Octopamine receptor gene expression in three lepidopteran species of insect

Peptides 41 (2013) 66–73

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Octopamine receptor gene expression in three lepidopteran species of insect Felix Lam a,b , Jeremy N. McNeil b , Cam Donly a,b,∗ a b

Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, Canada N5V 4T3 Department of Biology, University of Western Ontario, London, ON, Canada N6A 5B7

a r t i c l e

i n f o

Article history: Received 9 March 2012 Received in revised form 28 March 2012 Accepted 29 March 2012 Available online 6 April 2012 Keywords: Octopamine Receptors Tyramine Lepidopteran Insect qPCR

a b s t r a c t The invertebrate octopaminergic system affects many diverse processes and represents the counterpart to the vertebrate adrenergic/noradrenergic system with the classes of octopamine receptor (OAR) being homologous to those of vertebrate adrenergic receptors. However, there is still little information on the OARs present in different insect species, and the levels and distribution of these receptors throughout the body. cDNAs sharing high similarity with known insect OARs were cloned in three lepidopteran species: the cabbage looper, Trichoplusia ni; the true armyworm, Pseudaletia unipuncta; and the cabbage white, Pieris rapae. Seven major larval tissues and one adult tissue were examined in T. ni using quantitative real-time PCR to determine the relative expression levels of each receptor transcript across different tissues, as well as of all receptor transcripts within individual tissues. A subset of these tissues was also examined in P. unipuncta and P. rapae. All receptor transcripts were expressed in the nervous system of all three species, however, the distribution of the different receptor types varied between species. In all tissues, the OARbeta2 transcript was the most highly expressed, except in the Malpighian tubules where OARbeta1 was highest, and the midgut where there was no significant difference in receptor transcript levels. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction Biogenic amines play a prominent role in invertebrate physiology, and have quite diverse functions in a wide range of processes [4]. For example, the monoamine, octopamine functions as a neurotransmitter in the regulation of endocrine gland secretion, as a neuromodulator in modulating sensory inputs and behaviors such as learning and memory, and as a neurohormone in controlling lipid and carbohydrate metabolism [5,14,35]. Significant similarities between the octopaminergic signaling system in invertebrates and the adrenergic system of vertebrates have been reported. These include the structural and pharmacological properties of the respective receptors and transporters, as well as in the monoamine ␤-hydroxylases that catalyze formation of the amines. This suggests that the two systems have a common evolutionary origin prior to the divergence of the protostomes and deuterostomes [7,35]. In addition, the octopaminergic and adrenergic systems also parallel each other in their function and roles, as the ‘fight or flight’ responses in invertebrates and vertebrates are modulated by the octopamine/tyramine and adrenergic systems, respectively.

∗ Corresponding author. Tel.: +1 519 457 1470; fax: +1 519 457 3997. E-mail address: [email protected] (C. Donly).

There are two main classes of adrenergic receptors: ␣ and ␤ receptors, so their octopaminergic counterparts have been designated the ␣-adrenergic-like and ␤-adrenergic-like receptors, although the ␤-adrenergic-like receptors are composed of three subtypes [13]. In addition to these two main classes of octopamine receptor there is also a third class, the octopamine/tyramine (or tyraminergic) receptor (for a review see: [23]). The first two receptor types generally have a higher affinity for octopamine than tyramine, while in the third the inverse is true. Each of the three classes of receptor mediates its effects via specific secondmessenger systems [14]. Given the many roles that octopamine plays in coordinating complex physiological processes in insects, one would predict that the different receptors will have different distributions within the developmental stages of a given species, and between species as a function of their life history. To test this hypothesis we first examined octopamine receptor gene expression in various larval and adult tissues of the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae). We identified all five members of the three classes of OAR gene and developed quantitative polymerase chain reaction (qPCR) assays for each, allowing us to measure the relative expression level of each receptor gene across a variety of different tissues. Furthermore, we examined the relative expression levels of all the OAR gene transcripts within individual tissues so we were not only able to determine where in the insect any OAR gene was most highly expressed, but also to determine the particular OAR

