Biochemical Systematics and Ecology 89 (2020) 104001

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology
journal homepage: www.elsevier.com/locate/biochemsyseco

Carotenoids of dragonflies, from the perspective of comparative biochemical
and chemical ecological studies

T

Takashi Maokaa,∗, Naoki Kawaseb, Tetsuyuki Uedac, Ritsuo NishidadaResearch Institute for Production Development, 15 Shimogamo-morimoto-cho, Sakyo-ku, Kyoto, 606-0805, Japan
Minakuchi Kodomonomori Nature Museum, 10 Kita-naiki, Minakuchi-cho, Koka, 528-0051, Japanc
Ishikawa Prefecture University 305, Suematsu, Nonoichi-1, 921-8836, Japand
Kyoto University Yoshida-Honmachi, Sakyo-ku, Kyoto, 606-8501, Japanb

ARTICLE INFOABSTRACT

Keywords:
Carotenoid
Dragonfly
Food chain
Comparative biochemistry
Chemical ecology

Carotenoids of 20 species of dragonflies (including 14 species of Anisoptera and six species of Zygoptera) were
investigated from the viewpoints of comparative biochemistry and chemical ecology. In larvae, β-carotene, βcryptoxanthin, lutein, and fucoxanthin were found to be major carotenoids in both Anisoptera and Zygoptera.
These carotenoids were assumed to have originated from aquatic insects, water fleas, tadpoles, and small fish,
which dragonfly larvae feed on. Furthermore, β-caroten-2-ol and echinenone were also found in all species oflarvae investigated. In adult dragonflies, β-carotene was found to be a major carotenoid along with lutein,zeaxanthin, β-caroten-2-ol, and echinenone in both Anisoptera and Zygoptera. On the other hand, unique carotenoids, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene, were present inboth Anisoptera and Zygoptera dragonflies. These carotenoids were not found in larvae. Food chain studies ofdragonflies suggested that these carotenoids originated from aphids, and/or possibly from aphidophagous ladybird beetles and spiders, which dragonflies feed on. Lutein and zeaxanthin in adult dragonflies were also
assumed to have originated from flying insects they feed on, such as flies, mosquitoes, butterflies, moths, andplanthoppers, as well as spiders. β-Caroten-2-ol and echinenone were found in both dragonfly adults and larvae.
They were assumed to be metabolites of β-carotene in dragonflies themselves. Carotenoids of dragonflies wellreflect the food chain during their lifecycle.

1. Introduction
Carotenoids are tetraterpene pigments that are distributed in photosynthetic bacteria, some species of archaea and fungi, algae, plants,
and animals. About 850 naturally occurring carotenoids had been reported up until 2018 (Britton et al., 2004; Maoka, 2019).
Insects are the most diverse group of animals. Therefore, carotenoids in insects show structural diversity. For example, a series ofcarotenoids with 2-hydroxy, 2-keto-, and 3,4-didehydro-2-keto-β-end
groups are present in stick insects (Kayser, 1981a, 1981b; 1982;
Matsuno et al., 1990; Davidson et al., 1991) and some species of moth
(Kayser, 1975, 1976, 1979). Oxidative metabolites of luteins, philosamiaxanthin (3-hydroxy-β,ε-caroten-3′-one), fritschiellaxanthin, andpapirioerythrinon, are found in several species of moths and butterflies(Kayser, 1975; Harashima et al., 1972). Astaxanthin and adonirubin
were found in locust and mantis (Manuta, 1948). Fucoxanthin and fucoxanthinol were reported from larvae of mayfly and caddishfly∗(Matsuno et al., 1999). Several species of beetles contain phytoene,
phytofluene, ζ-carotene, neurosporene, lycopene, β-zeacarotene, γcarotene, and torulene, which are carotenoids in the torulene biosynthetic pathway (Britton et al., 1977a, 1977b). Furthermore, a series ofcarotenes with a unique γ-end group, such as γ,ψ-carotene, 3,4-didehydro-γ,ψ-carotene, β,γ-carotene, and γ,γ-carotene, are present in
aphids (Britton et al., 1977a, 1977b).
Dragonflies are insects belonging to the order Odonata and are oneof the familiar insects with symbols of courage, strength, victory, andhappiness in Japan (Asahina, 1974). They are mainly divided into two
suborders, Anisoptera and Zygoptera. Adults of Anisoptera dragonflies
are characterized by large, multifaceted eyes, two pairs of strong,transparent wings, sometimes with colored patches, and a conspicuously elongated body. Adults of Zygoptera dragonflies haveslimmer bodies, and most species fold the wings along the body when at
rest. Dragonflies are carnivorous throughout their lifecycle. Larvae inhabit aquatic environments such as rice paddies, ponds, swamps, andCorresponding author.
E-mail address: maoka@mbox.kyoto-inet.or.jp (T. Maoka).

https://doi.org/10.1016/j.bse.2020.104001
Received 9 December 2019; Received in revised form 24 December 2019; Accepted 5 January 2020
Available online 15 January 20200305-1978/ © 2020 Published by Elsevier Ltd.

Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

streams and feed on aquatic insects, water fleas, tadpoles, and small
fishes. After emergence, adults feed on several insects and spiders(Corbet, 1999).
It is well-known that carotenoids found in animals are either directly accumulated from feed or partly modified through metabolic
reactions. Several carotenoids in animals are indicators of taxonomy
and food-chain markers (Liaaen-Jensen, 1998., Maoka, 2011). Therefore, carotenoids of dragonflies were investigated from the viewpointsof comparative biochemistry and chemical ecology.
In the present paper, we describe carotenoids in the body tissue of
20 species of dragonflies, including 14 species of Anisoptera and six
species of Zygoptera.

About 3000 specimens adult Sympetrum frequens were extractedwith acetone at room temperature. They were then transferred to nhexane: Et2O (1:1, v/v) by addition of water. The n-hexane: Et2O phase
was washed with water and dehydrated on anhydrous sodium sulphateand evaporated. The red residue was separated by silica gel chromatography using n-hexane, Et2O, and acetone as eluting solvents.
Carotenoids eluted with n-hexane was further separated by preparative ODS HPLC to afford β-carotene, (6′S)-β,γ-carotene, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, and γ,γ-carotene.
Carotenoids eluted with n-hexane: Et2O (1:1) from silica gel chromatography were further separated by ODS HPLC to afford echinenoone,
β-cryptoxanthin, β-caroten-2-ol. Carotenoids eluted with ether fromsilica gel chromatography were further separated by ODS HPLC to afford lutein and zeaxanthin. Carotenoids eluted with acetone from silica
gel chromatography were further separated by ODS HPLC afforded tomyxol.
Individual carotenoids in aphid Acyrthosiphon pisum were separated
as the same manner.
The following 12 carotenoids, β-carotene, (6′S)-β,γ-carotene, γ,γcarotene, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, echinenone, β-caroten-2-ol, β-cryptoxanthin, lutein, zeaxanthin, and myxol,
were identified in adult Sympetrum frequens. Fucoxanthin was identified
in larva. These spectral data were described in supplementary information. Details of spectral data of (6′S)-β,γ-carotene and β-zeacarotene have not been reported (Britton et al., 2004). Therefore, spectral
data of (6′S)-β,γ-carotene and β-zeacarotene were described here.
(6′S)-β,γ-Carotene: ESI TOF MS (m/z) 536.4382 [M+] C40H56, Calcd
for 536.4404; UV–Vis (Et2O) 412, 442, 471 nm; 1H NMR δ (in CDCl3 at
500 MHz) 0.82 (3H, s, H-16′), 0.90 (3H, s, H-17′), 1.03 (6H, s, H-16,17),
1.33 (2H, dd, J = 14, 7 Hz, H-2′), ~1.46 (2H, m, H-2), ~1.63 (2H, m,
H-3), 1.72 (3H, s, H-18), 1.95 (3H, s, H-19′), 1.97 (9H, s, H-19, 20, 20′),
2.02 (2H, t, J = 7 Hz, H-4), 2.07 (2H, m, H-3′), 2.29 (2H, m, H-4′), 2.51
(1H, d, J = 9.5 Hz, H-6′), 4.57 (1H, br. s, H-18′), 4.73 (1H, br. s, H-18′),
5.84 (1H, dd, J = 15.5, 9.5 Hz, H-7′), 6.12 (1H, d, J = 11.5 Hz, H-10′),
6.13 (1H, d, J = 15.5 Hz, H-8′), 6.14 (1H, d, J = 16 Hz, H-8), 6.14 (1H,d, J = 10 Hz, H-10), 6.15 (1H, d, J = 16 Hz, H-7), 6.24 (2H, m, H-14,14′), 6.33 (1H, d, J = 15.5 Hz, H-12′), 6.35 (1H, d, J = 15.5 Hz, H-12),
6.62 (1H, dd, J = 15.5, 11.5 Hz, H-11′), 6.63 (2H, m, H-15, 15′), 6.65
(1H, dd, J = 15.5, 11.5 Hz, H-11′); CD (in Et2O): λ nm (Δε) 216 (+3.0),230 (0), 238 (−4.8), 249 (0), 270 (+4.6), 225 (0), 340 (−1.8), 375(−0.2). These spectral data were in agreement with published data
(Arpin et al., 1971; Britton et al., 2004).
β-Zeacarotene: ESI TOF MS (m/z) 538.4552 [M+] C40H58, Calcd for538.4539; UV–Vis (Et2O) 400, 426, 451 nm; 1H NMR δ (in CDCl3 at
500 MHz) 1.03 (6H, s, H-16,17), ~1.46 (2H, m, H-2), ~1.63 (2H, m, H3), 1.60 (3H, s, H-18′), 1.61 (3H, s, H-17′), 1.68 (3H, s, H-16′), 1.72
(3H, s, H-18), 1.82 (3H, s, H-19′), 1.97 (9H, s, H-19, 20, 20′), 2.02 (2H,t, J = 7 Hz, H-4), ~2.12 (8H, m, H-3′,4′,7′, 8′), 5.11 (2H, m, H-2′, 6′),5.95 (1H, d, J = 11 Hz, H-10′), 6.14 (1H, d, J = 16 Hz, H-8), 6.14 (1H,d, J = 10 Hz, H-10), 6.15 (1H, d, J = 16 Hz, H-7), 6.23 (1H, d,
J = 15.5 Hz, H-12′), 6.25 (2H, m, H-14, 14′), 6.35 (1H, d, J = 15.5 Hz,
H-12), 6.50 (1H, dd, J = 15.5, 11.5 Hz, H-11′), 6.63 (2H, m, H-15, 15′),
6.64 (1H, dd, J = 15.5, 11.5 Hz, H-11’). These spectral data were inagreement with published data (Britton et al., 2004).

