβr3 in Malpighian tubules, but levels of octβr3 are more detectable in the Malpighian tubules in 2 other lepidopteran species (Lam et al., 2013). We also found that tyrr2 was the most highly expressed gene, in contrast to three species of Lepidoptera where octβr1 is most highly expressed in Malpighian tubules (Lam et al., 2013). Bombyx mori also does not have high levels of expression of tyrr2 in this tissue (Huang et al., 2009). Tyramine has a well-established role in osmoregulation of D. melanogaster Malpighian tubules (Blumenthal, 2003(Blumenthal, , 2009, which may help explain the high prevalence of tyrr2 we see in N. vespilloides. Octopamine/tyramine receptor gene expression has also been reported in the Malpighian tubules of the rice stem borer Chilo suppressalis (likely tyrr1; Wu et al., 2013) and the cockroach P. americana (Rotte et al., 2009). Low levels of octβr2 as seen here have also been reported in C. suppressalis Malpighian tubules, although the expression of other octopaminergic receptor genes was not measured (Wu et al., 2012). octαr and tyrr1 were found at low levels of expression. Overall, our results reinforce the view that tyramine plays a role in the regulation of Malpighian tubule function.
We found that all 6 receptors were detectable in the fat body of N. vespilloides with octαr and tyrr2 the most highly expressed genes. Our results contrast with the pattern seen in T. ni that, with octβr2 the most highly expressed receptor gene. However, octαr is also highly expressed in Lepidoptera (Lam et al., 2013). In S. gregaria, octαr is expressed at higher levels than an octβr2 (Verlinden et al., 2010b), a pattern seen here between octαr and all of the octβr's. octβr2 was also found at very low expression levels in the fat body of C. suppressalis (Wu et al., 2012). octβr1, octβr3, and tyrr1 were all at low levels of expression in fat body in N. vespilloides. Octopamine influences fat body dynamics through two mechanisms. First, it promotes the release of adipokinetic hormone from the corpora cardiaca (Wang et al., 1990). Second, it directly promotes the release of fatty acids from the fat body (Wang et al., 1990). Blocking Octαr, but not Octβr's, activity inhibits octopamine's direct influence on the fat body (Wang et al., 1990). This is consistent with our finding of octαr, but not octβr's, in high prevalence in this tissue in N. vespilloides. The fact that we see a high level of expression of tyrr2 suggests a role for this amine in regulating fat body activity.
In summary, our results support the idea that there is a high degree of conservation for the gene expression profiles of the octopaminergic receptors in thoracic musculature. Oviducts show moderate conservation in the pattern of expression of octopamine receptors. In contrast, Malpighian tubules and fat body show more divergence between species in which receptor predominates. This suggests that while specialization in the function of receptors might occur, the exact action of each receptor in each tissue type may be species-dependent. Our results also suggest that different octopamine/tyramine receptors might play a prominent role in the regulation of function in oviducts, Malpighian tubules, and fat body.
for our burying beetles. We thank Paola Barriga, Kyle Benowitz, Ashley Duxbury, Lauren Hebb, Elizabeth McKinney, Patricia Moore, and Eileen Roy-Zokan for discussions and/or comments on the manuscript. The University of Georgia's Office of the Vice President for Research and the Provost of the University of Georgia provided support for this research. MK Douthit was supported by a University of Georgia Center for Undergraduate Research Opportunities summer fellowship.

Disclosure
The authors declare no conflicting interest.