0196-9781/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.03.034

F. Lam et al. / Peptides 41 (2013) 66–73

gene that was most highly expressed in a given tissue. Then, to evaluate how representative our findings are in general for Lepidoptera we compared the expression levels in tissues of interest in T .ni with the expression levels of orthologous octopamine receptors cloned from the true armyworm, Pseudaletia unipuncta (Haw.) (Lepidoptera: Noctuidae) and the cabbage white butterfly, Pieris rapae (L.) (Lepidoptera: Pieridae). 2. Materials and methods 2.1. Insect rearing T. ni and P. unipuncta were obtained from laboratory colonies at the Southern Crop Protection and Food Research Centre in London, Ontario, and the University of Western Ontario, respectively, while P. rapae were purchased from Carolina Biological Supply. T. ni larvae were reared on an artificial wheat germ diet [16], while P. unipuncta larvae were reared on an artificial pinto bean diet [36]. The diet for P. rapae was a proprietary formulation obtained from Carolina Biological Supply. The insects were raised at 24 ◦ C, 16L:8D until they reached the last larval instar, whereupon they were either harvested and dissected or left in rearing to provide adults. Both T. ni and P. unipuncta were raised in individual plastic cups, while P. rapae were raised in groups of five or six in larger plastic containers. 2.2. Cloning 2.2.1. RNA isolation and cDNA synthesis Total RNA was extracted from the heads of T. ni, P. rapae and P. unipuncta larvae using a Qiagen RNeasy Kit. Insect heads were removed, placed in 4 ◦ C Calpode’s insect saline solution (pH 7.2, 10.7 mM NaCl, 25.8 mM KCl, 90 mM glucose, 29 mM CaCl2 , 20 mM MgCl2 and 5 mM HEPES) and stored in RNAlater (Ambion) at 4 ◦ C prior to homogenization and RNA extraction. cDNA was then synthesized from the RNA using Superscript II reverse transcriptase with random primers (Invitrogen). 2.2.2. Degenerate PCR Degenerate primers were designed to amplify a cDNA fragment of each of the receptor types from T. ni, P. unipuncta and P. rapae using highly conserved regions of octopamine receptor sequences known from other insects, and these were used for PCR amplification from cDNA templates. Degenerate and unique primers were also used to clone portions of the three reference genes in this study from P. unipuncta and P. rapae (Supplementary Table 1). Polymerase chain reactions were performed using Platinum Taq DNA polymerase (Invitrogen) in the presence of primers, MgCl2 and dNTPs. The temperature profile consisted of a 2 min denaturation at 95 ◦ C, followed by 35 cycles of 30 s denaturation at 95 ◦ C, 30 s annealing at 50–58 ◦ C (in order to optimize degenerate primers) and 30 s elongation at 72 ◦ C, and a final 7 min elongation at 72 ◦ C. MgCl2 concentrations were varied from 1.5 to 3 mM, and primer concentrations from 100 to 500 nM, in order to optimize degenerate primer efficiency. 2.2.3. Molecular cloning PCR products were separated on 1.2% agarose gels to purify and isolate the desired fragments, extracted with the QIAquick Gel Extraction kit (Qiagen) and eluted with 1 mM Tris–HCl (pH 8.0). The amplified products were cloned in pGEM-T Easy plasmid vector and transformed into XL1-blue MRF’ electrocompetent Escherichia coli cells grown on LB agar plates containing ampicillin, X-Gal and IPTG. Plasmids were isolated using a Qiagen Plasmid Mini kit and sequenced at the Southern Crop Protection and Food

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Research Centre in London, Ontario. In this way, fragments of five unique receptor genes were identified from all three species. 2.3. Quantitative real-time PCR (qPCR) Experiments for relative expression of a single gene of interest (GOI) across different tissues were carried out using the Lightcycler TaqMan Master kit (Roche) on a Rotor-Gene 3000 thermal cycler (Corbett Life Science), while multiple GOI experiments within single tissues used the TaqMan Probes Master kit (Roche) on a CFX96 Real-time system (Bio-Rad). The qPCR run profile in both cases consisted of a 10 min 95 ◦ C activation step, followed by 45 cycles of a three-step process: a 10 s 95 ◦ C denaturation step, a 30 s annealing step at a primer pair-specific temperature, and a 1 s 72 ◦ C elongation step. For T. ni, unique primers and Taqman® (dual-labeled hydrolysis) probes were designed from the sequenced fragments of each receptor. The primers and probes for reference genes were as described in Labbe et al. [21]. In the case of P. unipuncta and P. rapae, primers and probes were designed for each of the receptor subtypes, as well as for all three reference genes (Supplementary Table 2). All qPCR experiments were run in triplicate, both with respect to technical and biological replicates. For qPCR experiments, nervous system (brain and nerve cord), midgut, hindgut, muscle, fat body, integument and Malpighian tubule tissues from fifth instar T. ni caterpillars, and oviduct tissue from 5- to 8-day-old virgin female moths were dissected in 4 ◦ C Calpode’s insect saline solution and then stored in RNAlater (Ambion) at 4 ◦ C prior to homogenization and RNA extraction as described in Section 2.2.1. Nervous system, Malpighian tubule and oviduct tissues were similarly dissected from P. unipuncta and P. rapae. Tissues for each biological replicate were taken from a sample of up to 25 individuals, depending on tissue type. The nervous system, being the smallest of the sampled tissues, required all 25 individuals to ensure an adequate amount of starting tissue for RNA extraction. Larger tissues, such as the midgut, required fewer animals, though no fewer than five individuals were sampled in any instance. RNA quality was confirmed via analysis with an RNA 6000 Nano kit on a 2100 Bioanalyzer (Agilent). cDNA was then synthesized as described in Section 2.2.1 from 2 ␮g of RNA. Template cDNA from each tissue was diluted 1 in 10 and at this dilution, PCR inhibition from the reverse transcription components was found to be insignificant. No template controls (NTC) were also included in each experiment. Data for relative expression of a single GOI across different tissues were analyzed using the modified Ct method [17] to generate a quantity for the expression of the GOI in each tissue relative to the expression of the GOI in the highest-expressed tissue. The data were then normalized using the expression of three reference genes [38]. The expression data were also corrected using experimentally obtained qPCR amplification efficiencies for each gene. In order to determine efficiency, each experiment included a serial dilution of a linearized plasmid containing the gene fragment from which the primers and probe were designed. These plasmids, obtained during the original molecular cloning of the cDNA, were linearized in overnight enzymatic digestion reactions. The products were then purified and isolated using agarose gel electrophoresis (1.2%) and a QIAquick gel extraction kit (Qiagen). Concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific). qPCR optimization experiments were run using a serial dilution of the linearized plasmid to determine optimal annealing temperature for the experiments. In the case of relative expression of multiple GOIs within a single tissue, the data are expressed relative to the highest expressed GOI in that tissue. Expression normalization was not necessary in these experiments as there is no sample variation to control for.

