Research paper
Identification and activity of monoamine oxidase in the orb-weaving spider Larinioides cornutus

https://doi.org/10.1016/j.ygcen.2020.113580Get rights and content

Highlights

  • Monoamine oxidase activity is present in an arachnid species.

  • De novo transcriptome assembly of a novel species identified isoforms of monoamine oxidase putative sequences.

  • These transcripts appear to have homologous binding sites to vertebrate orthologues.

  • Activity appears higher during the day for nocturnal species, as shown by fluorometric tests.

  • Higher affinity for octopamine and serotonin was observed.

Abstract

Monoamine oxidase (MAO) is a mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of monoamines in a wide array of organisms. While the enzyme monoamine oxidase has been studied extensively in its role in moderating behavior in mammals, there is a paucity of research investigating this role in invertebrates, where the latter utilizes this enzyme in a major pathway to degrade monoamines. There is especially a dismal lack of information on how MAO influences activity in invertebrates, particularly in account of the circadian cycle. Previous studies revealed MAO degrades serotonin and norepinephrine in arachnids, but did not investigate other critically important compounds like octopamine. Larinioides cornutus is a species of orb-weaving spider that exhibits diel fluctuations in behavior, specifically levels of aggression. The monoamines octopamine and serotonin have been shown to influence aggressive behaviors in L. cornutus, thus this species was used to investigate if MAO is a potential site of regulation throughout the day. Not only did gene expression of MAO orthologs and MAO activity fluctuate at different times of day, but the enzymatic activity was substrate-specific producing a higher level of degradation of octopamine as compared to serotonin in vitro. This study further supports evidence that MAO has an active role in monoamine inactivation in invertebrates and provides a first look at how MAO ultimately may be regulating behavior in an invertebrate.

Introduction

Monoamine oxidase (MAO) is an essential protein involved in the regulation of neurotransmitter levels and thus plays an instrumental role in the physiology, metabolism, and behavior of many organisms (Shih et al., 1999). This protein has two isoenzymes, MAO-A and MAO-B, which are characterized by their regional distribution, substrate preference, and physiological roles in mammals (Bach et al., 1988). Although these two isoenzymes share 70% structural identity, MAO-A has a high affinity for serotonin and norepinephrine while MAO-B primarily catabolizes 2-phenylethylamine (PEA) and aids in the degradation of trace amines and dopamine (Bach et al., 1988, Tsugeno and Ito, 1997). Furthermore, lack of MAO-A has largely been associated with impulsive and reactive aggression in humans (Alia-Klein et al., 2008, Godar et al., 2016), and in fact, several studies have shown the robust associations between low activity MAO-A variants and psychopathy and/or criminal behavior (Guo et al., 2008, Beaver and Holtfreter, 2009, Beaver et al., 2010, Beaver et al., 2013, Beaver et al., 2014). Similar findings have also been reported in rodents, revealing a proclivity to aggressive responses toward intruders when MAO-A activity is pharmacologically blockaded (Whitaker-Azmitia et al., 1994, Mejia et al., 2001, Shih, 2004). Lastly, MAO-A is also influenced by the circadian clock components (Bhaskaran and Radha, 1984), and it was shown that clock proteins Per2 and BMAL positively upregulate MAO-A gene expression, producing daily fluctuations in levels of the enzyme which ultimately impact the likelihood of aggressive behaviors throughout the day (Hampp et al., 2008).

Invertebrates use a wide array of enzymatic routes to degrade monoamines including N-acetylation, γ -glutamyl conjugation, sulphation, and beta-alanyl conjugation (Bodnaryk and Brunet, 1974, Hayashi et al., 1977, Hopkins et al., 1982, McCaman et al., 1985, Sloley et al., 1990, Sloley, 2004). Similar to what is seen in chordates, MAO is also utilized in some invertebrates including echinoderms (Nicotra and Naccarato, 1982, Nicotra et al., 1986), mollusks (Juorio and Killick, 1972, Juorio and Philips, 1976, Pani and Anctil, 1994, Pani and Croll, 1998, Pani and Croll, 2000), and some arachnids (Atkinson et al., 1974, Meyer and Jehnen, 1980, Wong and Kaufman, 1981, Sparks et al., 1994). However, while MAO activity has also been established within the Class Arachnida, the conclusions have not been consistent between ticks, mites, and spiders (Witt et al., 1961, Atkinson et al., 1974, Holden and Hadfield, 1975, Meyer and Jehnen, 1980, Sparks et al., 1994). Some studies in ticks reported the primary presence of MAO as a degredation pathway for dopamine and serotonin, with very little N-acetylated or γ -glutamyl conjugated amines (Wong and Kaufman, 1981, Kaufman and Sloley, 1996). However, these results in ticks nor spiders analyzed the activity with octopamine. MAO activity in ticks was also inhibited by MAO-B inhibitor deprenyl, but not MAO-A inhibitor clorgyline, similar to previous findings in other invertebrates like echinoderms (Kaufman and Sloley, 1996).

