The Detection and Quantitative Analysis of Endocannabinoids and Endogenous Fatty

University of South Florida Scholar Commons

Graduate Theses and Dissertations Graduate School

3-16-2015 The etD ection and Quantitative Analysis of Endocannabinoids and Endogenous Fatty Acid Amides in Apis Mellifera and Tribolium Castaneum Perry Robert Mitchell Jr. University of South Florida, [email protected]

Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Organic Chemistry Commons

Scholar Commons Citation Mitchell, Perry Robert Jr., "The eD tection and Quantitative Analysis of Endocannabinoids and Endogenous Fatty Acid Amides in Apis Mellifera and Tribolium Castaneum" (2015). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/5538

This Thesis is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected].

The Detection and Quantitative Analysis of Endocannabinoids and Endogenous Fatty

Acid Amides in Apis mellifera and Tribolium castaneum

Perry (PJ) Robert Mitchell, Jr.

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida

Major Professor: Edward Turos, Ph.D. Robert Potter, Ph.D. James Leahy, Ph.D.

Data of Approval: March 16, 2015

Keywords: cannabinoids, mass spectroscopy, N-acyldopamines, anandamide, honeybees, red flour beetles

DEDICATION

“Science is much more than a body of knowledge. It is a way of thinking.”

Carl Sagan, 1990.

To my step-mother, Kathy Mitchell, for teaching me how to finally tie my shoes. To my aunt Bessy Mitchell, who told me to “get off your a**.” To my surrogate mother and grandmother Karen Heidenreich and Charlotte Miller, for the years of support and giving me a family. To my surrogate brother David Walters, for jokingly “being the brother I’m glad I never had.” To my close friends: Tim

Holder, Sean Reed, Allyson and Scott Speizer, Stewart Sandler, Tim/Tiffany and

Stella Halbert, Billy Harrison and Susana S. Jacobs, for putting up with me for so long and still caring. To my brother Brian Northcutt, for continuing to hang in there.

In memory of Carl Sagan and Gene Roddenberry, whose inspiration and great works have made me the science nerd that I am today and always will be.

Ad astra, per ardua.

ACKNOWLEDGEMENTS

I would like to express my deepest and sincerest gratitude to my current and previous research advisor, Dr. Edward Turos and Dr. David Merkler, for the guidance, support and opportunity to allow me to conduct this research in completion of this work. I would like to thank my committee members for taking the time and energy to support me, Dr. James Leahy (for the inspiration) and

Associate Dean Dr. Robert Potter (for giving me a chance).

I would also like to thank my fellow graduate students and colleagues

Frankie Costanza and Mohammed Nassir Amin for their insight and friendship;

Daniel Dempsey, Kristen Jeffries and Matthew Battistini for the challenges and direct and indirect lessons they have taught me. To my students, who shoes I will always share and whom I hope to guide through the trials and tribulations of science. To Adrienne McCain, for all of her support and laughter. I would like to also thank the University of South Florida and the Department of Chemistry for the continued financial and institutional support, especially the USF Foundation for bestowing upon me the Graduate Fellowship in Diversity, for without which this would not have come to fruition.

Lastly, I would especially like to thank Dr. Pamela Epps, and those whom I have dedicated this work to, for helping me keep it all together. Without you all I would not have made it this far.

TABLE OF CONTENTS

List of Tables iii

List of Figures iv

Abstract vii

Chapter 1: Introduction, Background and Current Review of Endocannabinoids/Fatty Acid Amides 1 1.1 Introduction to Endocannabinoids (eCBs) and Fatty Acid Amides (FAAs) 1 1.2 Anandamide and Other NAEs 4 1.3 N-Acyldopamines: Physiological Role, Biosynthesis and Degradation 8 1.3.a Physiological Roles 9 1.3.b Biosynthesis and Degradation 14 1.4 Conclusions 18 1.5 References 18

Chapter 2: Detection and Quantification of Endogenous Endocannabinoids /Fatty Acid Amides in A. mellifera (honeybees) and T. Castaneum (red flour beetles) 33 2.1 Introduction and Background 33 2.2 Experimental Procedures and Methods 39 2.2.a A. mellifera (honeybee) Acquisition and Processing 39 2.2.b T. Castaneum (red flour beetle) Acquisition and Processing 41 2.2.c General Extraction Method 42 2.2.d General Purification Method 44 2.2.e Preparation of Standards/Standard Curves and Blanks for Carry-Over and Slip Additives 45 2.2.f HPLC-QTOF-MS and MS/MS Sample Analysis 46 2.2.g Sample Data Analysis – Qualitative Identification 49 2.2.h Sample Data Analysis – Quantitative Identification 52 2.2.i Sequence Homology with D. melanogaster AANAT 54 2.3 Results and Discussion 55 2.3.a Standard Samples/Curves & Spiked Samples in A. mellifera 55 2.3.b Endogenous Results – Farmed & Feral A. mellifera 61

2.3.c Endogenous Results – T. castaneum 72 2.3.d Genetic Homology Study of AANAT between D. Melanogaster , A. mellifera and T. Castanaeum 79 2.4 Conclusions and Recommendations 81 2.5 References 84

Appendix 88

LIST OF TABLES

Table 1.1: Families of endocannabinoids/fatty acid amides, major acyl forms and known endogenous organisms 4

Table 1.2: The major eCB/FAA groups and known receptors with biological effects 5

Table 2.1: Select endocannabinoids/fatty acid amides to be identified and quantified in A. mellifera and T. castaneum 40

Table 2.2: Summary of Standard Molecules Used for Standard Curves 56

Table 2.3: Summary of eCB/FAA Quantitative Results in Farmed A. mellifera 61

Table 2.4: Summary of eCB/FAA Quantitative Results in Feral A. mellifera 61

Table 2.5: Summary of eCB/FAA Quantitative Results in T. castaneum 72

iii

LIST OF FIGURES

Figure 1.1: Major NAE Derivatives, incl. anandamide (AEA) 3

Figure 1.2: The Four Major N-Acyldopamine eCB Derivatives 8

Figure 1.3: Proposed L-DOPA/Dopamine-Dependent NAD Biosynthetic Pathways 17

Figure 1.4: Proposed Tyramine/Tyrosine-Dependent NAD Biosynthetic Pathways 19

Figure 1.5: Proposed NAD Degradation Pathways 20

Figure 2.1: AANAT Sequence Homology between D. melanogaster, A. mellifera and T. castaneum 38

Figure 2.2: Select Routes of N-acyldopamine Oxidation 39

Figure 2.3: Example Data Set with Example Calculation 54

Figure 2.4: Example Standard Curve/MS Results for D8-NAG 59

Figure 2.5: Spiked OEA in A. mellifera Thorax 60

Figure 2.6: Aggregate Spectra/MS Results of Endogenous OEA and NPD in farmed A. mellifera 67

Figure 2.7: Aggregate Spectra/MS Results of Endogenous NPG and Palmitamide in farmed A. mellifera 68

Figure 2.8: Aggregate Spectra/MS Results of Endogenous NOSt in both Farmed and Feral A. mellifera 69

Figure 2.9: Targeted MS/MS Spectra of Standard AEA 70

Figure 2.10: Targeted MS/MS Spectra of Endog. AEA and Spiked OEA in A. mellifera 71

Figure 2.11: Aggregate Spectra/MS of Endogenous OEA and NOD in T. castaneum 76

Figure 2.12: Aggregate Spectra/MS of Endogenous NOG and Palmitamide in T. castaneum 77

Figure 2.13: Targeted MS/MS Spectra of Endogenous AEA in T. castaneum 78

Figure 2.14: Protein Sequence and Motif Homology of AANATs 80

Figure 2.15 Proposed Key Step in the Biosynthesis of an N-acyldopamine eCB/FAA 82

Figure A.1 Standard Curves and Standard MS results for N-arachidonylethanolamine, AEA (anandamide) 89

Figure A.2 Standard Curves and Standar d MS results for N-oleoylethanolamine, OEA 90

Figure A.3 Standard Curve and Standard MS Results for N-palmitoyldopamine, NPD 91

Figure A.4 Standard Curve and Standard MS Results for N-oleoyldopamine, NOD 92

Figure A.5 Standard Curve and Standard MS Results for N-arachidonyldopamine, NAD 93

Figure A.6 Standard Curve and Standard MS Results for N-palmitoylglycine, NPG 94

Figure A.7 Standard Curve and Standard MS Results for N-oleoylglycine, NOG 95

Figure A.8 Standard Curve and Standard MS Results for N-arachidonylglycine, NAG 96

Figure A.9 Standard Curve and Standard MS Results for the N-oleoylserotonin, NOSt 97

Figure A.10 Standard MS Results of primary fatty acid amides linoleamide, LOA, and palmitoleamide, POA 98

Figure A.11 Standard MS Results of primary fatty acid amides Oleamide, OA, and Palmitamide, PA 99

Figure A.12 Spiked AEA, Anandamide, in A. mellifera thorax 100

Figure A.13 Spiked NAD, N-arachidonyldopamine, in A. mellifera thorax 101

Figure A.14 Spiked NOG, N-oleoylglycine, in A. mellifera thorax 102

Figure A.15 Aggregate spectra and MS results of N-oleoyldopamine, NOD, in farmed A. Mellifera 103

Figure A.16 Aggregate spectra and MS of N-acylglycines NPG and NOG in farmed A. Mellifera 104

Figure A.17 Aggregate spectra and MS of N-acyl ethanolamines OEA and AEA in feral A. Mellifera 105

Figure A.18 Aggregate spectra and MS of N-acyldopamines NPD and NOD in feral A. Mellifera 106

Figure A.19 Aggregate spectra and MS of N-acylglycines NPG, NOG and NAG in feral A. Mellifera 107

Figure A.20 Aggregate spectra and MS of endogenous primary fatty acid Amides LOA and POA in feral A. Mellifera 108

Figure A.21 Aggregate spectra and MS of endogenous primary fatty acid amides OA and PA in feral A. Mellifera 109

Figure A.22 Aggregate spectra and MS of endogenous ethanolamine AEA in T. Castaneum 110

Figure A.23 Aggregate spectra and MS of endogenous acyl dopamines, NPD,NOD, and NAD, in T. Castaneum 111

Figure A.24 Aggregate spectra and MS of endogenous acyl glycines, NPG, NOG and NAG, in T. Castaneum 112

Figure A.25 Aggregate spectra and MS of endogenous primary fatty acid amides, POA, OA and PA, in T. Castaneum 113

ABSTRACT

Endocannabinoids, and the fatty acid amides from which they are a member,

have garnered greater scientific interest in the last two decades due to their

cannabimimetic properties . The most well-known of these is anandamide, which has

thus far been discovered in several species of animal ranging from C. elegans , fruit

flies, to bovine and humans . Because of the importance and increasing impact of

these compounds a brief overview is presented herein, with a major focus on the N-

acyldopamines due to the direct impact they potentially pose to human physiology .

Secondly, the detection and quantitative analysis of these molecules was

conducted in the recently fully genome sequenced honeybee and red flour beetle,

due in part to recent research showing the existence of these molecules in D.

melanogaster, to which no known cannabinoid receptors had been found to date .

Interest in these potentially new model organisms may provide additional insight

not only into the endocannabinoids but also as potential targets for protection of

honeybees and pest control of red flour beetles .

Utilizing established HPLC-MS methods for the detection and quantification of

these compounds provided a series of endogenous results for these molecules

within both farmed and feral honeybees and the red flour beetle . Additionally, a

protein sequence and motif homology study with a newly discovered

acyltransferase from Fruit flies shows strong evidence that a similar enzyme is

expressed in both honeybees and red flour beetles . Therefore providing future steps for the continuation of this research to better elucidate and quantify the endocannabinoids as well as determine the biosynthetic metabolism within these organisms .

vii

CHAPTER 1:

INTRODUCTION, BACKGROUND AND CURRENT REVIEW OF ENDOCANNABINOIDS/FATTY ACID AMIDES

1.1 Introduction to Endocannabinoids (eCBs) and Fatty Acid Amides (FAAs)

The detection of biological fatty acid amides, and their metabolites, dates

back to the initial discovery in horse urine by Justus von Liebig in 1829. (1) The

biological role of these bioactive lipids are still of importance within the biochemical

and medical fields and have been related to physiological conditions ranging from

diabetes and renal function to autism and infections. (2) Long-chain fatty acid

amides (FAAs), in which the long-chain primary fatty acid acyl group is the defining

characteristic, were first extracted and characterized starting in 1957. (3) Several

classes of FAAs are now currently known, including derivatives of important

biologically and neurologically-active compounds such as N-acylethanolamines and

N-acyldopamines (Table 1.1). (4)

Endocannabinoids (eCBs) are defined as endogenous cannabimimetic FAA- derived molecules that illicit physiological effects similar to those of the active molecules present in marijuana and similar cannabis plants, Δ 9-

tetrahydrocannabinol and cannabidiol, from which the term is derived. (5)

Additionally, endocannabinoids are structurally characterized as lipid mediators with

either long-chain saturated, unsaturated or polyunsaturated fatty acid moieties.

Like the related FAAs, eCBs can be classified as amide, ester or ether derivatives.

The detection of N-arachidonylethanolamine in 1992 in the porcine brain marked the initial discovery of a fatty acid amide compound shown to produce endocannabic effects (commonly known as anandamide, from the ancient Sanskrit word for “bliss”). (6) Anandamide (AEA) is thus considered the prototypical endocannabinoid.

Over the last two decades several additional endogenous eCB/FAA derivatives have been discovered in organisms ranging from C. elegans (7), fruit

flies (8), reptiles (9) bovine (10), sheep (11), mice (10), rats (12, 13) and humans

(14, 15) to even fungi (16). Additionally, there is evidence to suggest that eCBs are

also present in chocolate. (17) A select group of relevant eCB/FAA derivatives and

known N-acyl moieties are presented in Table 1.2.

Ongoing research into the endogenous biosynthesis and function of these

molecules continues to show their primary location in brain or central nervous

system tissue, such as the substantia nigra (15), specific neuronal cells (10),

cerebrospinal fluid (17), choroid plexus (10) and blood serum (19). The early

research into the function, potential receptor and bioactivity of anandamide used

extracted rat synaptic tissue (6) and live mice (18). It was the initial study by

Devane that suggested the potential receptor for AEA as being cannabinoids in vivo .

(6)

The primary receptors which have been extensively studied with eCBs as

potential ligands include mammalian cannabinoid receptors, CB 1/CB 2, and the transient receptor potential vanilloid type 1 channel, TRPV1. (5) Other research has shown that eCBs may also act as ligands to receptor proteins such as G-protein-

coupled receptor 119 (GPR119) and peroxisome proliferator-activated receptor

gamma and alpha (PPARγ/PPARα). (19, 20)

Though the exact function of these receptors is not completely known

numerous studies suggest several notable physiological effects including the

modulation of pain perception, sleep induction, body temperature, locomotion,

appetite and even fear and anxiety (Table 1.2). (21) It is primarily due to these

effects that the eCBs/FAAs are an ever-increasing research area of interest, both as

potential therapeutic targets and to better understand the physiology involved.

1.2 Anandamide and Other NAEs

Figure 1.1: Major NAE Derivatives, incl. anandamide (AEA).

As stated, anandamide is considered the prototypical endocannabinoid not only because it was the first discovered within the eCB metabolome but also due to the high research interest it garnered by its physiological roles to date, those roles being cannabimimetic in nature, and is only one of several within the N- acylethanolamine (NAE) family (Fig. 1.1).

Table 1.1: Families of endocannabinoids/fatty acid amides, major acyl forms and known endogenous organisms. R 2 represents the amino acid group.