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F. Lam et al. / Peptides 41 (2013) 66–73

Fig. 1. Phylogenetic tree comparison of cloned octopamine receptor gene fragments from Trichoplusia ni (Tni), Pseudaletia unipuncta (Pun) and Pieris rapae (Pra), along with known receptors from Drosophila melanogaster (Dme) and Bombyx mori (Bmo). Accession numbers for genes are found in Table 1. The sequences were aligned using ClustalW in CLC Sequence Viewer 6.4 (CLC bio, Aarhus, Denmark) with the software’s alignment parameters, and the phylogenetic tree calculated by the Neighbor Joining method (bootstrap = 100). The scale bar shows amino acid substitutions per site.

3. Results 3.1. Cloning and sequence analysis Five octopamine receptor gene fragments were isolated from each of T. ni, P. unipuncta, and P. rapae. Phylogenetic analysis

of the sequence fragments showed that they correspond to the 5 types of octopamine receptor proposed by Evans and Maqueira [13] (Fig. 1). The gene fragments share generally high translated amino acid identity with known and predicted octopamine receptors from other insects, as well as with each other (Table 1).

Table 1 Amino acid identity values between octopamine receptor gene fragments cloned from T. ni, P. unipuncta and P. rapae, and with known and predicted octopamine receptors from other insect species. Receptor

Accession No.

T. ni

P. unipuncta

P. rapae

OARalpha Bombyx mori BmOAR1 Manduca sexta MsOAR T. ni P. unipuncta P. rapae

NP 001091748 ABI33825 JN815175 JN815180 JN815189

93% 92% – 95% 87%

92% 90% 95% – 85%

87% 88% 87% 85% –

OA/TA Mamestra brassicae MbraOAR/TAR Heliothis virescens K50Hel Spodoptera littoralis TyrRI T. ni P. unipuncta P. rapae

AAK14402 Q25188 ACJ06651 JN815176 JN815181 JN815190

99% 99% 98% – 99% 99%

100% 99% 98% 99% – 98%

99% 98% 97% 99% 98% –

OARbeta1 Apis mellifera Drosophila melanogaster DmOctbeta1R Tribolium castaneum Pediculus humanus corporis GPRoar2 Harpegnathos saltator OAR beta-1R T. ni P. unipuncta P. rapae

XP 397139 NP 001163690 XP 974265 XP 002422997 EFN87040 JN815177 JN815182 JN815191

72% 64% 70% 71% 47% – 87% 91%

75% 74% 85% 80% 79% 87% – 95%

79% 69% 85% 83% 78% 91% 95% –

OARbeta2 Bombyx mori BmOAR2 Schistocerca gregaria SgOctbetaR T. ni P. unipuncta P. rapae

NP 001171666 ADD91575 JN815178 JN815183 JN815192

89% 65% – 92% 84%

90% 65% 92% – 85%

90% 63% 84% 85% –

OARbeta3 Drosophila melanogaster DmOctbeta3R Tribolium castaneum T. ni P. unipuncta P. rapae

NP 001034048 XP 974238 JN815179 JN815184 JN815193

68% 66% – 97% 62%

70% 77% 97% – 60%

55% 80% 62% 60% –

Identity values obtained using the NCBI BLAST program (http://blast.ncbi.nlm.nih.gov/).

F. Lam et al. / Peptides 41 (2013) 66–73

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8 *

Nervous system Midgut

7

Malpighian tubule Muscle Fat body

6

Integument Hindgut/rectum Oviduct

5 4

*

3 a

a b

c d

1

c d

*

*

b c d

2

b c c d

d

0 OARalpha

OA/TA

OARbeta1

OARbeta2

OARbeta3

Fig. 2. Normalized relative quantity of each octopamine receptor mRNA compared between major tissues of T. ni. The mRNA quantity is expressed relative to the tissue with the highest level of transcript for each gene. For OARalpha, the letters denote levels of statistical significance; tissues that share letters with other tissues have no significant difference between them, while for the remaining genes, asterisks indicate significant difference in expression level, according to a Tukey test (˛ < 0.05).

3.2. Quantitative real-time PCR

G3PDH in T. ni, the remaining two genes, S5 and eIF4˛, were used for all normalization.

The geNorm software package [38] was used to evaluate reference gene stability to ensure accurate normalization of results for the single gene experiments measuring expression of each gene in different tissues. The software analyzes the expression of the reference genes across different samples and gives a measure of the average pairwise variation of each gene with all other reference genes (the M value). Reference genes with lower M values have more stable expression across different tissues. In T. ni, the M values of the three reference genes were 1.319, 0.989 and 1.113 for G3PDH, S5 and eIF4˛, respectively. In P. unipuncta, and P. rapae the respective M values were 0.532, 0.720 and 0.526; and 0.462, 0.476, and 0.405. Due to the high M value for

3.2.1. Relative expression levels of individual OAR genes across T. ni tissues In T. ni, the alpha adrenergic-like receptor (OARalpha) gene was found to be expressed in all tissues examined, though the expression levels varied, being highest in the muscle and nervous system and lowest in the midgut (Fig. 2). There was also a moderate level of expression in the oviduct. The expression level of the octopamine/tyramine (OA/TA) receptor gene in T. ni was significantly higher in the nervous system than in the other tissues examined, which showed no significant differences. There was a significantly higher level of expression of the Beta 1 receptor

1.8 *

1.6

*

1.4 1.2 1.0 0.8

* *

*

*

*

OARalpha OA/TA

0.6

OARbeta1

0.4

OARbeta2

0.2

OARbeta3

0.0

Fig. 3. Relative expression levels of OAR gene transcripts compared within each T. ni tissue. Expression quantities are given relative to the gene with the highest expression in each tissue. Asterisks denote genes with a significantly different expression level.