However, studies in spiders revealed a prominent presence of the MAO enzyme throughout different brain regions of various species but showed a significant reduction in MAO activity using clorgyline and no effect with the MAO-B inhibitor pargyline (Meyer and Jehnen, 1980). While these invertebrate studies have been influential in establishing the presence and activity of MAO, very few studies have investigated MAO’s role in diel behavioral processes and enzymatic fluctuations outside of mammals (Kaushal, 2008, Basu, 2014).

The orb-weaver Larinioides cornutus is a holarctic species that has daily patterns of locomotor activity and aggression that appear to be under circadian control (Jones et al., 2011b). This species is mostly active at night and this pattern in locomotor activity persists in constant conditions (Jones and Moore, unpublished work). When threatened by a predator stimulus, the spider forms a tight-ball formation and exhibits a death-feigning behavior or a “huddle” response. This response can be the reflect of a diel and circadian pattern, with the spider huddling longer during the day while in a less aggressive state, and breaking out of this huddle more rapidly at night while in an apparent more aggressive state when the spider would otherwise be actively hunting prey (Jones et al., 2011b). These varied aggressive states can also be induced using exogenous amounts of the biogenic amines, serotonin and octopamine. When octopamine levels were artificially increased, the boldness level of the individual increased (Jones et al., 2011a). Thus, it appears the monoamines octopamine and serotonin play a role in these diel shifts in behavior. As some previous studies show MAO activity in oxidizing serotonin previously in arachnids (Kaufman and Sloley 1996), MAO may potentially be a source of regulation in the shift in behavioral states in L. cornutus.

This study aimed at first identifying monoamine oxidase transcripts and characteristics of the enzyme in the spider L. cornutus. We investigated substrate preference, the specificity of inhibition, and fluctuations in transcript expression and activity of the MAO enzyme during critical times of the day.

Section snippets

Identification of putative MAO transcripts from de novo assembled transcriptome

We isolated homolog sequences of L. cornutus MAO from the transcriptome assembly using a TBLASTX query of known MAO protein sequences from other organisms (Supplementary Table 1; See Methods Section for Transcriptome Sequencing Methodology). Several transcripts were initially pulled, but only a few showed significant changes in expression among the time points (Fig. 1A). We found that transcript DN53734_c3_g1_i3, showed high homology with the query sequences and significant fluctuations in

Discussion

While a significant amount of research has focused on the role MAO plays in behavior in vertebrates, very few studies have evaluated this same role in invertebrates. In order to address this question, MAO activity must be investigated in organisms that exhibit fluctuations in behavioral state and utilize the enzyme for inactivating of monoamines. Our study investigated the presence and expression of MAO at different time points throughout the day in L. cornutus. This spider exhibits diel shifts

Conclusions

This study demonstrated diel fluctuations of MAO gene expression and activity in an invertebrate, similar to what is observed in vertebrates (Hampp et al., 2008). While further studies are needed to investigate and confirm the transcript of interest is directly responsible for MAO activity, MAO appears to be a potential site of regulation of monoamine signaling and aggression in this spider based on our in vitro studies. There is evidence for other metabolic pathways for monoamines in arachnids

Female L. cornutus collection and conditioning for MAO experiments

Individual L. cornutus females were collected from Washington County, Tennessee, USA during the month of October in 2017. Individuals were housed in plastic containers for a minimum of 7 days prior to sacrifice and were fed live crickets and misted with distilled water every 2–3 days. Specimens were entrained to 12:12 light: dark cycle with constant humidity. Fifteen individuals were sacrificed via flash-freezing in liquid nitrogen every 6 h for 24 h for a total of four-time points (0100, 0700,

CRediT authorship contribution statement

Rebecca J. Wilson: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing - original draft, Writing - review & editing. Tahmina H. Ahmed: Methodology, Investigation, Formal analysis, Data curation, Writing - review & editing. Md Mahbubur Rahman: Methodology, Investigation, Formal analysis, Data curation, Writing - review & editing. Brian M. Cartwright: Methodology, Investigation, Formal analysis, Data curation, Writing - review & editing. Thomas C. Jones:

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was funded by a grant from the National Science Foundation (IOS-7278284) and in part by a grant from the East Tennessee State University Research Development Committee Major/Small/Interdisciplinary Grants Program. I would also like to thank my undergraduate research advisor Dr. George Uetz for inspiring me to do research with such a fascinating group of organisms.

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