Endogenous eCB/FAA Class Major N-acyl Moieties, R 1 Organisms

C. elegans Fruit Fly Palmitoyl (C14:0) Reptiles Oleoyl (C18:1 cis -Δ9) Rodents Arachidonoyl (C20:4 cis-Δ5,8,11,14 ) Cows & Pigs Cats & Rabbits N-Acylethanolamine (NAE) Human

Fruit Fly Palmitoyl (C14:0) Rodents Oleoyl (C18:1 cis -Δ9) Cows & Pigs Arachidonoyl (C20:4 cis-Δ5,8,11,14 ) Cats & Rabbits N-Acylamino acid (NAA) Human

Fruit Fly Palmitoyl (C14:0) Rodents Oleoyl (C18:1 cis -Δ9) Cows Arachidonoyl (C20:4 cis-Δ5,8,11,14 ) Human N-Acyldopamine (NAD)

Palmitoyl (C14:0) C. elegans Palmitoleoyl (C16:1 cis -Δ9) Fruit Fly 9 Oleoyl (C18:1 cis -Δ ) Rodents Linoleoyl (C18:2 cis,cis -Δ9,12 ) Cows & Pigs Primary Fatty Acid Amide Eicosenoyl (C20:1 cis -Δ13 ) Cats & Rabbits (PFAA) Arachidonoyl (C20:4 cis-Δ5,8,11,14 ) Human

Table 1.2: The major eCB/FAA groups and known receptors with biological effects Sleep, Pain, Anandamide N-Acylethanolamine CB /CB Body N-Oleoylethanolamine 1 2 (NAE) TRPV1 Temperature, N-Palmitoylethanolamine PPARγ/PPARα Appetite &

Anxiety

Pain, Body N-Acylamino acid N-Arachidonoylglycine CB /CB 1 2 Temperature & (NAA) N-Arachidonoyltaurine TRPV1 Anti- PPARγ/PPARα inflammatory

N-Acyldopamine N-Arachidonoyldopamine CB /CB 1 2 Sleep, Pain & (NAD) N-Oleoyldopamine TRPV1 Anxiety N-Palmitoyldopamine PPARγ/PPARα

Sleep, Pain, Eicosenoamide CB Primary Fatty Acid 1 Body Linoleamide PPAR Amide (PFAA) Temperature, Oleamide 5-HT 1A/2A Appetite & Palmitamide Anxiety

Fride and Mechoulum, colleagues of Devane, were the first to provide an in vivo analysis of the effects of anandamide within a mammalian organism (mice).

(18) In general, cannabis’ effects on mammals are dose-dependent, and thus range from simultaneous depression and stimulation at low doses to more extensive central depression at high doses. Using a classic tetrad series of behavioral tests with anandamide and Δ 8-THC for these cannabic effects it was shown that the two compounds produced remarkable similarity in the expected effects from the majority of test animals. Examples of these tests included antinociception

(decreased pain sensation), measured hypothermia and physical activity. (22) Such research also concluded that, as cannabic effects occur in the central nervous

system, anandamide readily crosses the blood-brain barrier and suggested possible

cellular transporters specific to these compounds.

As a result of the early research into the in vivo effects of anandamide it was

clear that specific receptors existed for these molecules. Receptors for the active

psychotropics in Cannabis had already been known, the cannabinoid receptors 1

and 2 (CB 1/CB 2); and the results by Fride and Mechoulom lent clear evidence that

anandamide and other endocannabinoids were endogenous ligands to these

receptors. (23, 24) What was still unclear at that time was the biological purpose

and biosynthesis of molecules like anandamide. Interestingly enough anandamide is

one of the least abundant of the NAEs in the mammalian brain, consisting less than

10% of the total endogenous amounts to date of NAEs known. Several other NAEs

were since detected and measured within various organisms since the initial

discovery by Devane et al., including N-palmitoylethanolamine (PEA), N- oleoylethanolamine (OEA) and various others ranging from N-stearoylethanolamine

(SEA) and N-linoleoylethanolamine (LEA) to N-dihomo-γ-linoleoylethanolamine

(DLEA) and N-docosatetraenoylethanolamine (DEA). (21)

In addition to the cannabinoid receptors, other receptors were discovered to be paired to endocannabinoids such as the transient receptor vanilloid type 1

(TRPV1), transient receptor potential of melastatin 8 (TRPM8), G-coupled-protein receptor 119 and peroxisome proliferator-activated receptors alpha and gamma

(PPARα,γ). (25, 26, 20,27, and 28) For anandamide the binding constants with these receptors vary greatly (K d = 61 – 11,000 nM), with higher affinity for CB 1/CB 2

and lower affinities for PPARγ. (21) A select few NAEs, like SEA, have exhibited

cannabimimetic effects without binding to any known cannabinoid receptors, thus

suggesting the possible existence of a heretofore unknown cannabinoid receptor

(potentially, “CB 3”). (29)

Furthermore, much of the research into the various eCB NAEs like AEA, OEA

and PEA have continued to suggest what has become known as the “entourage

effect,” in which interactions of these molecules can augment substrate-ligand

binding. (30) This effect may explain some of the observed competing activities of

these NAEs for the various receptors, and the large variations in endogenous

amounts, due to the promiscuity of NAEs for the NAE-degradation enzymes known

for anandamide; thus resulting in cellular concentration stabilization. (21, 30, and

31)

Despite the extensive research into the biological nature of anandamide and

the NAEs the biosynthetic and degradation routes for these molecules is complex

with few clear answers thus far. Several articles have provided sound arguments for

several biosynthetic routes, with the most widely accepted among them currently

being the N-acylphosphotidylethanolamine (NAPE) specific phospholipase-D dependent (NAPE-PLD) synthesis. The NAPE-PLD route catalyzes the cleavage of

NAPE into the corresponding NAE and phosphotidic acid. (32) Several additional routes related to these and similar enzymes have been proposed as well. A more thorough discussion can be accessed from Waluk, et al., 2014 (21).

The degradative routes for the NAEs are also equally as complex and varied.

Current literature strongly suggests the use of fatty acid amide hydrolase (FAAH) for the hydrolytic degradation of NAEs to ethanolamine and free fatty acid, while other research also suggests an oxidative pathway to the N-acylated amino acid N- acylglycine via fatty aldehyde dehydrogenase. (33, 34). Several of the metabolites

from these pathways have been implicated in additional bioactivity, suggesting

alternative routes of regulation. (35, 36, and 37) Indeed, research has progressed

with FAAH inhibition as a potential therapeutic route for the treatment of various

neuropathological states such as Alzheimer’s and Parkinson’s diseases. (38)

Recent research into the physiological roles of the NAEs has added to an

ever-increasing list beyond those previously mentioned. Some of these roles include

human reproduction and fertility (39, 40). Continued research also includes the use

of NAEs as anxiolytics and antidepressant therapeutics (41, 42).

1.3 N-Acyldopamines: Physiological Role, Biosynthesis and Degradation

Because of the substantial physiological role that dopamine occupies in

vertebrate species the N-acyldopamine-derived endocannabinoids are especially of interest in the scope of this review.

Figure 1.2: The Four Major N-Acyldopamine eCB Derivatives.

1.3.a. Physiological Role

As first reported by Huang, et al . in 2002, a small number of N- acyldopamines, such as N-arachidonoyldopamine (NADA), have been identified in

mammalian brain tissues, with the highest concentrations within the hippocampus,

striatum and cerabellum . (43) Additionally, NADA was shown to be a potent agonist

(EC 50 ≈ 50 nM) of the nonselective cation channel TRPV1 in both human and rats with similar efficacy to that of capsaicin, a non-endogenous mediator of TRPV1 . The

Huang group described the ability of NADA to induce TRPV1-mediated hyperalgesia

in rats intradermally (EC 50 = 1 .5±0 .3 μg) . Before direct studies of NADs with TRPV1

were conducted, previous research indicated their role in pain mediation as a

molecular transducer of chemical and physical nociceptive stimulus . (44) Shortly

before the endogenous detection of NADs, NADA was found to be a greater CB 1

agonist than anandamide, producing: hypothermia, hypo-locomotion, catalepsy,

and analgesia . It was also found, along with N-pinolenoyldopamine and N-

docosapentaenoyldopamine, to inhibit FAAH activity . (45)

Chu and colleagues of the Huang group had shown the existence of

endogenous levels of additional long-chain N-acyldopamines: N-stearoyldopamine

(STEARDA), N-palmitoyldopamine (PALDA) and N-oleoyldopamine (NOLDA) . (46) In

this study, it was shown that the saturated fatty acyl derivatives STEARDA and

PALDA had no effect on calcium influx or thermal hyperalgesia via the TRPV1

receptor . However, NOLDA, an unsaturated derivative, caused calcium influx and

thermal hyperalgesia via TRPV1 with similar potency to that of NADA (EC 50 ≈ 36 nM

and ≈ 17 nM, respectively) . Unlike NADA, NOLDA was shown to be a weak ligand

for CB 1. Like NADA, NOLDA was shown to be a ligand for the anandamide

membrane transporter but a poor substrate for FAAH (therefore suggesting an

additional route of degradation) . The dichotomy between the saturated and

unsaturated fatty acyl derivatives of N-acyldopamine suggests the requirement of an unsaturated acyl chain for optimal binding and function with TRPV receptors . The

key seminal research by Huang, Chu and colleagues had shown that endogenous N-

acyldopamines are putative substrates to TRPV1 receptors and opened the door to

research in the field of these molecules and their neurological function .

It has also been noted that TRPV1 is the only one in the family of TRPV

receptors that is activated by both endogenous long-chain acyl derivatives and

capsaicin . (47) PALDA and STEARDA were studied as enhancers of the binding of

other NADs to the TRPV1 receptor . It was found that the EC 50 of NADA on the

TRPV1 receptor was significantly lowered in the presence of PALDA and STEARDA

(from 90 nM to 30 nM) . This enhancement was also found to be true for anandamide on Ca 2+ levels in the studied HEK-293 cells overexpressing human

TRPV1 . At low pH, it was discovered that PALDA and STEARDA could significantly increase Ca 2+ levels in the cells . Hyperalgesia was then tested in the rat hind paw

using a radiant heat source . It was determined that the observed sensitivity to heat

was much higher in the rat hind paws treated with both N-arachidonoyldopamine

and STEARDA than either of the two molecules alone . STEARDA and PALDA had

thus been shown to modulate the activity of TRPV1 under specific conditions,

leading to a sensitization of the receptor with respect to these ligands . Thus, it was

suggested that there is an entourage effect in the mechanism of action of the N- 10

acyldopamines with TRPV1, which has also been seen with anandamide and the N-

acyldopamines upon TRPV1 . (30)

Early research by Pokorski, et al . showed the ability of NOLDA to cross the

blood brain barrier, thus suggesting its use as a transporter of dopamine into the

brain . (48)

Focus on the hyperalgesic/analgesic functions of NADs continued with the

TRPV and CB receptors . Bianchi, et al . examined whether NADA, NOLDA, and AEA affected TRPV1 receptors in HEK293 and 1321N1 brain astrocyte cells by measuring the levels of calcium influx . (49) NOLDA was shown to have a reduced effect compared to NADA and AEA in HEK293 cells and markedly so in 1321N1 cells, while decreases in pH increased this difference . Additional research indicates that NOLDA

could exert cardiac protective effects mediated by TRPV1 in wild-type mice when

compared to TRPV1-null mutant mice . (50) The anti-nociceptive potential of NADA, based on hyperalgesia research, was further established by studying the interaction of NADA and endomorphin-1 in rats at the spinal level . (51) NADA was shown to

have dose-dependent anti-hyperalgesia effects but was 5 .4 times less potent than

endomorphin -1. Additionally, both TRPV1 and CB 1 antagonists/inverse agonists reduced these effects at higher concentrations .

Continued research with NADs has now provided evidence that these

molecules are potential mediators of neurological function . (52) NADA’s action upon

dopaminergic neurons has been shown to be mediated by glutaminergic/GABAergic

transmission via TRPV1/CB 1 receptors, thus suggesting a role for NADs in synaptic transmission regulation . This study hypothesized that the activation of either TRPV1 11

or CB 1 by NADA was caused by increases in glutaminergic and GABAergic

transmission respectively . NADA may also show inhibitory aspects upon

dopaminergic neurons with glutaminergic transmission via the CB 1 receptor . Further work by Ferreira et al . in 2009, had shown that NADA, NOLDA, and PALDA were found to cause the uptake of calcium and the release of dopamine and glutamate in striatal nerve terminals, possibly by ligand-gated ion channels, which continued to occur in TRPV1-null mutant mice . (53) TRPV1 antagonists and the absence of potassium indicated no change in this effect, while Gd 3+ inhibited the effect with

high potency in low nanomolar ranges .

Earlier work with N-acetyldopamine has shown it to be an inhibitor of

sepiapterin reductase, an intermediate enzyme in the biosynthesis of

tetrahydrobiopterin (an enzyme cofactor for the biosynthesis of various

neurotransmitters) . (54) Recent research by Grabiec and colleagues has indicted

that NADA exerts neuro-protective effects via CB 1 receptors, but not with TRPV, after excitotoxic damage . (55) These effects were blocked by the AM251 CB 1

receptor antagonist and absent in CB 1-null mutant mice . This work hypothesized the role of NADs as novel drug targets to mitigate neuronal damage .

Recent research on T-type calcium channels with NADs has indicated their potential as anti-epileptics . (56) NADA was shown to potently inhibit recombinant

human T-type calcium channels (EC 50 = 300nM-1µM) . NOLDA gave similar results while PALDA had miniscule results . For a more rigorous review of N-acyl amino acids and neurotransmitters on these various other receptors, the reader is encouraged to refer to Conner, et al . 2010 . (56) 12

Explorations in NAD research have indicated their potential role as

neurobiological anti-inflammatory leads . (57, 58) NADs have been shown to induce increases in COX-2 expression within brain endothelial cells via regulation of PGE2 and PGD2, thereby mediating prostaglandin production . (59) Furthermore, Dang

and others reported that NADs and analogues are potent anti-inflammatory leads

against pro-inflammatory mediators/cytokines such as TNF-α. (60, 61) Additionally, it is noted that NADA and other endocannabinoids stimulate the transcription activity of PPARγ, a key enzyme in fatty acid metabolism . (58) As endocannabinoids

are known to be part of the eicosanoid biosynthetic pathway the action of NADs on

these systems may potentially be negative feedback regulation pathways .

Additional research with NADs outside of their traditional hyperalgesia effects has led to recent research into their potential role as thermogenic (anti-obesity) drugs . Research conducted by Mahmmoud and colleagues has shown NADA to be an uncoupling agent of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) . (62) Earlier

work by Sharkey and colleagues had indicted NAD’s roles as anti-emetics with

CB 1/TRPV1 receptors in ferrets . (63) The results showed that NADA reduced the effects of emesis and was similarly inhibited by the antagonist AM251 at 10 mg/kg .

Their research further indicated that endocannabinoids like NADs preferentially

inhibit emesis in the brain stem via CB 1 when compared to TRPV1 .

Non-cannabinoid and “orphaned” G-protein coupled receptors have also been

proposed as potential targets of NADs (in particular GPR55 and GRP119) and have

shown similar effects with other endocannabinoid receptors and their corresponding

effects, such as inflammation mediation . Additionally, NADA has been shown to

have activity with TRPM8, a receptor similar in function to TRPV1 .

1.3.b. Biosynthesis and Degradation

A primary question of NADs as eCBs in the mammalian system is the discrete

biosynthetic and degradation processes in vivo and is considered a primary goal of

NAD-eCB research .

For the biosynthesis of NADs the primary question is how and where the NAD

precursors acquire the acyl group . Huang and colleagues initially proposed the biosynthesis of NADs from the formation of acyl-CoAs via acyl-coA synthetase and their subsequent conjugation with dopamine via an acyl transferase (see Figure

1.3, Pathway IIb,c). (10, 43) Degradation by FAAH was proposed, for which known

metabolites exist (Fig . 1.5). Additional research by Huang and colleagues gained further evidence for these hypotheses as well as a possible biosynthetic route from

N-acyltyrosines (NATyro) using the pre-existing dopamine biosynthetic enzymes of

tyrosine decarboxylase (TDC), tyrosine hydroxylase (TH) and DOPA decarboxylase

(DDC) (Fig . 1.3 and 1.4) . Evidence was also shown for an alternative degradation pathway via methylation of NADs by catechol-O-methyltransferase (COMT) (Fig .

1.5). However, degradation by COMT was slow, suggesting metabolism by methylation as a partial or secondary route . In the Huang study, it was further

shown that radiolabeled arachidonic acid with dopamine or tyrosine both produced

radiolabeled NADA, including evidence of the formation of a radiolabeled NADA-like

molecule from tyrosine with the inhibition of tyrosine hydroxylase . Such research 14

suggests that both of the proposed pathway sets shown herein are possible (Figs .

1.3 and 1.4).