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F. Lam et al. / Peptides 41 (2013) 66–73

6

1.6 1.4

a

a

a

a

a

a

a

Nervous system Malpighian tubule

1.2

Nervous system

5

Malpighian tubule

Oviduct

Oviduct

4

1 0.8

a

3

b

0.6

2

0.4

b

0.2 0

a

c OARalpha

b

a

b b OA/TA

OARbeta1

OARbeta2

OARbeta3

a

a 1 0

b c OARalpha

Fig. 4. Normalized relative quantity of octopamine receptor mRNA compared between the nervous system, Malpighian tubule, and oviduct of P. rapae. The mRNA quantity is expressed relative to the tissue with the highest level of transcript for each gene. The letters denote levels of statistical significance; tissues that share letters with other tissues have no significant difference between them, according to a Tukey test (˛ < 0.05).

(OARbeta1) gene in the Malpighian tubule compared to other tissues while the Beta 2 receptor (OARbeta2) gene had significantly higher expression in the oviduct than elsewhere. Similar to the OA/TA receptor, the expression level of the Beta 3 receptor (OARbeta3) gene was significantly higher in the T. ni nervous system than in all other tissues examined. 3.2.2. Relative expression levels of multiple OAR genes within single T. ni tissues In the experiments testing relative T. ni OAR gene expression within each tissue, the OARbeta2 gene was found to be the most highly expressed OAR within every tissue, with the exception of the Malpighian tubules where the OARbeta1 gene was most highly expressed (Fig. 3). No significant difference was found between the expression levels of the OAR genes in the midgut. 3.2.3. Relative expression levels of individual OAR genes across P. rapae tissues In P. rapae, the OARalpha gene was expressed most strongly in the nervous system, followed by the oviduct (Fig. 4), with a very low relative level of expression in the Malpighian tubules, while the OA/TA receptor gene was expressed most highly in the nervous system. The OARbeta1 and OARbeta2 gene expression levels were found to be the highest in the nervous system and Malpighian tubules while the expression levels of the OARbeta3 gene did not differ significantly between the tissues examined. 3.2.4. Relative expression levels of individual OAR genes across P. unipuncta tissues In P. unipuncta, the OARalpha gene was expressed most strongly in the nervous system, followed by the Malpighian tubule, with little expression in the oviduct (Fig. 5). The expression of the OA/TA receptor gene was high in the nervous system, but significantly lower in the Malpighian tubules and oviduct. The OARbeta1 gene expression level was significantly higher in the Malpighian tubule than the nervous system and oviduct, while the OARbeta2 and OARbeta3 receptor genes were significantly higher in the nervous system than in the Malpighian tubules and oviduct. 4. Discussion We were able to isolate fragments in T. ni, P. unipuncta and P. rapae, corresponding to genes for all five of the octopamine receptor types outlined by Evans and Maqueira [13]. The three lepidopteran OARalpha gene fragments cloned shared very high identity with

a

b b b

OA/TA

b

a b b

b b

OARbeta1 OARbeta2 OARbeta3

Fig. 5. Normalized relative quantity of octopamine receptor mRNA compared between the nervous system, Malpighian tubule, and oviduct of P. unipuncta. The mRNA quantity is expressed relative to the tissue with the highest level of transcript for each gene. The letters denote levels of statistical significance; tissues that share letters with other tissues have no significant difference between them, according to a Tukey test (˛ < 0.05).

known alpha adrenergic-like receptors from other lepidopteran species, although less so for P. rapae than the other two species. This is not surprising as the other known genes are from the moths Bombyx mori and Manduca sexta, in Bombycoidea superfamily and therefore more closely related to the noctuid moths P. unipuncta and T. ni than the cabbage white,which is a butterfly in the Family Pieridae, We found very high identity (98–100%) between the OA/TA receptor sequences in the three species that we examined and those of previously cloned Mamestra brassicae, Heliothis virescens and Spodoptera littoralis, indicating that this gene is extremely well conserved in the Lepidoptera. There are only four cloned insect beta adrenergic-like receptors described in the literature, the three Drosophila receptors: DmOctBeta1, DmOctBeta2 and DmOctBeta3 and the BmOAR2 from B. mori. We were able to clone fragments of all three beta adrenergic-like receptor subtypes from T. ni, P. rapae and P. unipuncta. These receptor types generally appeared less well conserved than the alpha and OA/TA types (Table 1) although, this may be due to the fact that the BmOAR2 receptor from B. mori, is the only lepidopteran beta adrenergic-like receptor gene cloned. We also found that the sequence identities for the OARbeta3 fragments were affected by the presence of a large insertion of approximately 360 nucleotides that is absent in the Drosophila DmOctBeta3 and predicted Tribolium castaneum OARbeta3 genes. As a result these fragments group apart from the Drosophila orthologue in phylogenetic analyses (Fig. 1). Following a search of the silkworm genomic database, Chen et al. [9] concluded that B. mori lacked complete versions of both OARbeta1 and OARbeta3 orthologues, the former being completely absent, and the latter being a pseudogene lacking a start codon. It remains possible that one or more of the OARbeta genes we have identified in these other lepidopterans could also be pseudogenes and cloning of full length cDNAs will be necessary to determine this. However none of the genes is absent completely as in the case of B. mori OARbeta1. Since we found that the sequence identities for the OARbeta fragments are substantially lower than for the alpha and OA/TA receptors (Table 1), this suggests the beta receptors are more divergent and that individual receptors may have less highly conserved roles. The three beta adrenergic-like receptors share similar signaling properties and it is possible that in B. mori the roles of OARbeta1 and 3 may be carried out by OARbeta2. To determine the level of expression of all of these OAR genes, two different types of qPCR experiment were performed in this study. Initial experiments were conducted to determine the relative expression levels of each receptor gene across different