2. Materials and methods
2.1. Insects
The following adults and larvae of dragonflies were collected at
Koka City, Shiga Prefecture, Kyoto City, Kyoto Prefecture, and Komono
Town, Mie Prefecture in Japan during April to October.
Adult dragonflies; Lestidae; Lestes sponsa, Lestes temporalis,
Calopterygidae; Mnais costalis, Atrocalopteryx atrara, Calopteryx cornelia,
Coenagrionidae; Paracercion calamorum belonging to Zygoptera,
Aeshnidae; Sarasaeschna pryeri, Anax parthenope, Gomphidae; Sieboldius
albardae, Asiagomphus pryeri, A. melaenops, Cordulegastridae;
Anotogaster sieboldii, Libellulidae; Sympetrum darwinianum, S. infuscatum, S. frequens, Pantala flavescens, Lyriothemis pachygastra,
Orthetrum albistylum, O. japonicum, O. melania belonging to Anisoptera.
Larvae dragonflies; Zygoptera, Calopterygidae; Atrocalopteryx
atrara,Coenagrionidae;
Paracercion
calamorum,
Anisoptera,
Cordulegastridae; Anotogaster sieboldii, Libellulidae; Pantala flavescens,
Lyriothemis pachygastra, Sympetrum frequens, Orthetrum albistylum, O.
japonicum.
Aphids, planthoppers, ladybird beetles, and spiders were also collected at Koka City, Shiga Prefecture, Kyoto City, Kyoto Prefecture, andKomono Town, Mie Prefecture in Japan during April to October.
The scientific names of dragonflies in this paper are referenced from
Ozono et al. (2012).
2.2. Carotenoid extraction, analysis, isolation, and identification
The carotenoids were extracted from larvae and adults of dragonflies of each species with acetone at room temperature. They were thentransferred to n-hexane:Et2O (1:1, v/v) by addition of water. The nhexane: Et2O phase was washed with water and dehydrated on anhydrous sodium sulphate. The total carotenoid amount were calculatedusing coefficient of E 1%cm = 2400 at λ max (Britton, 1995).
Quantitative and qualitative carotenoid analysis of dragonflies,
aphids, plant hoppers, beetles, and spiders were carried out using ourroutine method using LC/PDA/MS (Maoka, 2016).
In the case of adult Sympetrum frequens and Pantala flavescens individual carotenoids were isolated by column chromatography followed by preparative HPLC and identified from UV-VIS, ESI TOF MS,
1
H NMR, and circular dichroism (CD) spectral data.
The UV-VIS spectra were recorded with a Hitachi U-2001 spectrophotometer (Hitach Field Navigator, Tokyo, Japan) in Et2O. The 1H
NMR (500 MHz) spectrum was measured with a Varian UNITY INOVA
500 spectrometer (Varian Corporation, Palo Alto, California USA) in
CDCl3 with TMS as an internal standard. The CD spectrum was recorded
in Et2O at room temperature with a Jasco J-500C spectropolarimeter(JASCO Corporation, Hachioji, Tokyo, Japan). Preparative HPLC was
performed with a Hitachi L-6000 intelligent pump and an L-4250 UVVIS detector (Hitach Field Navigator, Tokyo, Japan) set at 450 nm. The
column used was a 250 × 10 mm i.d., 5 μm Cosmosil 5C18-MS-II ODS(Nacalai Tesque, Kyoto, Japan) with CHCl3:MeOH (25:75, v/v) as asolvent at a flow rate of 2.0 mL/min.

3. Results and discussion
3.1. Carotenoids of dragonfly larvae
Carotenoid contents and compositions in eight species of dragonfly
larvae including six species of Anisoptera and two species of Zygoptera
are shown in Table 1. Carotenoid compositions of dragonfly larvae weresimilar to each other. β-Carotene was found to be the major carotenoid
along with fucoxanthin, lutein, and zeaxanthin. β-Caroten-2-ol, βcryptoxanthin, and echinenone were also found in all species of larvae
investigated. Dragonfly larvae inhabit aquatic environments and feed2

Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

Table 1
Carotenoids in eight species of dragonfly larvae.
SuborderZygoptera

FamilyCalopterygidae

Coenagrionidae

Cordulegastridae

Libellulidae

Genus

Atrocalopteryx

Paracercion

Anotogaster

PantalaLyriothemis

Sympetrum

Orthetrum

Orthetrum

Speciesatraracalamorumsieboldii

flavescenpachygastra

frequens

albistylum

japonicum

Total Carotenoid (μg/g)(μg/specimen)

2.2
20.2

3.75
0.33

2.44
4.23

10.5
2.2

0.8
2.3

0.66
3.75

2.8
0.5

0.9
2.2

Composition

%

%

%

%

%

%

%

%

β,β-Carotene
β,γ-Carotene
β-Zeacarotene
β,ψ-Carotene
γ,γ-Carotene
Torulene
Echinenone
β-Caroten-2-ol
β-Cryptoxanthin
Zeaxanthin
LuteinFucoxanthin
Myxol
Unidentifiied