Recently, Hu and colleagues further reported on these two possible

biosynthetic pathways for the NADs . (64) In their study it was proposed that NAD biosynthesis required TH in the dopaminergic terminals, thus further suggesting the presence of a putative NATyro or N-acyltyramine (NATyra) derivative . Though an

NATyro derivative was found within rat brains it was determined not to be an intermediate in NAD biosynthesis . This conclusion was due to inhibiting TH, which

showed no increase in NATyro but a subsequent decrease in NAD levels (27% of

control) . Additionally, this study showed that radiolabeled NATyro gave very low

conversion to N-acyl-L-DOPA (NADOPA) with TH incubation, compared to the

natural molecules, indicating that NATyro is a poor substrate for TH . Incidentally,

radiolabeled NADOPA was shown not to be a direct precursor of NAD in vivo , thereby implying the possibility that N-acylation does not occur in conjunction with

NATyro-dependent DOPA decarboxylase . Furthermore, mammalian brain incubations with dopamine and arachidonoyl-coA/arachidonic acid phospholipid esters gave no detectable levels of the expected NAD, thus suggesting that acylation occurs prior to the final conversion to dopamine . As a result of this data it was concluded that NAD biosynthesis likely does not occur via the acylation and metabolism of NATyro or NADOPA in conjunction with either TH or DDC (Fig 1.4,

Tyrosine-dependent Pathway Ib) nor is the acyl-coA-mediated acylation of

dopamine likely as well (Fig . 1.3, Pathways IIa and IIb) . Additional studies by

Bezuglov and colleagues upheld this hypothesis by showing that the biosynthesis of

NATyro was not detected in rat liver and nerve tissues, while NADs and N-

acylphenylalanine were detected . (65)

Hu, et al ., further studied the second proposed biosynthetic pathway

involving the direct condensation of a free fatty acid with dopamine (Fig 1.3,

Pathway IIc) . (64) Their study was conducted via rat brain incubations with

radiolabeled arachidonic acid or radiolabeled dopamine . The expected radiolabeled

NADA was detected, with no indication of NADA in buffer or boiled membranes

(control) . Based on those results, FAAH-inhibited rats and FAAH-knockout mice

were examined with controls to show marked increases in AEA and decreases in

NADA for both animals, supporting a possible FAAH-mediated NAD biosynthetic

pathway . Additionally, it was reported that NADA was a poor substrate for

recombinant FAAH while AEA gave high levels of hydrolysis to the free fatty acid .

However, small amounts of NADA production were detected with

AEA/dopamine/FAAH incubations despite steps taken to eliminate possible mass

spectrometry carry-over . It was thus concluded that the biosynthesis of NADs likely involves an enzyme-mediated condensation of a free fatty acid with dopamine rather than an acyl-coA . FAAH may be a possible conjugation enzyme and/or a

rate-limiting enzyme for the release of free fatty acid from the corresponding

primary fatty acid amide . It was noted, however, that FAAH is an integral membrane protein and that recombinant FAAH is unbound in vitro , likely affecting its chemistry . Earlier research could suggest the possibility of a negative feedback route between FAAH and NADA, if FAAH is indeed involved in the biosynthesis of

NADs . (45) 16

Recent research into the degradation of NADs has lent increased evidence

towards methylation by COMT, which has been found to be an inactivator of NADA

after interaction with the TRPV1 receptor (Fig . 1.5) . (66, 67)

Akimov and colleagues proposed the oxidation of NADs via pmNOX, and

more recently have shown the sulfation of NADs via aryl sulfotransferases (AST) .

(68, 69) It was determined that NADs (arachidonoyl-, oleoyl- and cervonoyl-) were

sulfated via AST in rat liver, spinal cord and brain homogenates with relatively

robust activities compared to COMT- and FAAH-mediated degradation pathways

previously proposed .

Concurrently, work by Rimmerman et . al . indicate that NADA is metabolized

by Cytochrome P450 to the observed omega/omega-1 hydroxylated NAD

metabolites . (70) These metabolites were suggested to have a wider range of effects with lower potency .

Pathway IIa NH 2 Acyl-CoA Synthetase Fatty acid-coA HO COOH OH L-DOPA DOPA Decarboxylase H N R2 Pathway IIb HO N-Acyldopamine Acyl-CoA Synthetase OH R2 = Acyl chain NH 2 Fatty acid-coA H HO OH Dopamine Acyl Transferase Free fatty acid Pathway IIc

Figure 1.3: Proposed L-DOPA/Dopamine-Dependent NAD Biosynthetic Pathways.

1.4 Conclusions

Endocannabinoid/fatty acid amide research is an ever-evolving and complex

field of research within biochemistry and molecular biology. Other families of these

molecules, such as the N-acylamino acids and primary fatty acid amides, have yielded research similar to those discussed herein but a thorough review is not within the scope of this literature. However, one key report by Dempsey, et al., recently produced compelling work on the biosynthesis of long-chain N- acylarylalkylamines (of which N-acyldopamines are a member) using an endogenous recombinant N-acyltransferase from Fruit flies. (71) Dempsey and colleagues’ work shows the likelihood that (a) an acyltransferase is indeed a major part of the biosynthesis for these molecules and (b) previously unknown fatty acid derivatives are still being discovered, such as the N-acylserotonin described.

Continued work such as this emphasizes the complexity of the endocannabinoid metabolome and implies the richness of this area for further therapeutic research . The possibilities that these various endocannabinoids may help treat illnesses such as Alzheimer’s, Parkinson’s, pain, anxiety, depression and even appetite implies a whole new realm of potential pharmaceuticals .

1.5 References

1. Liebig, J., Ueber die Säure welche in dem Harn der grasfressenden vierfüssigen Thiere enthalten ist. Annalen der Physik 1829, 93 (11), 389-99.

2. Lees, H. J.; Swann, J. R.; Wilson, I. D.; Nicholson, J. K.; Holmes, E., Hippurate: the natural history of a mammalian-microbial cometabolite. Journal of proteome research 2013, 12 (4), 1527-46.

Figure 1.4: Proposed Tyramine/Tyrosine-Dependent NAD Biosynthetic Pathways.

Figure 1.5: Proposed NAD Degradation Pathways.

3. Kuehl, F. A.; Jacob, T. A.; Ganley, O. H.; Ormond, R. E.; Meisinger, M. A. P., The identification of N-(2-hydroxymethyl)-palmitamide as a naturally occuring anti-inflammatory. Journal of the American Chemical Society 1957, 79 (20), 5577-78.

4. Farrell, E. K., Merkler, D.J., Biosynthesis, degradation and pharmacological importance of the fatty acid amides. Drug Discovery Today. 2008, 13 (13/14), 558-68.

5. Battista, N.; Di Tommaso, M.; Bari, M.; Maccarrone, M., The endocannabinoid system: an overview. Frontiers in Behavioral Neuroscience 2012, 6, 9.

6. Devane, W.; Hanus, L.; Breuer, A.; Pertwee, R.; Stevenson, L.; Griffin, G.; Gibson, D.; Mandelbaum, A.; Etinger, A.; Mechoulam, R., Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992, 258 (5090), 1946-49.

7. Lehtonen, M.; Reisner, K.; Auriola, S.; Wong, G.; Callaway, J. C., Mass-spectrometric identification of anandamide and 2- arachidonoylglycerol in nematodes. Chemistry & Biodiversity 2008, 5 (11), 2431-41.

8. Tortoriello, G.; Rhodes, B. P.; Takacs, S. M.; Stuart, J. M.; Basnet, A.; Raboune, S.; Widlanski, T. S.; Doherty, P.; Harkany, T.; Bradshaw, H. B., Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2- Monoacylglycerols and N-acyl Amides. PLoS ONE 2013, 8 (7).

9. Astarita, G.; Rourke, B. C.; Andersen, J. B.; Fu, J.; Kim, J. H.; Bennett, A. F.; Hicks, J. W.; Piomelli, D., Postprandial increase of oleoylethanolamide mobilization in small intestine of the Burmese python ( Python molurus ). American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 2006, 290 (5), R1407-R12. 10. Huang, S. M.; Bisogno, T.; Petros, T. J.; Chang, S. Y.; Zavitsanos, P. A.; Zipkin, R. E.; Sivakumar, R.; Coop, A.; Maeda, D. Y.; De Petrocellis, L.; Burstein, S.; Di Marzo, V.; Walker, J. M., Identification of a new class of molecules, the arachidonyl amino acids, and characterization of one member that inhibits pain. Journal of Biological Chemistry 2001, 276 (46), 42639-44.

11. Farrell, E. K.; Chen, Y.; Barazanji, M.; Jeffries, K. A.; Cameroamortegui, F.; Merkler, D. J., Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells. Journal of Lipid Research 2012, 53 (2), 247-56.

12. Kirkham, T. C.; Williams, C. M.; Fezza, F.; Di Marzo, V., Endocannabinoid levels in rat limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: stimulation of eating by 2- arachidonoyl glycerol. British Journal of Pharmacology 2002, 136 (4), 550-7.

13. Muccioli, G. G.; Stella, N., An optimized GC–MS method detects nanomolar amounts of anandamide in mouse brain. Analytical Biochemistry 2008, 373 (2), 220-28.

14. Durham, H. A.; Wood, J. T.; Vadivel, S. K.; Makriyannis, A.; Lammi- Keefe, C. J., Detection of the endocannabinoid metabolome in human plasma and breast milk. The FASEB Journal 2013, 27 (45.8).

15. Fride, E., The endocannabinoid-CB receptor system: Importance for development and in pediatric disease. Neuroendocrinology Letters 2004, 25 (1-2), 24-30.

16. Pacioni, G.; Rapino, C.; Zarivi, O.; Falconi, A.; Leonardi, M.; Battista, N.; Colafarina, S.; Sergi, M.; Bonfigli, A.; Miranda, M.; Barsacchi, D.; Maccarrone, M., Truffles contain endocannabinoid metabolic enzymes and anandamide. Phytochemistry 2015, 110 , 104-10.

17. di Tomaso, E.; Beltramo, M.; Piomelli, D., Brain cannabinoids in chocolate. Nature 1996, 382 (6593), 677-78.

18. Fride, E.; Mechoulam, R., Pharmacological activity of the cannabinoid receptor agonist, anandamide, a brain constituent. European Journal of Pharmacolology 1993, 231 (2), 313-4.

19. Fu, J.; Gaetani, S.; Oveisi, F.; Lo Verme, J.; Serrano, A.; Rodriguez De Fonseca, F.; Rosengarth, A.; Luecke, H.; Di Giacomo, B.; Tarzia, G.; Piomelli, D., Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 2003, 425 (6953), 90-3.

20. Hansen, H. S., Palmitoylethanolamide and other anandamide congeners. Proposed role in the diseased brain. Experimental Neurology 2010, 224 (1), 48-55.

21. Waluk, D. P.; Battistini, M. R.; Dempsey, D. R.; Farrell, E. K.; Jeffries, K. A.; Mitchell, P.; Hernandez, L. W.; McBride, J. C.; Merkler, D. J.; Hunt, M. C., Chapter 9 - mammalian fatty acid amides of the brain and CNS. In Omega-3 Fatty Acids in Brain and Neurological Health , Meester, R. R. W. D., Ed. Academic Press: Boston, 2014; pp 87-107.

22. Martin, B. R.; Compton, D. R.; Thomas, B. F.; Prescott, W. R.; Little, P. J.; Razdan, R. K.; Johnson, M. R.; Melvin, L. S.; Mechoulam, R.; Ward, S. J., Behavioral, biochemical, and molecular modeling evaluations of cannabinoid analogs. Pharmacology, Biochemistry, and Behavior 1991, 40 (3), 471-8.

23. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I., Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990, 346 (6284), 561-64.

24. Munro, S.; Thomas, K. L.; Abu-Shaar, M., Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993, 365 (6441), 61- 65.

25. Ross, R. A.; Gibson, T. M.; Brockie, H. C.; Leslie, M.; Pashmi, G.; Craib, S. J.; Di Marzo, V.; Pertwee, R. G., Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. British Journal of Pharmacology 2001, 132 (3), 631-40.

26. De Petrocellis, L.; Starowicz, K.; Moriello, A. S.; Vivese, M.; Orlando, P.; Di Marzo, V., Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): Effect of cAMP, cannabinoid CB1 receptors and endovanilloids. Experimental Cell Research 2007, 313 (9), 1911- 20.

27. Sun, Y.; Alexander, S. P. H.; Garle, M. J.; Gibson, C. L.; Hewitt, K.; Murphy, S. P.; Kendall, D. A.; Bennett, A. J., Cannabinoid activation of PPARα; a novel neuroprotective mechanism. British Journal of Pharmacology 2007, 152 (5), 734-43.

28. Rockwell, C. E.; Kaminski, N. E., A cyclooxygenase metabolite of anandamide causes inhibition of interleukin-2 secretion in murine splenocytes. Journal of Pharmacology and Experimental Therapeutics 2004, 311 (2), 683-90.

29. Maccarrone, M.; Cartoni, A.; Parolaro, D.; Margonelli, A.; Massi, P.; Bari, M.; Battista, N.; Finazzi-Agrò, A., Cannabimimetic Activity, Binding, and Degradation of Stearoylethanolamide within the Mouse Central Nervous System. Molecular and Cellular Neuroscience 2002, 21 (1), 126-40.

30. Smart, D.; Jonsson, K.-O.; Vandevoorde, S.; Lambert, D. M.; Fowler, C. J., ‘Entourage’ effects of N-acyl ethanolamines at human vanilloid receptors. Comparison of effects upon anandamide-induced vanilloid receptor activation and upon anandamide metabolism. British Journal of Pharmacology 2002, 136 (3), 452-58.

31. Lambert, D. M.; Di Marzo, V., The palmitoylethanolamide and oleamide enigmas : are these two fatty acid amides cannabimimetic? Current Medicinal Chemistry 1999, 6 (8), 757-73.

32. Magotti, P.; Bauer, I.; Igarashi, M.; Babagoli, M.; Marotta, R.; Piomelli, D.; Garau, G., Structure of Human N-Acylphosphatidylethanolamine- Hydrolyzing Phospholipase D: Regulation of Fatty Acid Ethanolamide Biosynthesis by Bile Acids. Structure 2015, 23 (3).

33. Burstein, S. H.; Rossetti, R. G.; Yagen, B.; Zurier, R. B., Oxidative metabolism of anandamide. Prostaglandins & Other Lipid Mediators 2000, 61 (1–2), 29-41.

34. Ivkovic, M.; Dempsey, D. R.; Handa, S.; Hilton, J. H.; Lowe Jr, E. W.; Merkler, D. J., N-acylethanolamines as novel alcohol dehydrogenase 3 substrates. Archives of Biochemistry and Biophysics 2011, 506 (2), 157-64.

35. Piscitelli, F.; Di Marzo, V., "Redundancy" of endocannabinoid inactivation: new challenges and opportunities for pain control. ACS Chemical Neuroscience 2012, 3 (5), 356-63.

36. Hermanson, D. J.; Hartley, N. D.; Gamble-George, J.; Brown, N.; Shonesy, B. C.; Kingsley, P. J.; Colbran, R. J.; Reese, J.; Marnett, L. J.; Patel, S., Substrate-selective COX-2 inhibition decreases anxiety via endocannabinoid activation. Nature Neuroscience 2013, 16 (9), 1291-98.

37. Glaser, S. T.; Kaczocha, M., Cyclooxygenase-2 mediates anandamide metabolism in the mouse brain. Journal of pharmacology and experimental therapeutics 2010, 335 (2), 380-8.

38. Hwang, J.; Adamson, C.; Butler, D.; Janero, D. R.; Makriyannis, A.; Bahr, B. A., Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality. Life Sciences 2010, 86 (15–16), 615-23.

39. Rapino, C.; Battista, N.; Bari, M.; Maccarrone, M., Endocannabinoids as biomarkers of human reproduction. Human Reproduction Update 2014, 20 (4), 501-16.

40. El-Talatini, M. R.; Taylor, A. H.; Konje, J. C., The relationship between plasma levels of the endocannabinoid, anandamide, sex steroids, and gonadotrophins during the menstrual cycle. Fertility and Sterility 2010, 93 (6), 1989-96.

41. Gaetani, S.; Dipasquale, P.; Romano, A.; Righetti, L.; Cassano, T.; Piomelli, D.; Cuomo, V., Chapter 5 The endocannabinoid system as a target for novel anxiolytic and antidepressant drugs. In International Review of Neurobiology , Bagetta, G. C. M. T. S. T.; Sakurada, S., Eds. Academic Press: 2009; Vol. Volume 85, pp 57-72.

42. Abadji, V.; Lin, S.; Taha, G.; Griffin, G.; Stevenson, L. A.; Pertwee, R. G.; Makriyannis, A., ( R)-methanandamide: A chiral novel anandamide possessing higher potency and metabolic stability. Journal of Medicinal Chemistry 1994, 37 (12), 1889-93.