F. Lam et al. / Peptides 41 (2013) 66–73

tissues in the insects. Further experiments were then performed to determine the relative levels of the five receptor genes in each tissue, allowing us to see which genes were most active in a given tissue. Taken together, the two experiments allow us to understand which receptors are likely to be responsible for various octopaminergic effects throughout a lepidopteran insect. 4.1. Relative expression of the OARalpha gene The OARalpha gene fragments cloned were expressed in different patterns by the three lepidopteran species. Malpighian tubule expression was more similar between the two moths species T. ni and P. unipuncta, than with the butterfly, P. rapae (Figs. 2, 4 and 5). Stanisc¸uaski et al. [37] showed that plant secondary compounds can target insect Malpighian tubules, causing toxicity via an impairment of water balance. Thus, one might have predicted that as specialists feeding on plants in the Brassicacae with characteristic defensive compounds, including the isothiocyanates, that may reduce survival and growth [1] T. ni and P. rapae would be more similar than the generalist herbivore, P. unipuncta. However, the expression data suggest that if there is an octopaminergic component to the differences in Malpighian tubule adaptation to host plant defenses in these insects, it does not involve OARalpha. In addition, experiments measuring expression of each of the OAR genes in individual tissues of T. ni (Fig. 3) showed that the expression of OARalpha in the Malpighian tubules is much lower than that of OARbeta1, which seems to be the dominant receptor in this tissue. A different pattern was seen in the oviduct, where expression was more similar in T. ni and P. rapae than in P. unipuncta. In Drosophila the alpha adrenergic-like receptor (OAMB) is required for ovulation and female fecundity [25] and the moderate expression level in the oviducts of T. ni and P. rapae females would support the role of OARalpha in ovulation of these two species. If ovulation in P. unipuncta is mediated by octopamine receptors, it would probably involve different receptor types. One possible explanation for the relative difference in oviduct expression in P. unipuncta compared with the other two species may lie in differences in egg laying, as T. ni and P. rapae females lay eggs singly while P. unipuncta does so in clusters. OARalpha gene expression was widely distributed in the remaining tissues of T. ni. This contrasts with the other OARs, which were each typically expressed in specific tissues. The highest expression of the OARalpha gene appears in the muscle, which suggests that OARalpha may play a role in octopaminergic modulation of muscle, possibly in muscle tension and relaxation as is seen in other insects [35]. Besides the direct modulation of skeletal and visceral (e.g. oviduct) muscle, octopamine also acts on the central nervous system to alter locomotory patterns [15]. Whether this explains why the OARalpha receptor is also expressed highly in the nervous system needs to be the subject of further research. Moderate levels of expression of the OARalpha gene were also found in the fat body, integument and hindgut/rectum samples, though in all these tissues OARbeta2 was seen to have a much higher level of expression (Fig. 3). 4.2. Relative expression of the OA/TA receptor gene Relative OA/TA receptor transcript levels were similar in the three insect species, with significant expression only being observed in the nervous system, but at this time it is difficult to say which neurological processes are involved. Studies using the Drosophila honoka mutant (reduced OA/TA receptor expression) found that the receptor played a role in olfactory sensory input and neuromuscular modulation [20,31]. The OA/TA receptor had been found in the Malpighian tubules of Drosophila [6] but we did