30.8

27.0

10.2

20.6

38.2

37.8

18.6

37.2

2.2
10.2
7.1
8.2
11.5
24.4

6.5
1.2
20.2
27.2
2.4
+
+
15.5

1.6
0.5
1.5
+
34.3
46.8

8.8
8.2
35.2
6.0
+
+
11.4
9.8

11.5
8.2
10.2
1.2
10.5
16.5

1.8
2.2
6.2
+
15.2
30.1

11.6
9.2
11.2
0.9
11.5
15.5

3.7

6.7

5.6
7.2
38.5
5.8
+
+
14.9
9.4

5.6

Anisoptera

5.1

2.9

originated from associated bacteria in these dragonflies. On the other
hand, fucoxanthin, which was present as one of the major carotenoids
in larvae of dragonflies, was not found in adult dragonflies.
Dragonflies belonging to the genera Sympetum and Pantala have a
red or orange body color. On the other hand, dragonflies belonging to
the genera Orthetrum and Asiagomphus have a pale blue or yellowishgreen body color. However, these carotenoid compositions were mostly
similar to each other. Therefore, it was revealed that carotenoids indragonflies did not directly reflect their body color. A red and yellow
body color of adult dragonflies was identified to be ommochrome
pigments (Futashasi et al., 2012). Characteristic carotenoids present indragonflies are shown Fig. 1.

on aquatic insects, water fleas, tadpoles, and small fishes. In freshwater
environments, herbivorous aquatic insects ingest β-carotene, lutein,zeaxanthin, fucoxanthin, from micro algae such as diatoms, green
algae, blue green algae, and cyanobacteria (Matsuno et al., 1999).
Dragonfly larvae appeared to accumulate these carotenoids from herbivorous aquatic insects and small aquatic animals. Myxol was identified in two species of larvae (Pantala flavescens and Orthetrum albistylum), accounting for about 10% of the total carotenoids.
Myxoxanthophyll (myxol chinovoside) is a characteristic carotenoid incyanobacteria (blue green algae) (Britton et al., 2004). Therefore,
myxol might be derived from myxoxanthophyll by hydrolysis of glycoside bonds in animals through the food-chain. It was reported thatseveral insects could convert β-carotene to β-caroten-2-ol and echinenone (Kayser, 1981a, 1981b; 1982, 1984). Therefore, β-caroten-2-oland echinenone may be oxidative metabolites of β-carotene in dragonflies.

3.3. Change in carotenoid composition in Sympetrum frequens through thelifecycle
In Japan, S. frequens (akatonbo, the “red dragonfly”) is a common
species distributed throughout Japan. Its larvae live in the rice fields
and feed on small aquatic animals. After emergence, these dragonflies
migrate to high mountains where they feed on several flying insects andgrow until descending to rice fields for breeding (Asahina, 1974). Fig. 2
shows changes in the carotenoid content during the lifecycle in S. frequens. As described previously, β,β-carotene, echinenone, β-caroten-2ol, β-cryptoxanthin, zeaxanthin, lutein, and fucoxanthin were present inlarvae. After emergence, dragonflies do not ingest food for a few days.
During this period, the carotenoid content of S. frequens markedly decreases. In teneral adults, the total carotenoid content was decreased to
1/5 of that found in larvae. Furthermore, fucoxanthin, one of the major
carotenoids in larvae, completely disappeared in the teneral adults.
Therefore, it was assumed that fucoxanthin, a characteristic carotenoid
in diatoms, remained within the digestive tract of the dragonfly larvae
and was not absorbed in their body. The carotenoid content of adults
increased with their growth. After migration to high mountain areas,
unique carotenoids, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene,
β,γ-carotene, and γ,γ-carotene, were found in S. frequens. The contentsof these carotenoids increased during the adult growth period. Therefore, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene,and γ,γ-carotene were considered to have originated from dietary insects during the adult stage.

3.2. Carotenoids of adults dragonflies
Carotenoids of 20 species of adult dragonflies (including 14 speciesof Anisoptera and six species of Zygoptera) are shown in Table 2 (Thelists of dragonfly species in Table 2 were presented in the order ofdiscussion). In adult dragonflies, β-carotene was found to be a major
carotenoid along with lutein, zeaxanthin, β-caroten-2-ol, and echinenone in both Anisoptera and Zygoptera. Unique carotenoids, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γcarotene, were present in adults of both Anisoptera and Zygoptera
dragonflies. These carotenoids were not found in larvae. β-Zeacarotene,β,ψ-carotene (γ-carotene), and torulene are carotenoids belonging totorulene biosynthetic pathways (Britton et al., 2004). β,γ-Carotene andγ,γ-carotene are carotenoids with a unique γ-end group (terminal methylene group in the C-5 end group), and have only been reported fromaphids, beetles that feed on aphids (Britton et al., 1977a, 1977b), anddiscomycete fungus Caloscyoha fulgens (Arpin et al., 1971). Furthermore, myxol was found in several species of adults dragonflies. In
Pantala flavescens, myxol comprised 23.4% of the total carotenoids.
Recently, myxol was isolated from several species of bacteria belonging
to Flavobacteriaceae (Yokoyama and Miki, 1995; Teramoto et al., 2003;
Shindo et al., 2006). Therefore, myxol in adult dragonflies may have3

Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

Table 2
Carotenoids in 20 species of adult dragonflies.
SuborderAnisoptera

FamilyLibellulidae

Genus

Sympetrum

Speciesdarwinianum

frequens

Total carotenoid (μg/g)(μg/specimen)

19.5
3.8

Aeshnidae
PantalaLyriothemis

Orthetrum

Sarasaeschna

Anaxinfuscatum

flavescens

pachygastra

albistylum

japonicum

melania

pryeriparthenope

18.1
4.29

11.2
4.4

23.5
4.2

1.7
5.8

14.4
2.7

7.1
1.98

9.2
3.6

8.3
4.1

14.0
12.7

39.7

33.4

41.6

20.9

25.9
13.9
2.2

16.1
10.1
7.3

8.1
9.7
6.7

7.1
2.2
7.2

46.0
2.9
1.1
13.0

29.3

52.4

45.2

75.3

5.8
2.3
3.6

1.8
4.7

62.1
4.4
5.1
1.7
0.7

9.2
4.2
1.6

1.9
1.6
3.6

1.5

1.3

0.4

2.6
2.2
4.0
1.1
2.3
1.4
3.2

3.5
4.2
5.6
2.6
4.0
6.2
5.7

3.7
4.4
5.0
3.4
8.6
1.2
7.2

1.0
5.3
6.7
3.8
11.8
5.2
2.2
23.4
3.2

1.0

5.6
5.7
2.9
9.9
2.4
7.6

+
+
3.9
2.5
6.5
2.1
18.9

+
+
2.6
1.2
9.3
0.6
2.6

5.9

1.3

Composition (%)β,β-Carotene
β,ε-Carotene
β,γ-Carotene
β-Zeacarotene
β,ψ-Carotene(γ-Carotene)
γ,γ-Carotene
Torulene
Echinenone
β-Caroten-2-ol
β-Cryptoxanthin
Zeaxanthin
LuteinMyxol
Unidentifieds

1.0
3.6
5.4
10.9
0.5
9.4

1.7
4.2
10.1
29.2
1.0
3.5
8.3

6.2

Suborder

7.0

0.6
1.1
2.7
1.5
11.5
1.4
7.2

Zygoptera

FamilyGomphidae

Cordulegastridae

LestidaeCalopterygidae

Genus

Sieboldius

Speciesalbardae

Total carotenoid (μg/g)(μg/specimen)

Coenagrionidae

Asiagomphus

Anotogaster

LestesMnais

Atrocalopteryx

Calopteryx

Paracercion

pryerimelaenops

sieboldii

sponsatemporalis

costalis

atraracornelia

calamorum

14.0
26.2

12.6
4.3

5.1
2.6

3.2
3.8

7.1
0.62

3.6
0.33

3.1
0.7

1.2
0.16

10.7
0.23

14.0
0.58

61.2

38.1

30.0

70.0

40

43.2

30.6

48.4

35.7

31.9

2.7
1.3
1.2

4.2
0.6
1.6

0.8
+
+

3.8
+
+

4.2
4.1
3.3

2.6
2.5
2.1

+
+
+

2.3
1.9
1.1

8.1
3.9
12.5

0.9
0.7
0.7

+
+
1.6
0.8
7.2
1.3
19.2

+
+
7.0
1.2
21.0
4.5
20.9

+
+
1.1
2.0
5.5
0.8
54.8

+
+
2.3
2
8.8
2
25.6

0.1
3.2
2.3
7.5
10.2
4.6
5.9

+
+
3.8
13.7
13.2
1.0
19.5

+
0.9
8.1
13.2
15.8
1.2
5.3

5.7
4.9
4.5
5.8

4.5
2.2
23.9
1.6
24.2

3.5

0.9

5.0

+
+
1.2
1.0
4.1
2.1
13.9
0.5
3.4

7.7

15.8

18.2

1.8

18.9

9.2

Composition (%)β,β-Carotene
β,ε-Carotene
β,γ-Carotene
β-Zeacarotene
β,ψ-Carotene(γ-Carotene)
γ,γ-Carotene
Torulene
Echinenone
β-Caroten-2-ol
β-Cryptoxanthin
Zeaxanthin
LuteinMyxol
Unidentifieds

3.4. Change of carotenoid composition in various body parts of Sympetrum
frequens

0.2

zeacarotene, β,ψ-carotene (γ-carotene), and β,γ-carotene were higher
than in other parts. The red color of feces in S. frequens was due to thepresence of carotenoids. This clearly suggested that these carotenoids
originated from their diet. Carotenoids were also found in eggs. Carotenoid compositions of eggs were also similar to those of adults.
Therefore, carotenoids were translocated to eggs non-selectively fromthe female dragonfly.

Table 3 shows the carotenoid content and composition in the head,thorax, abdomen, feces, and eggs of S. frequens. The carotenoid content
and composition of the head, thorax, and abdomen were similar to eachother. On the other hand, feces contained about 50 times more carotenoid than the head, thorax, and abdomen. Furthermore, levels of β-4Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

Fig. 1. Characteristic carotenoids in adults and larvae of dragonflies.