43. Huang, S. M.; Bisogno, T.; Trevisani, M.; Al-Hayani, A.; De Petrocellis, L.; Fezza, F.; Tognetto, M.; Petros, T. J.; Krey, J. F.; Chu, C. J.; Miller, J. D.; Davies, S. N.; Geppetti, P.; Walker, J. M.; Di Marzo, V., An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (12), 8400-05.

44. Tominaga, M.; Caterina, M. J.; Malmberg, A. B.; Rosen, T. A.; Gilbert, H.; Skinner, K.; Raumann, B. E.; Basbaum, A. I.; Julius, D., The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998, 21 (3), 531-43.

45. Bisogno, T., N-acyl-dopamines novel synthetic CB1 cannabinoid- receptor ligands and inhibitors of anandamide inactiviation with cannabimimetic activity in vitro and in vivo. Biochemical Journal 2000, (351), 817-24.

46. Chu, C. J.; Huang, S. M.; De Petrocellis, L.; Bisogno, T.; Ewing, S. A.; Miller, J. D.; Zipkin, R. E.; Daddario, N.; Appendino, G.; Di Marzo, V.; Walker, J. M., N-oleoyldopamine, a novel endogenous capsaicin-like lipid that produces hyperalgesia. Journal of Biological Chemistry 2003, 278 (16), 13633-39.

47. De Petrocellis, L.; Chu, C. J.; Moriello, A. S.; Kellner, J. C.; Walker, J. M.; Di Marzo, V., Actions of two naturally occurring saturated N- acyldopamines on transient receptor potential vanilloid 1 (TRPV1) channels. British Journal of Pharmacology 2004, 143 (2), 251-6.

48. Pokorski, M.; Matysiak, Z., Fatty acid acylation of dopamine in the carotid body. Medical Hypotheses 1998, 50 (2), 131-33.

49. Bianchi, B. R.; Lee, C. H.; Jarvis, M. F.; El Kouhen, R.; Moreland, R. B.; Faltynek, C. R.; Puttfarcken, P. S., Modulation of human TRPV1 receptor activity by extracellular protons and host cell expression system. European Journal of Pharmacology 2006, 537 (1-3), 20-30.

50. Zhong, B., N -oleoyldopamine, a novel endogenous capsaicin-like lipid, protects the heart against ischemia-reperfusion injury via activation of TRPV1. American Journal of Physiology-Heart and Circulatory Physiology 2008, (295), H728-H35.

51. Farkas, I.; Tuboly, G.; Benedek, G.; Horvath, G., The antinociceptive potency of N-arachidonoyl-dopamine (NADA) and its interaction with endomorphin-1 at the spinal level. Pharmacology, Biochemistry, and Behavior 2011, 99 (4), 731-7.

52. Marinelli, S.; Di Marzo, V.; Florenzano, F.; Fezza, F.; Viscomi, M. T.; van der Stelt, M.; Bernardi, G.; Molinari, M.; Maccarrone, M.; Mercuri, N. B., N-arachidonoyl-dopamine tunes synaptic transmission onto dopaminergic neurons by activating both cannabinoid and vanilloid receptors. Neuropsychopharmacology 2007, 32 (2), 298-308.

53. Ferreira, S. G.; Lomaglio, T.; Avelino, A.; Cruz, F.; Oliveira, C. R.; Cunha, R. A.; Kofalvi, A., N-acyldopamines control striatal input terminals via novel ligand-gated cation channels. Neuropharmacology 2009, 56 (3), 676-83.

54. Oxenkrug, G. F.; Requintina, P. J., N-acetyldopamine inhibits rat brain lipid peroxidation induced by lipopolysaccharide. Neuroprotective Agents 2005, 1053 , 394-99.

55. Grabiec, U.; Koch, M.; Kallendrusch, S.; Kraft, R.; Hill, K.; Merkwitz, C.; Ghadban, C.; Lutz, B.; Straiker, A.; Dehghani, F., The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB1). Neuropharmacology 2012, 62 (4), 1797-807.

56. Connor, M.; Vaughan, C. W.; Vandenberg, R. J., N-acyl amino acids and N-acyl neurotransmitter conjugates: neuromodulators and probes for new drug targets. British Journal of Pharmacology 2010, 160 (8), 1857-71.

57. Sancho, R., Immunosuppressive Activity of Endovanilloid N- arachidonoyldopamine Inhibits Activation of the NF-kB, NFAT, and Activator Protein 1 Signaling Pathways. Journal of Immunology 2004, (172), 2341-51.

58. De Petrocellis, L.; Di Marzo, V., Non-CB1, non-CB2 receptors for endocannabinoids, plant cannabinoids, and synthetic cannabimimetics: focus on G-protein-coupled receptors and transient receptor potential channels. Journal of Neuroimmune Pharmacology 2010, 5 (1), 103-21.

59. Navarrete, C. M.; Perez, M.; de Vinuesa, A. G.; Collado, J. A.; Fiebich, B. L.; Calzado, M. A.; Munoz, E., Endogenous N-acyl-dopamines induce COX-2 expression in brain endothelial cells by stabilizing mRNA through a p38 dependent pathway. Biochemical Pharmacology 2010, 79 (12), 1805-14.

60. Dang, H. T.; Kang, G. J.; Yoo, E. S.; Hong, J.; Choi, J. S.; Kim, H. S.; Chung, H. Y.; Jung, J. H., Evaluation of endogenous fatty acid amides and their synthetic analogues as potential anti-inflammatory leads. Bioorganic & Medicinal Chemistry 2011, 19 (4), 1520-27.

61. Yoo, J. M.; Park, E. S.; Kim, M. R.; Sok, D. E., Inhibitory effect of N- acyl dopamines on IgE-mediated allergic response in RBL-2H3 cells. Lipids 2013, 48 (4), 383-93.

62. Mahmmoud, Y. A.; Gaster, M., Uncoupling of sarcoplasmic reticulum Ca2+-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs. British Journal of Pharmacology 2012, 166 (7), 2060-69.

63. Sharkey, K. A.; Cristino, L.; Oland, L. D.; Van Sickle, M. D.; Starowicz, K.; Pittman, Q. J.; Guglielmotti, V.; Davison, J. S.; Di Marzo, V., Arvanil, anandamide and N-arachidonoyl-dopamine (NADA) inhibit emesis through cannabinoid CB1 and vanilloid TRPV1 receptors in the ferret. European Journal of Neuroscience 2007, 25 (9), 2773-82.

64. Hu, S. S.; Bradshaw, H. B.; Benton, V. M.; Chen, J. S.; Huang, S. M.; Minassi, A.; Bisogno, T.; Masuda, K.; Tan, B.; Roskoski, R., Jr.; Cravatt, B. F.; Di Marzo, V.; Walker, J. M., The biosynthesis of N- arachidonoyl dopamine (NADA), a putative endocannabinoid and endovanilloid, via conjugation of arachidonic acid with dopamine. Prostaglandins, Leukotrienes, and Essential Fatty Acids 2009, 81 (4), 291-301.

65. Bezuglov, V. V.; Gretskaya, N. M.; Blazhenova, A. V.; Andrianova, E. L.; Akimov, M. G.; Bobrov, M. Y.; Nazimov, I. V.; Kisel’, M. A.; Sharko, O. L.; Novikov, A. V.; Krasnov, N. V.; Shevchenko, V. P.; Shevchenko, K. V.; V’yunova, T. V.; Myasoedov, N. F., Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties. Russian Journal of Bioorganic Chemistry 2006, 32 (3), 231-39.

66. van der Stelt, M.; Di Marzo, V., Endovanilloids - Putative endogenous ligands of transient receptor potential vanilloid 1 channels. European Journal of Biochemistry 2004, 271 (10), 1827-34.

67. Cristino, L.; Starowicz, K.; De Petrocellis, L.; Morishita, J.; Ueda, N.; Guglielmotti, V.; Di Marzo, V., Immunohistochemical localization of anabolic and catabolic enzymes for anandamide and other putative endovanilloids in the hippocampus and cerebellar cortex of the mouse brain. Neuroscience 2008, 151 (4), 955-68.

68. Akimov, M. G.; Gretskaya, N. M.; Shevchenko, K. V.; Shevchenko, V. P.; Myasoedov, N. F.; Bobrov, M. Y.; Bezuglov, V. V., New aspects of biosynthesis and metabolism of N-acyldopamines in rat tissues. Russian Journal of Bioorganic Chemistry 2007, 33 (6), 602-06.

69. Akimov, M. G.; Nazimov, I. V.; Gretskaya, N. M.; Zinchenko, G. N.; Bezuglov, V. V., Sulfation of N-acyl dopamines in rat tissues. Biochemistry (Moscow) 2009, 74 (6), 681-85.

70. Rimmerman, N.; Bradshaw, H. B.; Basnet, A.; Tan, B.; Widlanski, T. S.; Walker, J. M., Microsomal omega-hydroxylated metabolites of N- arachidonoyl dopamine are active at recombinant human TRPV1 receptors. Prostaglandins & Other Lipid Mediators 2009, 88 (1-2), 10- 17.

71. Dempsey, D. R.; Jeffries, K. A.; Anderson, R. L.; Carpenter, A.-M.; Rodriquez Opsina, S.; Merkler, D. J., Identification of an arylalkylamine N-acyltransferase from Drosophila melanogaster that catalyzes the formation of long-chain N-acylserotonins. FEBS Letters 2014, 588 (4), 594-99.

CHAPTER 2:

THE DETECTION AND QUANTIFICATION OF ENDOGENOUS

ENDOCANNABINOIDS/FATTY ACID AMIDES IN A. MELLIFERA

(HONEYBEES) AND T. CASTANEUM (RED FLOUR BEETLES)

2.1 Introduction and Background

The common honeybee (Apis mellifera, Lat. “honey-bearing bee”) and the

red flour beetle (T. castaneum ) are widely considered the most beneficial and most

destructive cosmopolitan pests in the modern world, respectively. The honeybee is

the primary commercial pollinator in modern agriculture, as well as producing

agricultural products like honey and bee’s wax. (1, 2) The red flour beetle, on the

other hand, consumes up to a quarter of grain crops in the western world alone and

is considered one of the most destructive pests in agriculture. (3, 4, 5)

As a result, extensive research has occurred over many years with regards

to the roles of these two diametrically opposed insects in the modern world.

Starting in the 1990’s the phenomenon known as “sudden colony collapse” began a

pattern of wide-spread loss of commercialized honeybees and crops, resulting in

low supplies and increasingly high costs for honey and those crops requiring

pollination. (6, 7) Grain crop losses from the red flour beetle, and its close relative

the Confused Beetle ( Tribolium confusum ) have been estimated as high as 50% of

total global seasonal yield. (8)

For honeybees, the impact of the extensive loss of colonies worldwide is an

agricultural and commercial issue that cannot be adequately stressed within our

society. In the event of global catastrophic colony losses for this species a

cascading effect would result, causing not only massive agricultural and economic

devastation but also potentially worldwide famine. Such effects could result in the

deaths of millions. The current theory for the cause of sudden colony collapse is the

extensive use of pesticides, which likely causes massive die-back within colonies

including the queen. (2, 6) The mechanism for die-back events from pesticide usage is still unclear and hotly debated. Current speculation suggests that pesticides may be inhibiting or degrading the honeybee’s individual sensory perception and/or the chemical signaling process between colony-mates via the specie’s use of communal pheromonal communication.

In the context of the red flour beetle, conventional pesticides are inadequate

in controlling the species and any compounds that do are highly toxic upon

consumption of the treated crops. (9, 10, 11) Increases in pesticide usage have several severe environmental and health effects, while showing little results in controlling this pest species. A major concern is that as population density increases and global temperatures further rise controlling the species will become much more difficult and could result in even higher crop losses, especially in economically and technologically depressed regions. As a result, much like honeybees, wide-spread famine could ensue causing the deaths of millions and creating a devastating cascade effect worldwide in terms of economics, agriculture and possibly even lead to political strife.

Red flour beetle’s increasing resistance to pesticides is likely due to uneven

distribution, concentration and exposure. (9, 11, 10) Therefore, it is clear that both

safer and more effective forms of control are needed to combat red flour beetles

while limiting the potential hazards of pesticide use against honeybees. Because of

the importance in pheromone signaling in communal organisms like honeybees, and

the recent discovery of endocannabinoids and TRP ion channels tied to sensory

perception present within fruit flies, further research into the role of

endocannabinoids may prove highly relevant. (12, 13)

Honeybees present a unique constraint for research however, due in part to

the inability to completely contain and control them in a laboratory environment, as

honeybees require the constant ability to forage in the environment or the colony

would perish. (14) As such, several unknown factors that cannot be easily

documented and quantified can readily affect the organism and corresponding

results. While their food supply can be supplemented, it cannot be completely

controlled; therefore traditional feeding studies are not feasible. The lifecycle and

life stages of honeybees are also difficult to control and monitor within the certainty

accustomed to model organisms. Red flour beetles, however, are easily cultured,

controlled and manipulated in a laboratory setting and have proven to be an

excellent model organism. (15)

In 2001, however, McPartland and colleagues suggested that

endocannabinoids do not exist in insects. (16) In contrast however recent work by

Jeffries, et al. , have shown otherwise. (12, 17) The need for additional model organisms for continued studies in endocannabinoids were made even more relevant in insects due to the full genome sequencing of both honeybees in 2006

(18) and in red flour beetles in 2009. (3) As such genomic studies for the

endocannabic receptors and posited biosynthetic enzymes have shown significant

sequence homology within these insects between higher order animal species. (17)

Some of these key genomic similarities in the context of endocannabinoid

metabolism include the existence of an arylalkylamine N-acyltransferase (AANAT) enzyme recently characterized from fruit flies showing significant homology with similar enzymes in both honeybees and red flour beetles (Fig 2 .1) . (17)

Due in part to the research recently conducted in Fruit flies on the biosynthetic enzyme AANAT, and the sequence homology described herein, the usage of honeybees and red flour beetles to further elucidate the role, biosynthesis and degradation of endocannabinoids/fatty acid amides is clear .

Prior to any possible studies involving the AANAT-like enzymes present within

these organisms it is first necessary to determine the endogenous identities and

concentrations of the endocannabinoids/fatty acid amides present . Several procedures to accomplish this have been developed over the years by various groups, in organisms ranging from C. elegans , rats and mice, to cultured cells and

human tissue . (19, 20, 21) Much of these methods are only feasible with the use of

separation and spectroscopic techniques such as GC/LC/MS . These techniques are

not with limitations however, especially with lipid molecules such as the eCBs/FAAs,

and include oxidation, over-fragmentation, matrix effects and column

contamination, to name a few . (22, 23, 24)

Key limitations to note include inherent error with a shared mass

spectroscopy instrument and chromatography column such as cross-contamination,

column degradation and contamination by known labware slip additives Oleamide

and Palmitamide (25, 26). A significant limitation of note is the oxidation of N-

acyldopamines into the N-acyldopamine quinone/indole specie’s and has greatly

contributed to the elusiveness of detecting these specific molecules endogenously .

These reactions are widely documented and difficult to mitigate . Reasons for this difficulty are due to sample treatment and exposure to oxidative environments, such as air and light, that cause precipitation into di- and tri-polymers similar to melanin which are resistant to mass spectroscopy techniques (Figure 2 .2). For additional information please refer to the review by D’Ischia, et al . (22, 27)

Despite these limitations herein is presented research into the detection and quantification of a set of endocannabinoids/fatty acid amides (Table 2 .1) from A.

mellifera and T. castaneum via high performance liquid chromatography quadropole time of flight mass spectroscopy techniques (HPLC/QTOF-MS) along with genetic comparisons of the Fruit Fly arylalkylamine N-acyltransferase (AANAT) between these organisms .

Also presented herein is a proposed biosynthetic route to the eCBs/fatty acid

amides in light of the results shown and suggestions for further research . Overall,

this research provides additional evidence that endocannabinoids and fatty acid

amides are endogenous to these two organisms, despite the known cannabinoid

receptors showing no known expression in insects . In conjunction with the recent data from Fruit flies (12) such mounting results demand additional questions as to the nature of these compounds and the possibility of heretofore unknown cannabinoid-like receptors .