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not find it expressed in this tissue in any of the lepidopterans we studied. This could be due to the specific diet-related role of the receptor; in the case of Drosophila, tyramine from the bacteria in the rotting-fruit diet is presumed to stimulate diuresis through the OA/TA receptor. Furthermore, fermentation by yeast produces ethanol in the diet, potentially causing an osmotic imbalance that necessitates specific actions of the Malpighian tubules. Such roles would not be required in the lepidopterans under consideration in this study. The lack of OA/TA receptor gene expression in the muscle of T. ni was also unexpected given the evidence for its tyraminergic control (Fig. 2). The Drosophila mutant, honoka (with reduced OA/TA receptor expression), showed a reduced effect of tyramine on the excitatory junctional potential amplitude in the larval body wall muscle [20,31]. It appears from our results that if this effect occurs in the lepidopteran species examined, it would be achieved via the central nervous system and not through direct modulation of the muscle. Any effects of direct tyramine application on muscle are perhaps mediated by the tyramine-specific [8], instead of OA/TA, receptors. Differences in larval locomotion between lepidopterans and dipterans may also be important in the expression data differences observed in OA/TA muscle modulation. Caterpillars have three sets of legs, as well as several prolegs, while Drosophila larvae are legless and move by peristaltic movements followed by turning and head swinging (exploratory) behavior [34]. The OA/TA receptor is found in a wide range of tissues in other insects [6,30,40]. For example, in Locusta migratoria, RT-PCR and northern blotting revealed that this gene is expressed in the midgut, oviduct and nervous system [30], but it did appear that the expression is much higher in the nervous system than in the other tissues, which is consistent with our findings. The biological relevance of this low level of expression in non-nervous tissues is uncertain, however, given the similarity of the relative expression levels of the midgut and oviduct in T. ni to other tissues (such as the integument) for which no documented octopaminergic or tyraminergic control has been found, it appears that the OA/TA receptor in lepidopteran larvae is primarily located in the central nervous system. There remain many adult tissues to be assayed for the presence of the OA/TA receptor, and further study is needed in order to determine the extent of its distribution and the roles that it might play. 4.3. Relative expression of OARbeta genes Little is known regarding the expression of beta adrenergic-like receptors in insects, except that they are expressed at much higher levels in the head than the body of Drosophila adults [13]. In this study we observed that the expression of T. ni OARbeta1 receptor transcript was more than three-fold higher in the Malpighian tubules than in the nervous system and oviduct, a pattern that is also seen in P. unipuncta and P. rapae. Control of fluid secretion in Malpighian tubules by octopamine, and in some cases other biogenic amines, has been well documented in many insect species, increasing fluid secretion rates in the worker ant, Formica polyctena [12]; the house cricket, Acheta domesticus [11]; and the tobacco budworm, H. virescens [10]. In these studies, the diuretic effects of octopamine were compared with whole head extracts, with octopamine alone resulting in lower fluid secretion rates. Furthermore, the apparent mechanism of the effect also appeared to be different, as octopamine stimulated secretion in both starved and well fed larvae, while head extracts did not affect the feeding larvae, leading Chung and Keeley [10] to conclude that in the tobacco budmoth larvae biogenic amines mediated a stress-related diuretic effect. However, a stress-related effect was not seen in adults of the citrus swallowtail butterfly, Papilio demodocus [32]. This could be due to the fact that different stages of the life cycles were used

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in the two studies, and it is known that unlike adult tubules, the tubules of lepidopteran larvae exhibit high endogenous secretory activity and have no need for diuretic stimulation [10]. Still another study revealed the lack of octopaminergic action on the Malpighian tubules of the mosquito, Aedes aegypti [39], further highlighting the fact that octopaminergic control of Malpighian tubules is not uniform among insects. A high relative expression of the OARbeta1 receptor gene, the most highly expressed OAR in the T. ni Malpighian tubule when all genes are compared directly (Fig. 3), seen in all three of our test species indicates that this receptor is a good candidate for octopaminergic control in this tissue of lepidopteran larvae, rather than the OA/TA receptor that has been reported in other insects [6]. Given that this receptor gene is absent in B. mori [9], it is interesting to find it so highly expressed in the Malpighian tubules of these other lepidopterans. The experiments measuring relative T. ni OAR gene expression within each tissue suggest that OARbeta2 gene expression is almost ubiquitous in T. ni tissues. OARbeta2 had by far the highest relative expression in every tissue except the Malpighian tubules, in which OARbeta1 is the highest expressed OAR, and the midgut, where there was no significant difference between OAR gene expression levels. The experiments measuring expression of each gene in different tissues revealed that the T. ni OARbeta2 transcript was expressed highest in the oviduct, rather than in the head as reported for Drosophila. This suggests that the T. ni oviduct is potentially under the modulation of at least two different octopamine receptors: OARalpha and OARbeta2, though the expression of the latter is much higher. However, an examination of P. unipuncta and P. rapae failed to provide any clarity as OARbeta2 expression in the former appeared to be largely confined to the nervous system, and in the latter appeared high in both the nervous system and Malpighian tubules when compared to the oviduct. Furthermore, although locust oviducts treated with octopamine showed increased cAMP levels [22] this effect can be blocked with phentolamine, which is not an antagonist for beta adrenergic-like receptors and is in fact an agonist [9,27]. Further study is needed to determine if the high relative expression of OARbeta2 in T. ni oviduct is an artifact, or if it serves in a different way than reported for other insects. The high relative expression of OARbeta2 in the integument is surprising, given that we were unable to find any published demonstration of octopaminergic control of insect integument tissue. There is documented monoaminergic control in the cuticle of the blood-feeding insect Rhodnius prolixus [3,33], and the tick, Amblyomma hebraeum [19] involved in plasticization, a process which allows the cuticle to expand to accommodate a large blood meal. However, this is not something that would be required for the insects examined in this study. It is possible that octopamine could play a role in the plasticization of the cuticle during ecdysis, and further study is required to verify this proposition. As for the remaining tissues, octopamine plays a modulatory role on hindgut contractions, though the role may vary with species: increasing amplitude, but not frequency in L. migratoria [18], and increasing frequency, but not amplitude in A. aegypti [29]. Also, as mentioned previously, octopamine plays a role in lipid mobilization in fat body [2] and OARbeta2 expression in this tissue would likely be related to lipid mobilization and energy release. Finally, the relative expression levels for T. ni and P. unipuncta OARbeta3 were low in all tissues except the nervous system (Figs. 2 and 5). Of the three beta adrenergic-like receptors, this is the only one whose expression pattern agrees with that seen in Drosophila. More OARbeta3 expression was observed outside the nervous system of P. rapae, but the differences were statistically insignificant, so additional research is required to determine if this pattern is real or not.