Fig. 2. Changes in individual carotenoid contents ofSympetrum frequens during the lifecycle.
Foot note 3-OH-xanthophylls: β-Cryptoxanthin,
Lutein, Zeaxanthin.
Larvae; collected in rice paddy in June, Teneraladults; collected at around rice paddy in June, Young
adults; collected at Mt Gozaisyo in July, Mature
adults; collected at around rice paddy in October.

3.5. Origins of carotenoid torulene biosynthetic pathway and with a γ-end
group of dragonflies

cryptoxanthin, lutein, zeaxanthin, which originate from higher plants,
were found in flies, mosquitoes, butterflies, moths, plant hoppers, etc.
On the other hand, β-zeacarotene, β,ψ-carotene (γ-carotene), torulene,
β,γ-carotene, and γ,γ-carotene were found in aphids and a ladybird,
Coccinella septenpunctata, as shown in Table 4. The species C. septenpunctata feeds exclusively on aphids. Spiders are also known to feed on
aphids. Thus, carotenoids in the torulene biosynthetic pathway andwith a γ-end group may be present in these animals through the foodchain.

In order to reveal the origin of β-zeacarotene, β,ψ-carotene (γ-carotene), and torulene belonging to carotenoids in the torulene biosynthetic pathway (Goodwin, 1980) and β,γ-carotene and γ,γ-carotene
with a γ-end group, carotenoids in flying insects, such as flies, mosquitoes, butterflies, moths, aphids, planthoppers, and beetles, supposed
to be potential foods for dragonflies, were investigated. β-Carotene, β-5Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

they are considered to have originated from endocymbiotics microorganisms such as fungi (Liaaen-Jensen, 1998). Recent investigations
revealed that aphids synthesized carotenoid themselves by carotenoid
synthetic genes, which were horizontally transferred from fungi toaphids. Namely, the aphid genome itself encodes multiple enzymes for
carotenoid biosynthesis. Red colored aphids are known to have carotenoid desaturase genes and synthesize torulene from phytoene independently (Moran and Jarvik, 2010; Nováková and Moran, 2012;
Mandrioli et al., 2016; Ding B-Y et al., 2019). On the other hand, bluegreen aphids synthesize polycyclic quinones using genes of the endosymbiotic bacterium Richettsiella (Tsuchida et al., 2010). Therefore,
aphids make their own carotenoids and quinones by horizontal transfer
genes from fungi and symbiotic bacteria for coloration, depending on
the environmental signals. At present, our research group is conducting
functional analysis of carotenoid synthetic genes in aphids.
Figs. 1 and 2 show carotenoids in both larvae and adults of dragonflies. β-Zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene, which were synthesized by aphids, were accumulated from aphids directly or beetles and spiders through the foodchain. β-Carotene, β-cryptoxanthin, and lutein, which originated fromalgae and higher plants, were accumulated from dietary insects. βCaroten-2-ol and echinenone were found in both larval and adult dragonflies. These carotenoids were considered to be oxidative metabolitesof β-carotene in the dragonflies. Myxol might be derived from myxoxanthophyll, which originates from blue-green algae, through the foodchain or from an associated bacterium belonging to Flavobacteriaceae.
Fucoxanthin was found only in the larvae. This carotenoid originatedfrom diatoms.
In conclusion, carotenoids of dragonflies well reflect the food chain
during their lifecycle.

Table 3
Carotenoid content and composition in head, thorax, abdomen, feces, and eggs
of Sympetrum frequens.
Parts

Head

ThoraxAbdomen

Feces

Egg

Total Carotenoid μg/g
Total Carotenoid μg/specimen

9.4
0.34

5.4
0.7

10.5
0.6

590
4.9

0.68
0.003

Composition

%

%

%

%

%

β,β-carotene
β,γ-carotene
β-zeacarotene
β,ψ-carotene
γ,γ-carotene
toruleneechinenone
β-caroten-2-ol
β-cryptoxanthin
Unidentified
zeaxanthin
luteinOthers

56.8
6.8
2.7
2.3
1.0
0.5
3.5
3.4
7.1
3.7
2.0
4.9
5.4

46.1
9.2
9.0
9.0
1.1
1.0
3.4
4.1
5.4
2.4
0.8
5.5
3.2

52.8
9.5
4.8
11.5
1.1
1.1
2.5
3.0
4.9
2.8
1.1
3.4
1.6

14.0
9.6
16.2
34.0
4.6
5.7
1.9
3.9
1.9
5.5
0.4
0.4
1.9

46.9
4.4
3.4
11.9
0.8
0.8
4.3
6.2
8.7
1.2
0.8
3.0
7.5

Aphids are an important food of insects such as dragonflies and arecalled aeroplankton (Speight et al., 2008). Therefore, it was suggested
that carotenoids in the torulene biosynthetic pathway and those with aγ-end group in adults dragonflies originate from aphids and also possibly from aphidophagous ladybird beetles and spiders which dragonflies prey on.
The pea aphid displays red, yellow, green, and blue-green color
polymorphisms (Brown. 1975). Red and yellow body color pigments inthe aphid are carotenoids (Britton et al., 1977a, 1977b), while bluegreen pigments are polycyclic quinones (Brown. 1975; Tsuchida et al.,2010). β-Zeacarotene, β,ψ-carotene (γ-carotene), torulene, β,γ-carotene, and γ,γ-carotene were identified as major carotenoids in aphids(Britton et al., 1977a, 1977b). These carotenoids are known to be
characteristic carotenoids in fungi (Liaaen-Jensen, 1998). Therefore,

Acknowledgements
We thanks Mr. Katsuyuki Ino for collection of Anax partheonope in
Koka City.