BLASTp database. From NCBI Between Sequence Homology 2.1. AANATFigure D. D. melanogaster , A. mellifera

and

T. T. a taneumcas .

H HO N R O

N-acyldopamine-derived O N HO O Aminochrome N-acyldopamine O R HO N-acyldopamine-derived Dihydroxyindole HO N O R HO O

HO N N-acyldopamine-derived O N 5,6-Indolequinone N-acyldopamine-derived O Dihydroxyindole R O (Zwitterionic Resonance Form) R O

O N N-acyldopamine-derived R R 5,6-Indolquinone Trimer O N O O (Melanin-like Polymeric Precursor) R O HO N N HO O R HO R N-acyldopamine-derived HO OH N Dihydroxyindole Dimer O O O Figure 2.2: Select routes of N-acyldopamine oxidation to quinone/indole intermediates and final di- and tri-mer products. R represents the long-chain fatty acyl moiety. Based on chemistry as described by D’Ischia (22)

2.2 Experimental Methods and Procedures

All materials and chemicals used herein were acquired from Sigma-Aldrich,

Co. or Fisher Scientific unless otherwise noted. All fatty acid amide standards were

purchased from Caymen Chemical, Co.

2.2.a. A. mellifera (honeybee) Acquisition and Processing

Honeybees were donated by the USF Botanical Gardens in cooperation with

Dr. Brent Weisman (USF Department of Anthropology). Two separate batches of

honeybees were acquired from the botanical gardens. The first batch, labeled as

“Farmed”, was obtained in late Fall 2013 and comprised approximately of 500 adult 39 female worker bees (50 grams total mass). The second batch, labeled “Feral”, was acquired by Dr. Weisman from a local rogue colony approximately 15-20 miles from

USF-Tampa campus. The feral bees comprised of approximately 200 adult female workers (20 grams total weight).

Table 2.1: Select endocannabinoids/fatty acid amides to be identified and quantified in A. mellifera and T. castaneum within this study Compound Abbreviation Molecular Formula

N- Oleoylethanolamine OEAC20 H39 NO 2

N- Arachidonoylethanolamine AEA C22 H37 NO 2

N- Palmitoyldopamine NPD C24 H41 NO 3

N-Palmitoyldopamine Quinone NPDq C 24 H 39 NO 4

N- Oleoyldopamine NOD C26 H43 NO 3

N-Oleoyldopamine Quinone NODq C 26 H 41 NO 4

N- Arachidonoyldopamine NAD C28 H41 NO 3

N-Arachidonoyldopamine Quinone NADq C 28 H 39 NO 4

N- Oleoylserotonin NOS C28 H44 N2O2

N-Oleoylserotonin Oxid Prod NOSq C 28 H 44 N 2 O 3

N -Palmitoylglycine NPG C18 H35 NO 3

N- Oleoylglycine NOG C20 H37 NO 3

N- Arachidonoylglycine NAG C22 H35 NO 3

Linoleamide LOA C18 H33 NO

Palmitoleamide POA C16 H33 NO

Palmitamide PA C16 H31 NO Oleamide OA C18 H35 NO

Upon immediate acquisition of both batches all samples were placed on dry ice for transport to the lab facility for processing. Processing of these samples included full immobilization of the organisms in dry ice for 5-10 minutes,

40 transferred to deep freeze containers and weighed then flash frozen over liquid nitrogen for 10-15 minutes. After samples were thoroughly frozen the containers were tightly sealed and shaken vigorously for 5 minutes to break apart the organisms into three separate body parts: head, thorax and abdomen. Incidentally, this process also removed the legs, wings and antennae of the organism as well as any loose pollen or other particulate matter, wherein this material would collect on the wall of the container or settle to the bottom allowing easy separation from the organism segments. These segments were then separated out, while on ice, into individual deep freeze vials and weighed. Each vial of segments were then purged with nitrogen for 5 minutes, sealed and labeled and then stored at -80° C until needed.

2.2.b. T. castaneum (red flour beetle) Acquisition and Processing

Two samples of red flour beetle GA-1 strain were donated by Dr. Susan

Brown of the Kansas State University Tribolium Genetics Program in early spring of

2014. These samples were taken from the existing colony where the full genome was previously sequenced. The samples were shipped overnight in sealed plastic bottles containing approximately 50-75 adult beetles on 5 grams of growth media

(organic wheat flour with 5% w/w brewer’s yeast).

Upon receipt of the shipment the containers were immediately inspected. No pupil, larval or egg stages of the organism were detected. Both samples were placed in two sterilized large mason jars with a wire mesh screen lid containing 10 grams each of freshly prepared growth media (9.5 g. of pre-sifted organic whole wheat flash-frozen flour and 0.5 g. brewer’s yeast, % w/w). Each colony jar was

41 then placed in an incubating oven at 35° C under ambient humidity conditions.

Documentation obtained from the KSU:TGP determined that these conditions were optimal for obtaining a maximum amount of the organism in the least amount of time necessary (15, 28). Under these conditions a single generation from eggs to adults will occur within 21 days. Each colony jar was inspected at least twice a day to maintain temperature and to determine overall well-being of the colonies.

After approximately 2 weeks the colony jars were split, in which all adult beetles were removed and placed into new jars with 10 g. fresh media. The remaining jars, which now contain eggs and newly hatched larvae, are also maintained at 35° C under ambient humidity conditions and closely monitored. This cycle was repeated one additional time to produce 8 individual colony jars of either adults (4 jars) or eggs/larvae (4 jars) comprising 3 distinct generations. With a stable colony now established 2 of the adult colony jars were processed. All adult beetles were removed after egg-laying and larvae development was verified, placed into deep freeze vials and weighed then flash frozen over liquid nitrogen for 5 minutes. These vials were then purged with nitrogen, sealed and labeled then placed under storage at -80° C until needed. Two jars of adults yield approximately

2.5-3 g. total weight (or ~250-300 beetles).

2.2.c. General Extraction Method

The extraction and purification methods used for both organisms were previously or concurrently described by Farrell, et al., (21) Folch, et al., (29)

Jeffries, et al., (12) and Sultana and Johnson (30), with only slight modifications.

For the honeybees Apis mellifera , both farmed and feral, samples of each

segment (2.5 g. heads, 5.0 g. thorax and 10.0 g. abdomen) were taken and placed

in a mortar and pestle over ice with 1-5 g. clean sand and 1-5 mL of HPLC grade

methanol. The sample was then ground into a uniform paste, diluted with additional

methanol, allowed to settle from the sand and the remaining solution separated

into centrifuge vials. This solution containing ground organism segment suspended

in 10-15 mL of methanol was then placed on ice and ultra-sonicated for 20 minutes

in 1 minute on/off cycles to further rupture and homogenize cellular material for a

more extensive extraction. After sonication the solution was then briefly vortexed

for 1 minute then centrifuged at ~10000xG for 5 minutes. The supernatant was

then placed into large glass test tubes, capped with rubber stoppers, wrapped in

aluminium foil and set aside. The remaining pellet was then re-suspended in 10-15

mL of 1:1:0.1 of methanol:chloroform:water, ultra-sonicated on ice for an

additional 10 minutes, briefly vortexed for 1 minute and centrifuged for 5 minutes.

This secondary supernatant was then added to the first supernatant and set aside.

The remaining pellet was re-suspended again using 5-10 mL. of a 2:1

methanol:chloroform mixture containing 0.07 M KCl/0.11 M H 3PO 4 (stock 0.5 M

KCl/0.8 M H 3PO 4), further ultra-sonicated on ice for 5 minutes, briefly vortexed for

2 minutes and centrifuged for 10 minutes. The sample separated into three distinct

layers: the bottom organic layer, a middle highly viscous interphase layer

containing cellular material, and a top aqueous layer. The bottom organic layer was

separated and added to the first two supernatant layers. All three layers, now

combined, were dried overnight (~10-15 hours) under a steady stream of nitrogen.

The remaining residue under nitrogen was then either stored at -80° C until needed

or immediately re-dissolved in 200 ul of N-hexane for subsequent purification.

For the red flour beetles the same extraction method was used with only 2.5 grams total of the whole organism.

2.2.d. General Purification Method

The resultant residue from the extraction process is relatively crude and requires solid-phase extraction prior to any further analyses.

A small 10 mL. glass solid-phase extraction column with a porous frit was prepared using 0.5 g. of silica and a sand cap. This silica plug column was then rinsed with 2-3 mL of N-hexanes in preparation for the addition of the crude sample. The crude sample, re-dissolved in 200 ul. of N-hexane, was now carefully added to the sand bed and allowed to settle into the silica before addition of any further solvent. The crude sample was then washed and extracted using a mobile phase solvent gradient comprising of 4 mL. N-hexane, 1 mL (v/v) each of 99:1 hexane:acetic acid, 90:10 hexane:ethyl acetate, 80:20 hexane:ethyl acetate, 70:30 hexane:ethyl acetate, 2.0 mL of 2:1 chloroform:isopropanol and finally 1 mL. of methanol. Each gradient was collected separately in glass test tubes, capped and dried under nitrogen. The 70:30 hexane:ethyl acetate, cholorform:isopropanol and methanol fractions containing the extracted fatty acid amides were combined prior to drying. Each fraction sample was then stored under nitrogen at -80° C until further analysis. A total of 7 organism/body segments were used for the purification and extraction process: Farmed honeybee heads, thorax and abdomen; feral

honeybee heads, thorax and abdomen and whole-body red flour beetles. Each of

these samples were extracted and purified individually and separately.

2.2.e. Preparation of Standards/Standard Curves and Blanks for Carry-Over and

Slip Additives

Standard molecules were grouped into 5 groups: acyl-dopamines, acyl-

glycines, acyl-serotonin, primary fatty acid amides and the internal standard D 8-N- arachidonoylglycine. The saturated/unsaturated N-acyl moiety lengths varied from

C16 (palmitoyl), C 18 (oleoyl, linoleoyl), and C 20 (arachidonyl). Each compound was

initially individually assayed to determine optimal concentration, retention times

and expected ion patterns. For the use as a standard molecule to mimic an

endogenous environment each compound was then combined into a dilution of 25

uM in 1 mL HPLC-grade methanol (62.5 ug/mL, 0.025 nmoles on-column injection),

including the internal standard. All standards were assayed in triplicate with

retention time deviations less than 0.2 mins. prior to each assay elution profile for

all sample sets. (12)

For the purpose of standard curves used in quantitative analysis each

compound was serial diluted to the range of 0.025 – 0.25 nM (0.0025-0.025

nmoles injected on column). This range was determined to be the linear dynamic

range of detection, with the lower limit being approximately 0.00125 nmoles

injected on column due to significant scattering and the upper limit greater than

0.025 nmoles injected on column due to a non-linear response curve. Additionally,

each sample used for standard curve production included 0.025 nM of the internal

standard to correct for any instrument and day to day usage deviations. Standard

curves for oleamide, palmitamide, palmitoleamide and linoleamide were done

previously and only the slope of the linear region was available.

Blanks for use in between HPLC-MS samples were prepared using 1.0 mL of

pure HPLC-grade methanol assayed in triplicate. Sample blanks were also used

prior to and after all assays were completed to flush the HPLC-MS system. To

compensate for the presence of the slip additives oleamide and palmitamide 10 mL

of HPLC-grade methanol was used in the same procedure as the extraction and

purification method described herein, with 10 uL of the final concentrated sample

diluted into 1 mL for HPLC analysis after the completion of all other assays.

2.2.f. HPLC-QTOF-MS and MS/MS Sample Analysis

In preparation for analysis via mass spectroscopy each fraction was re-

dissolved in 100 µl. of HPLC methanol, from which 10 µl was then diluted into 1 mL.

total of methanol spiked with 25 µm of the internal standard N-arachidonoylglycine- d8 (1.50 µl at 170 µm). This internal standard was used to monitor and normalize

instrument deviation in tandem with a calibration sample of only the internal

standard assayed prior to all analyses. Additionally, a sample mixture of standard

molecules at 25 µm in 1 mL. of methanol was also prepared and used during all

sample analyses. Each organism/body segment sample is injected and analyzed 4

times and is considered an endogenous sample set. Therefore, a total of 18 unique

sample sets were used from farmed honeybees (5 heads, 7 abdomen, and 6

thorax), 20 unique sample sets for feral honeybees (12 heads, 4 each abdomen and

thorax) and 5 unique sample sets of red flour beetles (whole body). In total, data

acquisition for the detection and quantification of endogenous fatty acid amides

produced about 200 individual data files from the organisms alone, not including

standards, standard curve production, blanks and previous extractions and

purification sample assays that did not yield results. Additionally, a sample of A.

mellifera thorax extract was spiked with 25 pmols of pure standard mixture to

determine extract matrix effects on retention time deviations and detection.

All sample analyses were performed on an Agilent 1260 HPLC system fitted

with a Kinetex reverse-phase column (C 18 , 2.6 µm, 100 Å, 50 mm x 2.1 mm). The mobile phase comprised a binary mixture of Solvent A and Solvent B (water and acetonitrile, each with 0.1% formic acid, respectively) set to a linear gradient over time. Three time segments of this elution profile gradient over a total of 11.5 minutes were programmed for all sample assays, with a 1 minute pre- and 2 minute post-assay system flush. Time segment 1 was from 1-5 minutes with a linear increase of Solvent B from 10% to 100%. Time segment 2 was from 5-8 minutes with an isocratic mobile phase of Solvent B at 100%. Time segment 3, from 8-9.5 minutes, returned the mobile phase to 10% of Solvent B. Flow rate throughout the analysis time was 0.6 mL/min at ambient temperature with a programmed pressure limit of 600 bar. Sample injection was 10 µl per sample with a draw speed of 100 µL/min and an eject speed of 200 µL/min. A needle wash of 3 seconds with methanol was performed prior to sample injection into the HPLC system.

All compounds of interest were determined to elute between 5 and 7 minutes. To minimize mass spectrometer source contamination only this 5-7 minute portion of the analysis time was analyzed and recorded via MS. The remaining time periods were diverted to waste containment on the instrument and

chromatographic responses were not recorded in the majority of the samples used

for quantitative analysis.

Mass spectrometric analysis of all samples was performed using an Agilent

6540 Quadropole Time-of-Flight Mass Spectrometer coupled to the HPLC system.

Ion source was a Dual Agilent Jet Stream Electrospray Ionization set to positive ion

mode. Additional system parameters are as follows: Nitrogen source gas, 100-3000

m/z range; 2.00 spectra/sec acquisition scan rate in extended dynamic range mode

(2 GHz); 300° C. gas temperature; 8.0 l/min. gas flow rate; 35 psig nebulizer

pressure; 350° C. sheath gas temperature; 11 l/min. sheath gas flow rate; 3500

Vcap; 1000 V nozzle voltage; 175 V fragmentor and 65 V skimmer. Internal

reference samples inherent to any analysis performed with this instrument occurred

at +121.05087300 and +922.00979800 m/z . System calibration and auto-tuning was performed prior to any and all analysis.

For the purpose of confirming the presence of endogenous eCBs both AEA and OEA standards, and both A. mellifera and T. castaneum extracts, were used for

targeted tandem mass spec-mass spec product ion analysis. Each sample was

prepared as previously described herein. Pure standard of AEA was used to

determine the optimal collision energy for product ion fragmentation and the

retention time prior to determining the endogenous presence of AEA in both

organism extract samples. Optimal collision energy was experimentally determined

to be 25 eV, with testing also performed at 15 eV for AEA, and retention times of

approximately 5.6 and 6.0 minutes for AEA and OEA, respectively. OEA was spiked

into both organism extract samples to determine assay functionality and

confirmation of the retention time within each extraction matrix. Only the

endogenous AEA was assayed in A. mellifera thorax and T. castaneum whole body extracts. Parent-to-product ion fragmentation of AEA was m/z 349.18 ‰ 62.06 and

for OEA m/z 324.98 ‰ 62.06.

Prior to sample assays and after completing all assay elutions a column flush program was used to remove any contamination, standard molecule carry-over and slip additives from the column. This program consisted of the previously described method herein using an extended solvent linear gradient and isocratic period totaling 20 minutes (10 minutes each), with all solvent elution diverted to waste.

This flushing program was performed in duplicate.

2.2.g. Sample Data Analysis – Qualitative Identification

Each sample injection produced a Total Ion Count (TIC) chromatogram,

showing the full response of all detected ions within the 100-3000 m/z range for

the recorded time period, as an individual data file with an appropriate name to

reflect the organism/body segment, date and replication number. These individual

files were then analyzed using Agilent MassHunter qualitative analysis v. B. 05.00

software.