4.4. Tissues lacking OAR gene expression Surprisingly, no significant octopamine receptor gene expression was measured in P. unipuncta oviduct. It seems improbable that this is due to degradation as all three reference genes were expressed highly. The reproductive physiology of P. unipuncta is known to be highly dependent on environmental conditions such as temperature and photoperiodic conditions [28], so perhaps the low relative levels of octopamine receptor gene expression in the oviduct are related to rearing conditions. It would be interesting to determine whether or not ovulation in P. unipuncta is modulated by octopamine as in other insects, or if the low relative level of expression is in fact biologically relevant. The control of the locust oviduct by biogenic amines has been studied extensively with the conclusion that octopamine plays a very prominent role in the control of oviduct muscle contractions (for a review see: [24]). The studies also found that tyramine mimics many of the effects of octopamine and may be an important agent of oviduct control on its own. This mimicry suggests that both tyramine and octopamine act upon the same receptor: the octopamine/tyramine receptor. The absence of a high relative expression level of the octopamine/tyramine receptor in any of the species examined here might indicate that oviduct control is achieved in a different manner in the Lepidoptera. OARalpha appears to be a good candidate for oviduct modulation in T. ni and P. rapae as it appears to be expressed in both species, although OARbeta2 is very highly expressed in T. ni (Fig. 2) and would appear to be involved as well. The low relative expression levels of each of the OAR transcripts in the midgut of the three species was unexpected given that there is evidence for octopaminergic control of both long-lasting contractions and spontaneous rhythmic contractions of the midgut in Carausius morosus [26]. However, the latter effect was only seen in midgut preparations that included the ingluvial (ventricular) ganglion, indicating that the octopaminergic control is achieved neuronally rather than through midgut receptors. The former effect, consisting of a single long-lasting contraction, might suggest the presence of octopamine receptors in the midgut, but it was only seen at high doses. Furthermore, the effect was also elicited by serotonin at much lower concentrations than octopamine. In addition, dopamine, noradrenaline and acetylcholine also caused a contraction at high concentrations, leading the authors to suggest that the effect might be a non-specific reaction to high, non-physiological doses. Our experiments to determine the relative expression levels of OARs in the midgut did not suggest a candidate for midgut octopaminergic control, as the generally low level of expression of each gene resulted in high biological variation.

5. Conclusions We found representatives of all five OAR types in three lepidopteran species. These receptors showed expression patterns when measured in various tissues that are potentially related to their specific physiological roles. In particular, the OARbeta2 gene appears to be highly expressed in most major tissues of T. ni, and OARbeta1 seems to play a role in Malpighian tubule modulation. Future work, particularly involving in situ hybridization should help to determine the cellular locations of these receptors and reveal specific cell types and regions within tissues in which the receptors play a role. Additionally, immunohistochemical experiments to determine the cellular locations of the receptor proteins themselves would be useful in further understanding octopamine signaling. A comprehensive picture of the various octopamine receptors present in different tissues will help to determine their specific roles, alone or in combination, in the control of insect physiological processes.

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Acknowledgements The authors would like to thank Ida van Grinsven for DNA sequencing. This work was funded by an A-base grant from Agriculture and Agri-Food Canada (RBPI 1840). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.peptides.2012.03.034. References [1] Agrawal AA, Kurashige NS. A role for isothiocyanates in plant resistance against the specialist herbivore Pieris rapae. J Chem Ecol 2003;29:1403–15. [2] Arrese E, Soulages J. Insect fat body: energy, metabolism, and regulation. Annu Rev Entomol 2010;55:207–25. [3] Barrett FM, Orchard I, Tebrugge V. Characteristics of serotonin-induced cyclic AMP elevation in the integument and anterior midgut of the blood-feeding bug, Rhodnius prolixus. J Insect Physiol 1992;39:581–7. [4] Blenau W. Cellular actions of biogenic amines. Arch Insect Biochem Physiol 2005;59:99–102. [5] Blenau W, Baumann A. Molecular and pharmacological properties of insect biogenic amine receptors; lessons from Drosophila melanogaster and Apis mellifera. Arch Insect Biochem Physiol 2001;48:13–38. [6] Blumenthal EM. Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule. Am J Physiol Cell Physiol 2003;284:C712–28. [7] Caveney S, Cladman W, Verellen L, Donly C. Ancestry of neuronal monoamine transporters in the Metazoa. J Exp Biol 2006;209:4858–68. [8] Cazzamali G, Klaerke DA, Grimmelikhuijzen CJP. A new family of insect tyramine receptors. Biochem Biophys Res Commun 2005;338:1189–96. [9] Chen X, Ohta H, Ozoe F, Miyazawa K, Huang J, Ozoe Y. Functional and pharmacological characterization of a beta-adrenergic-like octopamine receptor from the silkworm Bombyx mori. Insect Biochem Mol Biol 2010;40:476–86. [10] Chung J, Keeley LL. Evidence and bioassay for diuretic factors in the nervous system of larval Heliothis virescens. J Comp Physiol B 1989;159:359–70. [11] Coast GM. Stimulation of fluid secretion by single isolated Malpighian tubules of the house cricket, Acheta domesticus. Physiol Entomol 1989;14:21–30. [12] De Decker N, Hayes TK, Van Kerkhove E, Steels P. Stimulatory and inhibitory effects of endogenous factors in head extracts of Formica polyctena (Hymenoptera) on the fluid secretion of Malpighian tubules. J Insect Physiol 1994;40:1025–36. [13] Evans PD, Maqueira B. Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors. Invert Neurosci 2005;5:111–8. [14] Farooqui T. Octopamine-mediated neuromodulation of insect senses. Neurochem Res 2007;32:1511–29. [15] Fox LE, Soll DR, Wu CF. Coordination and modulation of locomotion pattern generators in Drosophila larvae: effects of altered biogenic amine levels by the tyramine beta hydroxylase mutation. J Neurosci 2006;26:1486–98. [16] Guy RH, Leppla NC, Rye JR, Green CW, Barrette SL, Hollien KA. Trichoplusia ni. In: Singh P, Moore RF, editors. Handbook of Insect Rearing II. Amsterdam: Elsevier; 1985. p. 487–94. [17] Hellemans J, Mortier G, De Paepe A, Speleman F, Vadesomplele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR. Genome Biol 2007;8. R19.1-R19.14.