Table 4
Carotenoids in insects and spiders.
Order

Ephemeroptera

Orthoptera

Hemiptera

Hemiptera

Coleoptera

DipteraTrichoptera

Lepidoptera

Araneae

FamilyEphemeridae

Acridoidea

Aphidoidea

Pentatomidae

Coccinellidae

Chironomidae

Stenopsychidae

Papilionidae

Oxyopidae

Nephilidae

β,β-carotene
β,γ-carotene
β-zeacarotene
β,ψ-carotene
γ,γ-carotene
torulene
β-caroten-2-ol
β-cryptoxanthinzeaxanthin
luteinechinenone
Canthaxanthin
Astaxanthin
Fucoxanthin
myxol

+++
–
–
–
–
–
–
–
+
+
–
–
–
*

+++
–
–
–
–
–
–
–
+
+
+
+
+
–
–

+++
++
+
+
+
+
–
–
–
–
–
–
–
–
–

+++
–
–
–
–
–
–
–
+
+
–
–
–
–
–

+++
++
+
+
+
+

+++
–
–
–
–
–
–
–
+
+
+
+
–
–
–

+++
–
–
–
–
–

+++

++
++
+
+
+
+

++
++
+
+
+
+

+
+
+
+
+
+
+

+
+
+
+
+
+
+

–

+
+
–
*
*

++
++
++

+++ >20%.
++ >10%.
Examined species, Aphidoidea; Acyrthosiphon pisum, Brevicoryne brassicae, Megoura crassicauda, Uroleucon formosanum Pentatomidae; Hecalus prasinus, Nephotettix
cincticeps、Coccinellidae; Coccinella spetempunctata, Propylea japonica, Acridoidea; Locusta migratoria, Chironomidae; Chironomus sp.
Ephemeroptera; Ephemera strigata, Trichoptera; Stenopsyche marmorata, Lepidoptera; Papilio xuthus, Araneae; Oxyopes sertatus, Nephila clavata.