All analyzed endogenous samples were compared to both the chromatogram

retention times and mass spectrometer ion results of known pure standards

acquired from Caymen Chemicals, Inc. assayed concurrently with each sample

series. Acceptable variances for comparisons in terms of retention time and m/z were determined to be ±0.2 minutes and ±25.0 m/z . Additionally, dopamine and

serotonin derivatives are known to produce oxidized compounds in solution and in

contact with atmosphere. These quinones should also be determined and, if any are

detected, added to the total amount of the reduced form of the molecule.

Endogenous determination of each molecule was determined using the

following method with the Agilent MassHunter software, based on initial

determinations from the first set of standards using procedures contained within

the software, optimized based on greater usage and understanding of the software

and principles of mass spectroscopy.

After executing each sample injection data file the molecular formula for the

respective molecule of interest was input into the Find by Formula- Options tab

located under the Find Compounds by Formula section of the Methods Explorer

menu. The Matches per Formula option was set to 3, with the option to match for

isomeric compounds turned on. Match tolerances were set to ±100 ppm masses

and ±0.35 retention time. Expansion of values for chromatographic extraction was

set to systemic ±35.0 ppm with limited EIC extraction range left to default values.

The expansion values were varied as needed to obtain optimal results as

determined by the known standard analysis as this often varied by molecule.

Positive ions were set to H + and Na + only. Settings under the Results tab were left to default values, ensuring that Extract EIC and Extract clean spectrum are marked as on. Under the Results Filter tab all options were marked off except the option to only match formulas to generated compounds. After confirmation of these options the operation was executed by selecting all of the relevant samples in the subsequent menu. The results produced is the individual Extracted Ion Count chromatograms (EIC), a sortable table of the EICs showing the area of the peak(s) for the compound with retention times, and various other values, with a

50 corresponding mass spectrogram showing the ion and isotope distribution for each

EIC. The table is then sorted by retention time, highlighting the standards used, and any EICs listed outside of the acceptable variance are removed. In the event that the standards produce multiple EICs of various retention times only those EICs with similar retention times to the original standard sets assayed, and those with the expected higher peak areas, were used with all others disregarded. These disregarded results for the standards are considered on-column contaminants and/or molecules similar enough to match the given formula within the programmed tolerances. The remaining EICs and mass spectrograms are then compared to the standard. Ion and isotope abundance are compared in the context of the acceptable tolerances set as well as the overall “spread” of the isotopic ion abundance. Systemic variances of these spectrograms are endemic to this process due to the overall crude and complex nature of the endogenous samples as well as the oxidation of some of these molecules. (12, 22, and 31). Further details will be discussed in later sections below. As an additional cross-reference between potential endogenous samples and the standards a comparison of the target mass- to-charge ratio difference should also be taken into account. Finally, the software also outputs a Score value which takes into account several variables including target m/z differences, predicted ion masses and isotopic abundance pattern. Due to these parameters, this value is highly dependent upon sample purity and concentration and should not be readily considered for determination of compound identification. However, a high Score value, in conjunction with an acceptable retention time as compared to the standard, does not necessarily detract from a positive identification of a compound. (12)

After a sample series is identified as an endogenous compound the file is

then saved as a tab delimited Excel file from the Export option ensuring that

retention time and peak area are stored. This file can then be copy and pasted into

an existing Excel file for further data analysis and calculations.

2.2.h. Sample Data Analysis – Quantitative Analysis

After qualitative identification of the sample series is complete and all relevant data is stored and sorted, the area for each compound determined is then calculated into a corresponding endogenous amount within the organism/body segment.

For each sample series confirmation of the internal standard of N-

arachidonoylglycine-d8 must be determined prior to any quantitative analysis as

well as a compound’s corresponding standard curve. Each endogenous sample

series average area of the curve must be adjusted using the internal standard

average area of the curve present in the sample series with a pure sample injection

of the internal standard. First, the average area of the curve for the internal

standard co-injected with the endogenous sample is weighted against an injection

assay of the pure internal standard prior to any endogenous samples for that day’s

analyses. This ratio of pure internal standard to co-injected I.S. is then multiplied

into the average and standard deviations of all endogenous compound area of the

curves. This is done to compensate for day-to-day instrument deviations, potential

changes in column conditions and other similar variables such as solvent changes,

variances in temperature, etc. For any oxidation products detected in the acyl

dopamine series the area of the curve for the quinones are added to the area of the

52 reduced molecule prior to this correction, though are generally negligible when detected.

The compensated averages and standard deviations are then calculated into corresponding pmol/gram amounts using the slope of a standard curve for the corresponding compound. This calculation is determined prior to any analysis and is a series of stoichiometric conversions using the amount of the sample diluted for injection, the total volume diluted into, the calculated amount delivered to the column, the amount of the organism/body segment used for the sample and finally the slope of the standard curve. In general mathematical principles, all of these calculations can be summed into one value that is then divided by the slope and multiplied by the corrected average of the area of the curve for the endogenous compound. (12, 17, 21) This calculation is repeated for the standard deviation as well. Both values are in pmol/gram of organism. Care must be taken to ensure that these calculations are correct. An example of a data set and the calculation process is shown in Figure 2.3 below.

Finally, after each sample series is calculated into an average pmol/gram ±

S.D. value these averages are further averaged together based on the total number of sample series’ assayed, with any on-column contamination amounts subtracted out using the same methods applied to the corresponding blanks assayed in between each sample series. The resultant aggregate average and standard deviation now reflects the final endogenous amounts of the corresponding compound detected.

D8-NAG 373000000 Blanks 33991 C22H27D8NO3 OEA 49365 RT = 5.7 C20H39NO2 16519 Std 193693 326.3,327.3 13836 220561 RT = 5.9 28773 193306 28496.8 199244 14354.02 avg 201701 Avg 3.1 S.D 1.5

D8 OEA D8 OEA D8 OEA D8 OEA D8 OEA 6.12 576215 289080 6.11 152504 12938 6.11 A 161391 12931 6.11 B 221110 31256 6.11 C 136139 17801 519932 274425 153136 14501 144256 12088 178403 33159 133453 15714 438249 244082 145915 14738 141792 13300 136422 26155 165551 12267 299239 134886 97229 14037 128876 12119 150179 23829 165551 13360 645015 219384 146837 13984 201984 14351 165215 20106 136098 15139 avg 495730.0 92948.6 Avg 139124.2 20357.42 Avg 155659.8 16845.14 avg 170265.8 31743.18 avg 147358.4 20352.99 Correction 0.4 24325.8 1.45 1003.876 1.30 1219.328 1.18 6307.603 1.37 2940.734

Avg 99.68 21.83 18.06 34.04 21.83 S.D 2.61 1.08 1.31 6.76 3.15

Total of all 36.0 Avg determinations 7.9 S.D

. = ., / ×

. . .. = . . . .

.. = × 400000 = 99.68 pmol/gram

Figure 2.3: Example data set taken directly from results file (OEA, Tribolium ) and an example calculation for the quantitative analysis of OEA used for all endogenous endocannabinoids/fatty acid amides. This same calculation is used for the standard deviation of pmol/gram and for the carry-over/contamination amounts if detected, which are subtracted from the average pmol/gram.

2.2.i. Sequence Homology with D. melanogaster AANAT

Expression of fatty acid amides endogenous to A. mellifera and T. castaneum

requires the existence of arylalkylamine N-acyltransferases (AANATs) such as those

previously determined within Drosophila melanogaster . (17) Utilizing the NCBI

database (http://www.ncbi.nlm.nih.gov/), sequence homology comparisons

between the known D. melanogaster NAT CG9486 (NP_609005.1) with possible

NATs in both A. mellifera (taxid: 7460) and T. castaneum (taxid:7070). An online protein BLASTp sequence analysis using the NCBI database software of CG9486 with these organisms produce homology sequences with two isoforms of dopamine

N-acetyltransferases in both organisms, NP_001139380.1 and NP_001139379.1

(Tribolium isoforms 1 and 2, respectively); XP_392876.2 and XP_006566050.1 ( A. mellifera isoformX1 and X2, respectively). All sequence comparisons showed greater than 30% identity with coverage greater than 93%. Additionally, sequence comparison of protein residues was also performed showing conserved residues between all three organisms. (17) MEME motif studies were conducted by uploading the above amino acid sequences into the MEME tool located at http://meme.nbcr.net/meme/ and exporting as an htmL document.

2.3 Results and Discussion

2.3.a. Standard Samples, Standard Curves and Spiked Standards in Apis

mellifera Thorax

The N-acylated derivatives of ethanolamines (NAEs), dopamines (NADs),

serotonin (NOSt), glycines (NOGly) and primary fatty acid amides (PFAAs)

investigated in this study are summarized in Table 2 .2 above . Stated retention time is the average from all standard mixes assayed concurrently with each sample series, including any oxidation side-products when detected . Assays of individual

separate standards is not shown, as these were not indicative of the molecules

within the endogenous sample series tested due to matrix effects (dynamic sample

mixture, solvent effects, precipitation, etc .) and other uncontrollable and unknown

conditions .

Table 2.2. Summary of standard molecules used. Included is the abbreviated name, average retention time ±0.2 minutes, mass-to-charge ratio of the proton and sodium adducts and the molecular formula. Also included are the known possible oxidation products, when applicable.

N.D. is not detected with adequate reproducibility

Only the standards as a total mix were used to best mimic the complex

matrix environment of the endogenous samples . The stated mass-to-charge ratios

are that for the protonated ionized parent molecule, [M+H] +, and that of the

sodium adduct of the ionized parent molecule, [M+Na] +, respectively . These values are derived directly from the standards of these molecules as observed .

Construction of the standard curves proceeded as previously described.

Examples of these standard curves, and the corresponding mass spectral results,

are presented below for the internal standard D 8-NAG (Fig . 2.4). All other standard

curves and mass spectral results are presented in the appendix of this chapter .

Documented retention times for each standard sample shown are within the

aforementioned ±0.2 minute tolerance, with the average retention time for each

compound labeled within the graph . Percent error of the standard curves is less

than 5% for each data point shown . X-axis intercept values are not shown as each

sample gave values less than 10% of the calculated slope and thus would calculate

outside of the acceptable limits of significant figures . Calculated slope values shown

all provided R 2 linearity values >0 .95 . Mass-to-charge ratio values for both proton

and sodium adducts are within 25 ppm of the expected values per those calculated

via the software . The presence of oxidation products in the N-acyldopamine series

is also compensated for, as previously discussed in the methods portion .

Standard curves for the primary fatty acid amides are not shown and slope

values were provided by Jeffries, et al ., (12) using the same assay methods, stock

samples and column . Only the individually-assayed standards are provided

(Appendices) .

In an effort to control for matrix effects, cellular oxidation and other variables

due to assaying relatively impure and highly complex mixtures from these

organisms, a brief series of controlled spiked samples were performed (OEA

example, Figure 2 .5). Two sample sets of farmed A. mellifera thorax were

extracted and purified with the addition of known amounts of OEA, AEA, NAD and

NOG added at the beginning of the extraction process . An additional sample series

included these compounds just prior to purification only . Each set was assayed as

described previously then compared to the standard mix of these compounds that

were concurrently assayed . Only the samples spiked prior to extraction is shown,

as the samples spiked prior to purification did not complete the assay process

correctly (data not shown), likely due to high concentrations saturating instrument

detection or instrument error and time constraints . Side-by-side comparisons of

the spiked samples with the standard mix clearly shows a pattern of retention time

deviation within the acceptable tolerance of ±0 .2 minutes, likely due to the

aforementioned matrix effects, instrument variation and column conditions .

Additionally, these results also show changes in the detected m/z values and the

changes on ion distributions, likely a result of decreased concentration directly

affecting instrument analysis and computation of those values . Such errors and limitations were proven to be highly pervasive within the entirety of the data collected . Suggestions for the mitigation or elimination of these errors include use of a column in better condition and more extensive sample clean-up prior to assaying . Column/instrument contamination and compound cross-over of previous

samples also occurred, likely also due to the complex nature of the extracted

samples and instrument limitations

Figure 2.4. Example Standard Curve and MS results for D 8-NAG I.S.

59 thorax). and time retention on condition column 2.5 Figure . Spiked OEA in OEA Spiked . A. mellifera A.

thorax showing sample matrix effects, instrument de instrument effects, matrix sample showing thorax m/z aito (et sadr OA Rgt ageae sp aggregate Right: OEA, standard (Left: variation viation and and viation iked iked

2.3.b. Endogenous Results – Farmed and Feral A. mellifera

Table 2.3. Summary of quantitative results for eCBS/FAAs in farmed A. mellifera . Amounts are reported as average ± S.D in pmol/g. of organism from 20 individual assays of heads, 28 from thorax and 24 from abdomen. Farmed A.M. (pmol/g) Molecule Heads Thorax Abdomen N- Oleoylethanolamine 30 ± 8 6 ± 7 437 ± 122 N- Arachidonoylethanolamine 41 ± 3.0 6 ± 5 10 ± 6

N- Palmitoyldopamine 4 ± 0.5 11 ± 9 26 ± 10 N- Oleoyldopamine * 19 ± 6 28 ± 9 N- Arachidonoyldopamine ***

N -Palmitoylglycine 125 ± 41 16 ± 9 52 ± 18 N- Oleoylglycine 31 ± 1 6 ± 2 4 ± 2 N- Arachidonoylglycine 25 ± 3 14 ± 3 3 ± 1

N- Oleoylserotonin ***

Linoleamide 0.2 ± 0.5 11 ± 6 44 ± 15 Palmitoleamide 143 ± 200 114 ± 83 356 ± 288 Palmitamide 17 ± 5 12 ± 5 20 ± 13 Oleamide 179 ± 43 51 ± 15 85 ± 37 *Compound not detected with adequate reproducibility

Table 2.4. Summary of quantitative results for eCBs/FAAs in feral A. mellifera. Amounts are reported as average ± S.D in pmol/g. of organism from 48 individual assays of heads and 16 each from thorax and abdomen. Feral A.M. (pmol/g) Molecule Heads Thorax Abdomen N- Oleoylethanolamine 35 ± 16 31 ± 16 1918 ± 110 N- Arachidonoylethanolamine 27 ± 18 17 ± 9 *

N- Palmitoyldopamine 76 ± 29 20 ± 21 140 ± 6 N- Oleoyldopamine 46 ± 24 * * N- Arachidonoyldopamine ***

N -Palmitoylglycine 36 ± 29 50 ± 29 220 ± 72 N- Oleoylglycine 32 ± 4 18 ± 14 32 ± 14 N- Arachidonoylglycine 12 ± 7 * 21 ± 9

N- Oleoylserotonin 45 ± 26 59 ± 38 362 ± 170

Linoleamide 45 ± 21 24 ± 25 153 ± 39 Palmitoleamide 398 ± 141 911 ± 389 1423 ± 639 Palmitamide 127 ± 19 13 ± 6 80 ± 12 Oleamide 163 ± 57 948 ± 634 438 ± 164 *Compound not detected with adequate reproducibility 61

Samples for the detection of the endogenous eCB/FAAs proceeded as

previously described with the awareness of the aforementioned limitations observed

with the standards and spiked assays . Sample reproduction yielding results within acceptable values was difficult to acquire and is again likely due to the aforementioned limitations . Attempts at mitigating these errors and limitations provided limited success . The total number of assayed samples for the entirety of this study was prohibitively high (>200) considering the nature and preparation of the samples and the instruments used .

For clarity, the aggregate of all body segments from farmed A. mellifera is

shown as an overlapping spectrum per each compound, with select mass spectral

results shown also for clarity . All utilized data was comparable however for detected

molecules . Several examples are shown in Figures 2 .6-2.8, with the remaining supplied in the appendix of this chapter .

Reproducible results, or sufficient results to provide a statistically acceptable

value, were unobtainable for NAD in all body segments for both farmed and feral A.

mellifera . Any detected results were likely due to anandamide “shadow” peaks .

Therefore, the endogenous detection of this compound is inconclusive in both

groups of this organism . It is unlikely that the similarity of these results is due to

direct environmental or genetic differences between the two groups; rather it is

more likely that NAD exists below the detectable threshold of the assay technique

and instrument . Overall concentration of NAD within each organism may vary due

to unknown factors at the time of collection . An example of such a variable may be body temperature, as it has been speculated that NAD may be part of body 62

temperature regulation in other animals, though no evidence to date thus far

suggests this is the case in insects . Another factor is that NAD in low endogenous

concentrations may have readily oxidized to the quinone/indole forms, aggregated

and precipitated out either during the extraction and purification process or prior to

assaying of the diluted samples, thus preventing reliable and reproducible detection

by mass spectroscopy . Without a reliable method of measuring and quantifying this oxidation phenomenon overcoming this limitation using these techniques is difficult at best . One method that could provide quantifiable results would be the use of an

oxidizing agent such as sodium periodic acid with known amounts of heavy-labeled

compounds in vitro then analyzed via mass spectroscopy . (22) However, periodic

acid usage in HPLC-MS systems is generally unacceptable do to damaging the

instrument .