73

[18] Huddart H, Oldfield AC. Spontaneous activity of foregut and hindgut visceral muscle of the locust, Locusta migratoria – II. The effect of biogenic amines. Comp Biochem Physiol 1982;73C:303–11. [19] Kaufman WR, Flynn PC, Reynolds SE. Cuticular plasticization in the tick, Amblyomma hebraeum (Acari: Ixodidae): possible roles of monoamines and cuticular pH. J Exp Biol 2010;213:2820–31. [20] Kutsukake M, Komatsu A, Yamamoto D, Ishiwa-Chigusa S. A tyramine receptor gene mutation causes a defective olfactory behaviour in Drosophila melanogaster. Gene 2000;245:31–42. [21] Labbe R, Caveney S, Donly C. Expression of multidrug resistance proteins is localized principally to the Malpighian tubules in larvae of the cabbage looper moth, Trichoplusia ni. J Exp Biol 2011;214:937–44. [22] Lange AB, Orchard I. Identified octopaminergic neurons modulate contractions of locust visceral muscle via adenosine 3 ,5 -monophosphate (cyclic AMP). Brain Res 1986;363:340–9. [23] Lange AB. Tyramine: from octopamine precursor to neuroactive chemical in insects. Gen Comp Endocrinol 2009;162:18–26. [24] Lange AB. The female reproductive system and control of oviposition in Locusta migratoria migratorioides. Can J Zool 2009;87:649–61. [25] Lee H, Seong C, Kim Y, Davis RL, Han K. Octopamine receptor OAMB is required for ovulation in Drosophila melanogaster. Dev Biol 2003;264:179–90. [26] Luffy D, Dorn A. Immunohistochemical demonstration in the stomatogastric nervous system and effects of putative neurotransmitters on the motility of the isolated midgut of the stick insect, Carausius morosus. J Insect Physiol 1992;38:287–99. [27] Maqueira B, Chatwin H, Evans PD. Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors. J Neurochem 2005;94:547–60. [28] McNeil JN, Tobe SS. Flights of fancy: possible roles of allatostatin and allatotropin in migration and reproductive success of Pseudaletia unipuncta. Peptides 2001;22:271–7. [29] Messer AC, Brown MR. Non-linear dynamics of neurochemical modulation of mosquito oviduct and hindgut contractions. J Exp Biol 1995;198:2325–36. [30] Molaei G, Paluzzi JP, Bendena WG, Lange AB. Isolation, cloning, and tissue expression of a putative octopamine/tyramine receptor from locust visceral muscle tissues. Arch Insect Biochem Physiol 2005;59:132–49. [31] Nagaya Y, Kutsukake M, Chigusa SI, Komatsu A. A trace amine, tyramine, functions as a neuromodulator in Drosophila melanogaster. Neurosci Lett 2002;329:324–8. [32] Nicolson SW, Millar RP. Effects of biogenic amines and hormones on butterfly Malpighian tubules: dopamine stimulates fluid secretion. J Insect Physiol 1983;29:611–5. [33] Orchard I, Lange AB, Barrett FM. Serotonergic supply to the epidermis of Rhodnius prolixus: evidence for serotonin as the plasticising factor. J Insect Physiol 1988;34:873–9. [34] Rodriguez Moncalvo VG, Campos AR. Role of serotonergic neurons in the Drosophila larval response to light. BMC Neurosci 2009;10:66. [35] Roeder T. Tyramine and octopamine: ruling behaviour and metabolism. Annu Rev Entomol 2005;50:447–77. [36] Shorey HH, Hale RC. Mass rearing of the larvae of nine Noctuid species on a simple artificial medium. J Econ Entomol 1965;58:522–4. [37] Stanisc¸uaski F, Te Brugge V, Calini CR, Orchard I. In vitro effect of Canavalia ensiformis ureases and the derived peptide Jaburetox-2Ec on Rhodnius prolixus Malpighian tubules. J Insect Physiol 2009;55:255–63. [38] Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3:7. [39] Veenstra JA. Effects of 5-hydroxytryptamine on the Malpighian tubules of Aedes aegypti. J Insect Physiol 1988;34:299–304. [40] von Nicksch-Resenegk E, Krieger J, Kubick S, Laage R, Strobel J, Strotmann J, et al. Cloning of biogenic amine receptors from moths (Bombyx mori and Heliothis virescens). Insect Biochem Mol Biol 1996;26:817–27.