6

Biochemical Systematics and Ecology 89 (2020) 104001

T. Maoka, et al.

Appendix A. Supplementary data

caroten-2-ol and β,ε-caroten-2-one. Z. Naturforschung 36C, 755–764.
Kayser, H., 1982. Carotenoid biogenesis in the stick insect. Carausius morosus, during alarval instar. Z. Naturforschung 37C, 13–18.
Kayser, H., Aareskjold, K., Borch, G., Liaaen-Jensen, S., 1984. Partly racemized 2-hydroxy-β-type carotenoids from the insects Cerula vinula and Ectatosoma tiaratum.
Insect Biochem. 14, 51–54.
Liaaen-Jensen, S., 1998. Carotenoids in food chain. In: In: Britton, G., Liaaen-Jensen, S.,
Pfander, H. (Eds.), Carotenoids: Biosynthesis and Metabolism, vol. 3. Birkhäuser,
Basel, Switzerland, pp. 359–371.
Matsuno, T., Maoka, T., Toriiminami, Y., 1990. Carotenoids in the Japanese stick insect
Neophirosea japonica. Comp. Biochem. Physiol. 95B, 583–587.
Matsuno, T., Ohkubo, M., Toriiminami, Y., Tsushima, M., Sakaguchi, S., Minamai, T.,
Maoka, T., 1999. Carotenoids in food chain between freshwater fish and aquatic
insects. Comp. Biochem. Physiol. 124B, 341–345.
Maoka, T., 2011. Carotenoids in marine animals. Mar. Drugs 9, 278–293.
Maoka, T., 2019. Carotenoids as natural functional pigments. J. Nat. Med. https://doi.
org/10.1007/s11418-019-01364-x.
Maoka, T., 2016. Structural stugies of carotenoids in plants, animals, and food products.
In: Agnieska Kaczor, Malgorzata Baranska (Eds.), Carotenoids Nutrition, Analysis andTechnology. Wiley Blackwell, UK, pp. 103–129.
Manuta, C., 1948. Astaxanthin in insects and other terrestrial arthropoda. Nature 162,
298.
Mandrioli, M., Rivi, V., Nardelli, A., Manicardi, G.C., 2016. Genomic and cytogenetic
localization of the carotenoid genes in the aphid genome cytogenet. Genome Res.
149, 207–217.
Moran, N.A., Jarvik, T., 2010. Lateral transfer of genes from fungi underlies carotenoid
production in aphids. Science 328, 624–627. https://doi.org/10.1126/science.
1187113.
Nováková, E., Moran, N.A., 2012. Diversification of genes for carotenoid biosynthesis inaphids following an ancient transfer from a fungus. Mol. Biol. Evol. 29, 313–323.
https://doi.org/10.1093/molbev/msr206.
Ozono, A., Kawashima, I., Futahashi, R., 2012. Dragonflies of Japan. 531pp. Bun-ichi
Sogo-syuppan Co. ltd., Tokyo ([in Japanese with English summary]).
Shindo, K., Kikuta, K., Suzuki, A., Katsuta, A., Kasai, H., Yasumoto-Hirsose, M., Matsuo,Y., Misawa, N., Takaichi, S., 2006. Rare carotenoids, (3R)-saporoxanthin and(3R,2’S)-myxol, isolated from novel marine baxteria (Flavobacteriaceae) and theirantioxidative activities. Appl Microbiol Biochem. https://doi.org/10.1007/s00253006-0774-y.
Speight, M.R., Hunter, M.D., Watt, A.D., 2008. Ecology of Insect Concepts andApplications, Second. Wiley-Blackwell, Oxford UK.
Teramoto, M., Takaichi, S., Inomata, Y., Ikenaga, H., Misawa, N., 2003. Structural andfunctional analysis of a lycopene β-monocyclase gene isolated from a unique marine
bacterium that produces myxol. FEBS Lett. 545, 120–126.
Tsuchida, T., Koga, R., Mitsuyo, M., Tsunoda, T., Maoka, T., Matsumoto, S., Simon, J.-C.,
Fukatsu, T., 2010. Symbiotic bacterium modifies aphid body color. Science 330,
1102–1104.
Yokoyama, A., Miki, W., 1995. Isolation of myxol from a marine bacterium Flabobacter sp.
Associated a marine sponge. Fish. Sci. 61, 684–689.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bse.2020.104001.
References
Arpin, N., Fiasson, J.L., Baucher-Dangya-Caye, M.P., Francis, G.W., Liaaen-Jensen, S.,
1971. A new C40H56 carotene with a terminal methyene group. Phytochemistry 10,
1595–1601.
Asahina, S., 1974. The development of odonatology in the Far Esast. Odonatologica 3,
5–12.
Britton, G., Liaaen-Jensen, S., Pfander, H., 2004. Carotenoids Hand Book. Birkhäuser,
Basel, Switzerland.
Britton, G., Lockley, W.J.S., Harriman, G.E., Goodwin, T.W., 1977a. Pigmentation of theladybird beetle Coccinella septempunctata by carotenoids not of plant origin. Nature
266, 49–50.
Britton, G., Goodwin, T.W., Harriman, G.E., Lockley, W.J.S., 1977b. Carotenoids of theladybird beetle, Coccinella septempuncata. Insect Biochem. 7, 337–345.
Britton, G., 1995. UV/Visible spectrometry. In: In: Britton, G., Liaaen-Jensen, S., Pfander,
H. (Eds.), Carotenoids 1B. Brikhauser Verlag, Basel, pp. 13–62.
Brown, K.S., 1975. The chemistry of aphids and scale insects. Chem. Soc. Rev. 4, 263–288.
Corbet, P.S., 1999. Dragonflies: Behavior and Ecology of Odonata. Cornell University
Press, New York, pp. 829.
Davidson, B.S., Eisener, T., Meinwald, J., 1991. 3,4-Didehydro-β,β-caroten-2-one, a new
carotenoid from the eggs of the stick insect Anisomorpha buprestoides. Tetrahedron
Lett. 32, 5651–5654.
Ding, B.-Y., Niu1, J., Shang, F., Yang, L., Chang, T.-Y., Wang, J.-J., 2019. Characterizationof the geranylgeranyl diphosphate ayhthease gene in Acythosiphon pisum (Hemiptera;
Aphididae) and its association with carotenoid biosynthesis. Front. Physiol. 10, 1398.
https://doi.org/10.3389/fphys.2019.01398.
Futashasi, R., Kurita, R., Mano, H., Fukatsu, T., 2012. Redox alters yellow dragonfly in to
red. Proc. Natl. Acad. Sci. U.S.A. 109, 12626–12631.
Goodwin, T.W., 1980. Biosynthesis of carotenoids. In: Goodwin, T.W. (Ed.), The
Biochemistry of the Carotenoids. Chapman and Hall, London, pp. 33–76 1980.
Harashima, K., Ohno, T., Sawachika, T., Hidaka, T., Ohnishi, E., 1972. Carotenoids inorange pupae of the swallowtail, Papilio xuthus. Insect Biochem. 2, 29–48.
Kayser, H., 1975. Isolation of 3-hydroxy-β,ε-caroten-3'-one from two moth species, Actias
selene and Cerura vinula. J. Comp. Physiol. 104, 27–31.
Kayser, H., 1976. Isolation of β,β-carotene-2-ol from an insect, Cerura vinula(Lepidoptera). Z. Naturforschung 31C, 121–128.
Kayser, H., 1979. Presences and biosynthetic implications of β, β-carotene-2-one in themoth Cerura vinula. Z. Naturforschung 34C, 483–484.
Kayser, H., 1981a. Carotenoids in insect. In: Britton, G., Goodwin, T.W. (Eds.),
Carotenoids Chemistry and Biochemistry. Pergamon Press, Oxford, pp. 195–210.
Kayser, H., 1981b. Carotenoids in the stick insect, Ectatosoma tiaratum isolation of β,ε-7