NOSt was undetected in all three farmed body segments, unlike in the feral

body segments as presented in Table 2 .4. The reasons for this discrepancy is similar to those discussed for NAD, though the nature and function of NOSt is currently unknown in insects and may or may not be related to direct environmental or biological factors, such as body temperature regulation as posited for NAD . Serotonin is known to be a digestive system signaling and mediation neurotransmitter in higher animals, such as mammals, and has only very recently been determined to have similar roles in A. mellifera . (32) Since the highest

detected levels of NOSt from this study is in the abdomen (362 ± 170 pmol/g),

such data is therefore plausible . Concurrently, Verhoeckx and colleagues have

detected endogenous levels of N-acylserotonin within mice and pigs primarily

localized in the intestine . Their data suggests direct links to the endogenous levels

of these molecules with serotonin and a diet supplemented with fatty acids . (33)

Such research further sheds light, and lends strong evidence, to the currently

proposed biosynthetic and degradation pathways having been mentioned,

specifically the direct conjugation of fatty acids to fatty acid amides and the

degradation/regulation via FAAH (Figs . 1.3-1.5) . An additional source for this discrepancy may also be the existence of microbiota within or on the feral honeybees which may be contributing to these endogenous levels . Much of the

recent work with N-acylserotonins is providing very promising and exciting results

within the field of endocannabinoid research .

In general, a strict comparison of the endogenous levels of the remaining

compounds gives comparable results within an acceptable order of magnitude .

Much of the variations shown for many of these molecules can generally be

attributed to factors including variability in the extraction and purification process,

instrument variation, and unknown biological factors contributing to the expression

of these compounds in vivo . N-oleoyldopamine, however, in comparison between farmed and feral A. mellifera does show discrepant results . Between farmed and

feral groups the overall detection of NOD in specific body segments is not

consistent . For farmed bees NOD is detected with sufficient reproducibility in thorax and abdomen, while being absent or inconsistent in the heads . In contrast, the

opposite results occurred for the feral population . It is generally assumed such

discrepencies with N-acyldopamines specifically is due, at least in part, to their

predilection for oxidiation to polymeric structures which are difficult to detect and

quantifiy . (22) Additionally, because of the inherently high deviations from the

methods employed such discrepancies in detection can be expected . Exceptions would be any recurring patterns such as these occurring in other compounds between the two populations . Indeed, these high deviations in detected levels are

higher than the average levels calculated . An example of this is the primary fatty acid amides in both farmed and feral populations . As such it is debatable whether

these endogenous values are indeed valid . However, the PFAAs are generally

considered a separate class of molecules that have been extensively researched in

their own right and are tangentially related to the eCBs/FAAs in the scope of this

work . PFAAs presented herein are shown for the purpose of giving context and

broader relevance whilst connecting to established endogenous molecules .

Additionally, specific PFAAs, such as Oleamide and Palmitamide, are known slip-

additives in many laboratory plastic-ware and must be compensated for, as

described previously . (26, 25)

A final note on the quantification of the endogenous eCB/FAA levels using

standard curves are variations in the column and instrument over time between the

measurement of the standard curves and the measurement of the endogenous

levels . System response and column conditions are likely to change with extensive

usage, therefore a strict comparison of the response for standards with endogenous

levels over a separated period of time are increasingly likely to show deviation .

Generally speaking, standard curves should be repeated every three to six months,

depending on usage, to further compensate for such deviations . At the time of the

endogenous assays the provided standard curves were nearing the six month 65

limitation . Repeating the standard curves was not possible due to lack of access to

the HPLC-QTOF/MS upon completion of the endogenous assays .

The determination of these compounds being truly endogenous to these organisms would not be complete without a tandem mass spectroscopy assay . A targeted MS/MS assay is employed to show the expected product ion fragmentation from a given parent ion, such as [M+H] +. For this purpose, a targeted MS/MS

comparative study between standard anandamide, spiked OEA and endogenous

anandamide was conducted in A. mellifera thorax . Standard AEA was used to

determine the retention time (~5 .6 minutes) and optimal fragmentation voltage

(25 eV) using the standard assay techniques described previously . OEA was used in

conjunction as a spiked standard for the same purpose and to observe any potential

deviations due to matrix effects, etc . (~6 .0 minutes, 25 eV) . Both compounds gave

the expected product ion fragment of m/z 62 corresponding to the ethanolamine

fragment (Fig . 2.9 and 2 .10) . The remaining fragments, long-chain aldehydes of

m/z 289 .2 and 267 .2 for AEA and OEA respectively, are likely either obscured

during detection by other background ions or exist as uncharged species . Though

not predicted, the fragment at m/z 287 .2 may be this remainder existing as an

[M] + species .

Though limited, these results are fairly indicative of the endogenous nature

of the eCBs/FAAs described within this study and provides some validation of these

results in the Apis mellifera species .

Figure 2.6. Aggregate spectra and MS results of endogenous OEA and NPD in farmed A. mellifera .

Figure 2.7. Aggregate spectra and MS results of endogenous acyl glycine NPG and Palmitamide in farmed A. mellifera .

Figure 2.8. Aggregate spectra and MS of suspected endogenous N-oleoylserotonin in both farmed and feral populations of A. mellifera . .

O OH N Anandamide (AEA) H m/z [M+H]+: 348.28 (100.0%), 349.29 (24.3%), 350.29 (3.2%)

O OH + H2N

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenal 2-aminoethan-1-ol m/z [M+H]+: 289.25 (100.0%), 290.25 (22.0%), 291.25 (2.5%) m/z [M+H]+: 62.05 (100.0%), 63.06 (2.3%)

O OH N Anandamide (AEA) H m/z [M+H]+: 348.28 (100.0%), 349.29 (24.3%), 350.29 (3.2%)

O OH + H2N

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenal 2-aminoethan-1-ol m/z [M+H]+: 289.25 (100.0%), 290.25 (22.0%), 291.25 (2.5%) m/z [M+H]+: 62.05 (100.0%), 63.06 (2.3%)

Figure 2.9. Targeted MS/MS spectra of standard AEA at 15 eV collision energy showing the absence of the expected product ion fragment, m/z 62.06. Targeted MS/MS spectra of standard AEA at 25 eV collision energy showing the presence of the expected primary product ion fragment, m/z 62.06 and corresponding decrease in the parent ion, m/z 348.2. Inset shows the predicted fragmentation pattern for AEA.

O OH N Anandamide (AEA) H m/z [M+H]+: 348.28 (100.0%), 349.29 (24.3%), 350.29 (3.2%)

O OH + H2N

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenal 2-aminoethan-1-ol m/z [M+H]+: 289.25 (100.0%), 290.25 (22.0%), 291.25 (2.5%) m/z [M+H]+: 62.05 (100.0%), 63.06 (2.3%)

Figure 2.10. Targeted MS/MS spectra of AEA in A. mellifera extract at 25 eV collision energy showing the expected primary product ion fragment, m/z 62.06, and targeted MS/MS spectra of spiked OEA in A. mellifera extract at 25 eV collision energy showing the same expected product ion fragment. Inset shows the predicted fragmentation pattern for AEA and OEA, respectively.

2.3.c. Endogenous Results – T. castaneum

Table 2.5. Summary of quantitative results for eCBs/FAAs in T. castaneum . Amounts are reported as average ± S.D in pmol/g of organism from 20 individual assays of whole bodies. Tribolium (pmol/g) Molecule Whole Body N- Oleoylethanolamine 36 ± 8 N- Arachidonoylethanolamine 42 ± 24

N- Palmitoyldopamine 53 ± 25 N- Oleoyldopamine 21 ± 6 N- Arachidonoyldopamine 26 ± 14

N -Palmitoylglycine 25 ± 12 N- Oleoylglycine 16 ± 9 N- Arachidonoylglycine 18 ± 17

N- Oleoylserotonin *

Linoleamide * Palmitoleamide 178 ± 83 Palmitamide 53 ± 37 Oleamide 102 ± 45 *Compound not detected with adequate reproducibility

As for the honeybees, samples for the detection of the endogenous eCB/FAAs

in T. castaneum proceeded as previously described with the awareness of the aforementioned limitations observed with the standards and spiked assays. Sample reproduction yielding results within acceptable values was equally as challenging for

T. castaneum as it was for A. mellifera; again likely due to the aforementioned

limitations. Attempts at mitigating these errors and limitations provided limited

success for T. castaneum as well. The total number of assayed samples was also prohibitively high (>30) considering the nature and preparation of the samples and the instruments used, though not as high as compared to A. mellifera due to the

beetles being whole body extracts and not three separate segments.

For clarity, the aggregate of all assays for each molecule from T. castaneum

is shown as overlapping spectra, with select mass spectral results also shown for 72

clarity. All utilized data was comparable however for detected molecules. Several

examples are shown in Figures 2.11-2.12, with the remaining supplied in the

appendix of this chapter.

Reproducible results, or sufficient results to provide a statistically acceptable

value, were unobtainable for NOSt in T. castaneum and is inconclusive for this molecule. Like A. mellifera , it is unlikely that the inability to adequately detect and

quantify NOSt is due to any environmental or biological issue, but rather is likely

due to the endogenous levels existing below the detectability limit of the

instrument. Another potential factor, like that of NAD, is that NOSt in such low

endogenous concentrations may have readily oxidized to a form similar to the

quinones/indoles produced by N-acyldopamine oxidation. If so, NOSt may have aggregated and precipitated out either during the extraction and purification process or prior to assaying of the diluted samples, thus preventing reliable and reproducible detection by mass spectroscopy. Similarly, without a reliable method of measuring and quantifying this oxidation phenomenon overcoming this limitation using these techniques is difficult at best. The suggested method as described for the NAD, the use of periodic acid to induce oxidation of heavy-labeled compounds in conjunction with HPLC-MS, is again generally unacceptable do to damaging the instrument.

Additionally, the detected levels of NAG, 18 ± 17 pmol/g, suggests that this compound may not exist endogenously, statistically-speaking. It is likely that NAG does exist endogenously in comparison to A. mellifera and also by taking into account that the detected value for NAG does not follow the observed trend in the data for the other molecules present. The cause for such an outlier is suggested to

be similar to the absence of NOSt, that being low endogenous levels existing below

the detection limit of the instrument in conjunction with various other limitations

aforementioned.

The data collected for Linoleamide was inconclusive due to any endogenous

values having been subtracted out when compensating for carry-over and/or the

existence of slip additive determined from control samples.

Unlike honeybees however, the presence of NAD is detected and measured in

the red flour beetles (26 ± 14 pmol/g). Such data may suggest that the levels of

NAD are higher in this organism than in A. mellifera for it to be at such a detectable

level.

In general, a strict comparison of the endogenous levels of all assayed

compounds gives comparable results within an acceptable order of magnitude, with

relatively low deviations in comparison to the results presented for A. mellifera.

Again, variations shown for many of these molecules can generally be attributed to factors including variability in the extraction and purification process, instrument variation, and unknown biological factors contributing to the expression of these compounds in vivo .

It should be noted that the statistical variations of the data as a whole, with the exceptions previously noted, is comparatively low to that of A. mellifera (and to

a lesser degree the results published for D. melanogaster by Jeffries, et al .). A

likely cause for these comparatively positive results to A. mellifera is that all assays

used to derive these values were conducted during the same assay session in a

relatively short time span (6-8 hours for Tribolium versus 12-14 hours for

Mellifera ). In the context of the prior argument that the deviations of these values

are in part due to possibly outdated standard curves, and to day-to-day instrument

variations, these results suggest that instrument variation has a greater impact.

As for A. mellifera , confirmation of the endogenous nature of these molecules

requires a targeted MS/MS assay . Similar to that performed for A. mellifera , spiked

OEA and endogenous AEA were studied using the aforementioned MS/MS technique in Tribolium whole-body extract . Standard AEA was again used to determine the

retention time (~5 .6 minutes) and optimal fragmentation voltage (25 eV) using the standard assay technique . OEA was used as a spiked standard for the same purpose and to observe any potential deviations due to matrix effects, etc . (~6 .0 minutes, 25 eV) . Both compounds gave the expected product ion fragment of m/z

62 corresponding to the ethanolamine fragment (Fig . 2.13) . The remaining fragments, again long-chain aldehydes of m/z 289 .2 and 267 .2 for AEA and OEA

respectively, are likely either obscured during detection by other background ions

or exist as uncharged species, just as in the A. mellifera MS/MS results . Also

similar to A. mellifera results, the fragment at m/z 287 .2 may be this remainder existing as an [M] + species .

For T. castaneum , these results are fairly indicative of the endogenous

existence of the eCBs/FAAs described within this study and provides validation of

these results in the T. castaneum species .

Figure 2.11. Aggregate spectra and MS of endogenous OEA and the acyl dopamine NOD in T. castaneum .

Figure 2.12. Aggregate spectra and MS of endogenous the N-acylglycine, NOG, and of the PFAM Palmitamide in T. castaneum .

O OH N Anandamide (AEA) H m/z [M+H]+: 348.28 (100.0%), 349.29 (24.3%), 350.29 (3.2%)

O OH + H2N

(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenal 2-aminoethan-1-ol m/z [M+H]+: 289.25 (100.0%), 290.25 (22.0%), 291.25 (2.5%) m/z [M+H]+: 62.05 (100.0%), 63.06 (2.3%)

Figure 2.13. Targeted MS/MS spectra of endogenous AEA in T. castaneum extract at 25 eV collision energy showing the expected product ion fragment, m/z 62.06, and of spiked OEA with the same expected product ion.

2.3.d. Genetic Homology Study of AANAT between D. melanogaster , A. mellifera

and T. Castanaeum .

The final portion of this research into the existence and quantification of the endogenous endocannabinoids/fatty acid amides is a brief genetic study into the existence of a putative arylalkylamine N-acyltransferase within both T. castaneum and A. mellifera. An AANAT recently discovered and shown to produce the requisite chemistry in D. melanogaster (17) is compared to two similar homologs in both organisms using the NCBI database BLASTp tool as described previously (Fig.

2.14). From the sequence comparison of the protein residues several conserved regions were detected, showing significant sequence homology between all three organisms. Furthermore, to aid in this comparison, a motif-sequence analysis study of AANAT homology using MEME Suite (Multiple EM Motif Elucidation, http://meme.nbcr.net/meme/) was also conducted on the truncated forms of these

AANATs from all three organisms (Fig. 2.14, lower portion). These motifs are the protein sequence regions predicted to produce distinct secondary structures of a fully transcribed mature and active enzyme. (34) This prediction goes beyond simple residue sequence comparison by adding a second layer of homology determination between these enzymes. Additionally, motif prediction can determine if an already-known catalytic site shares both sequence homology and secondary structure homology, such as in the case of the AANAT from D. melanogaster

(wherein the Glu-47 residue was shown to be essential in the catalytic site) (35), as it is the secondary structure that is key to the enzyme’s function. As can be seen in

Figure 2.14 there is significant sequence and corresponding motif homology

between all four of the AANATs, as compared to the now known AANAT from D.

melanogaster .

Figure 2.14. Top: Protein residue sequence homology of AANATs showing conserved residues/domains between D. melanogaster , DM, with A. mellifera isoforms, AM1 and AM2, and T. castaneum isoforms, TC1 and TC2. Bottom: Comparison of sequence homology in the context of predicted secondary structure motifs between T. castaneum, A. mellifera and D. melanogaster AANATs (specifically dopamine N- acyltransfereases). Height of each motif block indicates relative homology between each organism.

2.4 Conclusions and Recommendations

Because of the recent completion of genome sequencing for both the

honeybee (Apis mellifera ) and red flour beetle (T. castaneum ) their use as new model organisms are now feasible, within limitations. In conjunction, the environmental, agricultural and potential human impact of these organisms has made research such as this vital in recent years. With the recent discovery of endocannabinoids and fatty acid amides in Fruit flies, and a likely enzyme in the biosynthetic process, research into the endocannabinoid system (or the

“endocannabinome”) of insects is now of growing interest. To that end, this work set out to identify and quantify the eCBs/FAAs in these two novel organisms in conjunction with a brief genetic comparison using the known Fruit Fly enzyme shown to conduct the requisite biosynthetic production of these molecules.

Based on the results presented within this work, though limited, there is now fair evidence to support the theory that (a) the eCBs/FAAs are indeed endogenous molecules within these two additional insect species as compared to Fruit flies and

(b) genetic comparison studies show that a known biosynthetic enzyme very likely does exist within these insects to produce these molecules.

With the discovery of the Fruit Fly AANAT, and the homology of the AANAT within mellifera and Tribolium , a specific biosynthetic pathway can thus be proposed, as presented in Figure 2.15 below using N-acyldopamine as an example based on Chapter 1.

Additionally, a new hypothesis for the in vivo /physiological activity of the

NADs based on their known quinone/indole oxidation products is now proposed.

Due to the similarities of these quinone/indole products to serotonin, the observed

effects of the NADs as have been presented may be due to these quinone/indole

forms and not the reduced form of the N-acyldopamines. If so, the observed levels

of activity may not fully reflect the actual physiological amounts and may actually

be significantly higher. If true, the physiological impact and significance of the

NADs may also be higher.

Figure 2.15. The proposed key step in the biosynthesis of an N-acyldopamine endocannabinoid/fatty acid amide based on AANAT sequence and motif homology for D. melanogaster, A. mellifera and T. castaneum.

Recommendation for further research as a continuation of this work includes

refining the quantification of the endogenous levels, performing a heavy-labeled

fatty acid/fatty acid-coA feeding study, RNAi or AANAT gene expression knockout

with Tribolium , and the expression, purification and kinetic evaluation of Tribolium -

derived AANAT and FAAH from e. coli . The apparent absence of known receptors for

these molecules in insects also must be addressed.

Refining the endogenous levels of the eCBs/FAAs in both organisms should

proceed with a separate column isolated from any other use, preferably utilizing

ion-trap mass spectroscopy in order to “pool” the ions for better quantification and further MS/MS studies using other fatty acid amides.

Heavy-labeled fatty acid studies with Tribolium would be relatively straight

forward and should yield significant results, similar to those presented by Jeffries,

et al . for melanogaster (12). RNAi and gene knockout studies in Tribolium are

feasible, though time-consuming, very work-intensive and may not yield results 82

superior to a heavy-labeled feeding study thus far. A heavy-labeled feeding study

with mellifera would be challenging, but may be possible using heavy-labeled glucose as a food supplement to isolated hives over a potentially significant period of time. Such experiments may yield similar results to a feeding study with

Tribolium ; however, the time frame needed may be difficult to achieve without saturating the organism with heavy-labeled carbon atoms yet also long enough to ensure adequate assimilation into the fatty acid biosynthetic route. A separate possibility would be to use heavy-labeled nitrogen supplemented into the honeybee and red flour beetle diets, making tracing the heavy-labeled molecules potentially easier because of the existence of a very distinct heavy atom. Proper

RNAi/knockouts of mellifera is not currently feasible considering the nature, needs and behavior of the organism.

A very clear next step in furthering this work would be the expression, purification and evaluation of a Tribolium or mellifera -based AANAT, similar to that

done in Fruit flies by Dempsey, et al . (17, 35). Doing so can further confirm the

biosynthesis of these compounds in insects using these enzymes. Additionally, the

design, expression, purification and evaluation of any possible Tribolium/mellifera - based Fatty Acid Amide Hydrolase (FAAH) enzyme should also be undertaken to begin elucidation of the degradation pathways in insects.

Furthermore, both AANAT-knockout and FAAH-knockout Tribolium are feasible and may yield superior results to heavy-labeled feeding studies and RNAi experiments alone, by showing increases and decreases in endogenous levels while further specifying the nature and mechanism of the biosynthesis and degradation of eCBS/FAAs in these organisms.

Lastly, the need to determine the receptor(s) paired to these compounds in insects is a vital step in the overall progress of this research field, and may be the most challenging to date. The receptor-ligand pairing in both organisms is key to potentially utilizing the eCBs/FAAs in controlling or protecting Tribolium and mellifera species, respectively.

2.5 References

1. James, R. R.; Pitts-Singer, T. L., Bee pollination in agricultural ecosystems . Oxford University Press: Oxford; New York, 2008.

2. Oldroyd, B. P., What's Killing American honeybees? PLoS Biol 2007, 5 (6).

3. Richards, S.; Bucher, G.; al., e., The genome of the model beetle and pest Tribolium castaneum . Nature 2008, 452 (7190), 949-55.

4. Willis ER, R. L., The attraction of Tribolium castaneum to flour. Journal of Economic Entomology 1950, (43).

5. Grünwald, S.; Adam, I.; Gurmai, A.-M.; Bauer, L.; Boll, M.; Wenzel, U., The red flour beetle Tribolium castaneum as a Model to Monitor Food Safety and Functionality. In Yellow Biotechnology I , Vilcinskas, A., Ed. Springer Berlin Heidelberg: 2013; Vol. 135, pp 111-22.

6. van der Valk, H., Koomen, I., Aspects Determining the Risk of Pesticides to Wild Bees. Agriculture. Food and Agriculture Organization of the United Nations : Rome, Italy; 2013.

7. Krell, R., Value-Added Products from Beekeeping. Nations. Food and Agriculture Organization : Rome, Italy; 1996.

8. Sallam, M. N., Insect Damage: Post-Harvest Operations. Ecology. Food and Agriculture Organization of the United Nations : Rome, Italy; 1999.

9. Abdelgaleil, S. M.; Mohamed, M. E.; Badawy, M. I.; El-arami, S. A., Fumigant and Contact Toxicities of Monoterpenes to Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) and their Inhibitory Effects on Acetylcholinesterase Activity. Journal of Chemical Ecolology 2009, 35 (5), 518-25.

10. Wool, D.; Noiman, S.; Manheim, O.; Cohen, E., Malathion resistance in Tribolium strains and their hybrids: Inheritance patterns and possible enzymatic mechanisms ( Coleoptera, Tenebrionidae ). Biochemical Genetics 1982, 20 (7-8), 621-36.

11. Collins, P. J., A new resistance to pyrethroids in Tribolium castaneum (herbst). Pesticide Science 1990, 28 (1), 101-15. 12. Jeffries, K. A.; Dempsey, D. R.; Behari, A. L.; Anderson, R. L.; Merkler, D. J., Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism. FEBS Letters 2014, 588 (9), 1596-602.

13. Kohno, K.; Sokabe, T.; Tominaga, M.; Kadowaki, T., Honeybee thermal/chemical sensor, AmHsTRPA, reveals neofunctionalization and loss of transient receptor potential channel genes. Journal of Neuroscience 2010, 30 (37), 12219-29.

14. Dearden, P. K.; Duncan, E. J.; Wilson, M. J., The honeybee Apis mellifera . Cold Spring Harbor Protocols 2009, 2009 (6).

15. Bucher, G. The Beetle Book. Univ. of Göttingen, http://wwwuser.gwdg.de/~gbucher1/protocols.htmL.

16. McPartland, J.; Di Marzo, V.; De Petrocellis, L.; Mercer, A.; Glass, M., Cannabinoid receptors are absent in insects. Journal of Comparative Neurology 2001, 436 (4), 423-9.

17. Dempsey, D. R.; Jeffries, K. A.; Anderson, R. L.; Carpenter, A.-M.; Rodriquez Opsina, S.; Merkler, D. J., Identification of an arylalkylamine N-acyltransferase from Drosophila melanogaster that catalyzes the formation of long-chain N-acylserotonins. FEBS Letters 2014, 588 (4), 594-99.

18. Consortium, H. B. G., Insights into social insects from the genome of the honeybee Apis mellifera . Nature 2006, 443 (7114), 931-49.

19. Lehtonen, M.; Reisner, K.; Auriola, S.; Wong, G.; Callaway, J. C., Mass-spectrometric identification of anandamide and 2- arachidonoylglycerol in nematodes. Chemistry & biodiversity 2008, 5 (11), 2431-41.

20. Chu, C. J.; Huang, S. M.; De Petrocellis, L.; Bisogno, T.; Ewing, S. A.; Miller, J. D.; Zipkin, R. E.; Daddario, N.; Appendino, G.; Di Marzo, V.; Walker, J. M., N-oleoyldopamine, a novel endogenous capsaicin-like lipid that produces hyperalgesia. Journal of Biological Chemistry 2003, 278 (16), 13633-39.

21. Farrell, E. K.; Chen, Y.; Barazanji, M.; Jeffries, K. A.; Cameroamortegui, F.; Merkler, D. J., Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells. Journal of Lipid Research 2012, 53 (2), 247-56.

22. D'Ischia, M.; et al., 5,6-Dihydroxyindoles and Indole-5,6-diones. Advances in Heterocyclic Chemistry 2005, 89 , 1-63.

23. Davison, A.; Milan, A.; Dutton, J., Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry. Annals of Clinical Biochemistry 2013, 50 (3), 274.

24. Perez-Victoria, I.; Zafra, A.; Morales, J. C., Positive-ion ESI mass spectrometry of regioisomeric nonreducing oligosaccharide fatty acid monoesters: in-source fragmentation of sodium adducts. Journal of Mass Spectrometry 2008, 43 (5), 633-8.

25. McDonald, G. R.; Hudson, A. L.; Dunn, S. M.; You, H.; Baker, G. B.; Whittal, R. M.; Martin, J. W.; Jha, A.; Edmondson, D. E.; Holt, A., Bioactive contaminants leach from disposable laboratory plasticware. Science 2008, 322 (5903), 917.

26. Olivieri, A.; Degenhardt, O. S.; McDonald, G. R.; Narang, D.; Paulsen, I. M.; Kozuska, J. L.; Holt, A., On the disruption of biochemical and biological assays by chemicals leaching from disposable laboratory plasticware. Canadian Journal of Physiology and Pharmacology 2012, 90 (6), 697-703.

27. Herlinger, E.; Jameson, R. F.; Linert, W., Spontaneous autoxidation of dopamine. Journal of the Chemical Society, Perkin Transactions 2 1995, (2), 259-63.

28. Brown, S. J.; Shippy, T. D.; Miller, S.; Bolognesi, R.; Beeman, R. W.; Lorenzen, M. D.; Bucher, G.; Wimmer, E. A.; Klingler, M., The red flour beetle, Tribolium castaneum (Coleoptera ): A model for studies of development and pest biology. Cold Spring Harbor Protocols 2009, 2009 (8).

29. Folch, J.; Lees, M.; Sloane Stanley, G. H., A simple method for the isolation and purification of total lipides from animal tissues. The Journal of Biological Chemistry 1957, 226 (1), 497-509. 30. Sultana, T.; Johnson, M. E., Sample preparation and gas chromatography of primary fatty acid amides. Journal of Chromatography. A 2006, 1101 (1-2), 278-85.

31. Zoerner, A. A.; Gutzki, F. M.; Batkai, S.; May, M.; Rakers, C.; Engeli, S.; Jordan, J.; Tsikas, D., Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective. Biochimica et Biophysica Acta 2011, 1811 (11), 706-23.

32. French, A. S.; Simcock, K. L.; Rolke, D.; Gartside, S. E.; Blenau, W.; Wright, G. A., The role of serotonin in feeding and gut contractions in the honeybee. Journal of Insect Physiology 2014, 61 , 8-15.

33. Verhoeckx, K. C.; Voortman, T.; Balvers, M. G.; Hendriks, H. F.; H, M. W.; Witkamp, R. F., Presence, formation and putative biological activities of N-acyl serotonins, a novel class of fatty-acid derived mediators, in the intestinal tract. Biochimica et Biophysica Acta 2011, 1811 (10), 578-86.

34. Bailey, T. L.; Williams, N.; Misleh, C.; Li, W. W., MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Research 2006, 34 (Web Server issue), W369-W73.

35. Dempsey, D. R.; Jeffries, K. A.; Bond, J. D.; Carpenter, A. M.; Rodriguez-Ospina, S.; Breydo, L.; Caswell, K. K.; Merkler, D. J., Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases. Biochemistry 2014, 53 (49), 7777-93.

APPENDIX

Figure A.1 . Standard Curves a nd Standard MS results for N-arachidonylethanolamine, AEA (anandamide)

Figure A.2 . Standard Curves and Standard MS results for the N-acyletha nolamine OEA (oleoylethanolamine)

Figure A.3 . Standard Curve and Standard MS Results for the N-acyldopamine NPD. Note the presence of the quinone oxidation product in NPD at ~5.8 mins. 91

Figure A.4. Standard Curve and Standard MS Results for the N-acyldopamine NOD. Note the presence of the “shadow” or overlapping peaks between NOD/NAD at ~5.9 mins. 92

Figure A.5. Standard Curve and Standard MS Results for the N-acyldopamine NOD. Note the presence of the “shadow” or overlapping peaks between NOD/NAD at ~5.9 mins.

Figure A.6 . Standard Curve and Standard MS Results for N-palmitoylglycine, NPG. Note the distinct absence of compound overlap and oxidation products and the superior linearity as compared to the N-acyldopamine series. 94

Figure A.7 . Standard Curve and Standard MS Results for N-oleoylglycine, NOG .

Note the distinct absence of compound overlap and oxidation products and the superior linearity as compared to the N-acyldopamine series. 95

Figure A.8 . Standard Cu rve and Standard MS Results for N-arachidonylglycine NAG. Note the distinct absence of compound overlap and oxidation products and the superior linearity as compared to the N-acyldopamine series. 96

Figure A.9 . Standard Curve and MS Results for N-oleoylserotonin, NOS. Note the clearly defined oxidation and/or overlapping “shadow” peaks with NOD and NAD at ~5.9 and ~6.2 minutes, respectively. 97

Figure A.10 . Standard MS Res ults of primary fatty acid amides linoleamide LOA and palmitoleamide POA 98

Figure A.11 . Standard MS Res ults of primary fatty acid amides oleamide OA and palmitamide PA 99 eito ad oun odto o rtnin ie an time spiked thorax). retention on condition column and deviation Figu e Are 1. Spiked .12.

A EA , anandamide,

i n . melliferaA. d m/z

hrx hwn sml mti efcs instrument effects, matrix sample showing thorax aito (et sadr AA Rgt aggregate Right: AEA, standard (Left: variation

100 a retent on condition column and deviation instrument AFigure ggregate spikedthorax). 1. Spiked .13. N - arachidonoyldopamine,

A in NAD o tm and time ion . melliferaA. m/z

hrx hwn sml mti effects, matrix sample showing thorax aito (et sadr ND Right: NAD, standard (Left: variation

101 h sie NG ape n hrx oprd o h sta the to compared thorax in sample NOG spiked the a time retention on conditions column and deviation AFigure 1. Spiked.14.

N - oleoylglycine,

O in NOG . melliferaA. nd nd m/z dr sample ndard

hrx hwn sml mti efcs instrument effects, matrix sample showing thorax variation. Note the extensive band-broadening of band-broadening extensive the Note variation. Lf: tnad O, ih: aggregate Right: NOG, standard (Left:

102

Figure A.15. Aggregate spectra and MS results of endogenous AEA in farmed

Figure A.15. Aggregate spectra and MS results of N-oleoyldopamine, NOD, in farmed A. mellifera .

103

Figure A.16. Aggregate spectra and MS of N-acylglycines NPG and NOG in farmed A. Mellifera 104

Figure A.17. Aggregate spectra and MS of N-acyl ethanolamines OEA and AEA in feral A. Mellifera

105

Figure A.18. Aggregate spectra and MS of N-acyldopamines NPD and NOD in feral A. Mellifera . NAD is a false positive of AEA.

106

Figure A.19. Aggregate spectra and MS of N-acylglycines, NPG, NOG and NAG, in feral A. mellifera .

107

Figure A.20. Aggregate spectra and MS of endogenous primary fatty acid amides LOA and POA in feral A. mellifera.

108

Figure A.21. Aggregate spectra and MS of endogenous primary fatty acid amides OA and PA in feral A. mellifera .

109

Figure A.22. Aggregate spectra and MS of endogenous ethanolamine AEA in T. castaneum.

110

Figure A.23. Aggregate spectra and MS of endogenous acyl dopamines, NPD, NOD, and NAD, in T. castaneum .

111

Figure A.24. Aggregate spectra and MS of endogenous acyl glycines, NPG, NOG and NAG, in T. castaneum .

112

Figure A.25. Aggregate spectra and MS of endogenous primary fatty acid amides, POA, OA and PA, in T. castaneum .

113