Morphological and molecular characterizations of the Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera:
Chrysomelidae)
Ju Il Kim, Jee Sun Min, Min Kwon, June-Yeol Choi, Si Hyeock Lee
PII:
DOI:
Reference:

S1226-8615(13)00083-6
doi: 10.1016/j.aspen.2013.08.008
ASPEN 448

To appear in:

Journal of Asia-Pacific Entomology

Received date:
Revised date:
Accepted date:

13 October 2012
16 July 2013
26 August 2013

Please cite this article as: Kim, Ju Il, Min, Jee Sun, Kwon, Min, Choi, June-Yeol, Lee,
Si Hyeock, Morphological and molecular characterizations of the Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera: Chrysomelidae), Journal
of Asia-Pacific Entomology (2013), doi: 10.1016/j.aspen.2013.08.008

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Send comments and proofs to:

Journal of Asia-Pacific Entomology

Mr. Ju Il Kim

Draft of Oct. 6, 2012

Highland Agriculture Research Center

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For publication in:

National Institute of Crop Science

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Rural Development Administration

Pyeong-chang, Korea 232-955

Tel: +82-33-330-1991
Fax: +82-33-330-1519

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Email: forweek@korea.kr

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Morphological and molecular characterizations of the

Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon
brassicae (Coleoptera: Chrysomelidae)

Highland Agriculture Research Center, NICS, RDA, Pyeong-chang, Korea 232-955

Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea 151-742

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Ju Il Kima,1, Jee Sun Minb, Min Kwon a, June-Yeol Choic and Si Hyeock Leeb

Crop Environment Division, NICS, RDA, Suwon, 441-857, Korea

Footnotes
1

To

whom

correspondence

should

be

addressed.

Fax:

82-33-330-1519,

forweek@korea.kr
2

Abbreviation used:

DLB, Daikon leaf beetle, Phaedon brassicae Baly (Coleoptera: Chrysomelidae)

E-mail:

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Abstract

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A gregarine parasite (Eugregarinida: Gregarinidae) was observed in the population of

daikon leaf beetle, Phaedon brassicae Baly, (Coleoptera: Chrysomelidae) in Korea.

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Gregarines are well known species-specific parasites of various Arthropoda. This

Gregarina sp. also confirmed a species-specific parasite in P. brassicae. Based on
1.727kb of 18S rDNA sequence (FJ481523), this gregarine species was grouped in
eugregarine and a 5.258kb of full length rDNA replicon was cloned (JF412715). We

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also observed interaction of tropozoite or gamonto of gregarine and epithelium of a

host midgut using a light microscope and a scanning electron microscope. Although the

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developmental period of the infected host is delayed half a day in every larval stage,
there was no significant difference in the developmental period of P. brassicae whether
Gregarina sp. was infected or not. Gregarina sp. was a kind of facultative parasite from

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P. brassicae. This is the first report of a gregarine parasite in P. brassicae.

Key words: gregarine, Phaedon brassicae, parasite, bio-pesticide, development

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regulation

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Introduction
daikon

leaf

beetle

(DLB),

Phaedon

brassicae

Baly

(Coleoptera:

The

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Chrysomelidae) is widely distributed in east and south Asia, including Korea, Japan,
China, Vietnam and India (Wang et al., 2007). This species mainly feed on cruciferous

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vegetables. In Daegwallyeong area in Korea, all stages of DLB were observed from
June to September. Generally, DLB demonstrated a very low resistant level against
almost all kinds of insecticides against coleoptera. However, because of their high
reproducibility (a female lays more than 10 eggs per day), long lifespan in the adult

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stage (more than a month) and short development time (egg to adult, about 20 days at
25C), DLB is one of the major pests in organic cabbage cultivation.

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Gregarines are protozoan parasites in the phylum Apicomplexa and are also
species-specific parasites of a wide range of invertebrates from phyla Arthropoda
(including

crustaceans,

insects,

myriapods),

Annelida

(including

polychaetes,

oligochaetes and leeches), Nemertea, Mollusca, Echinodermata, and Urochordata.

Apicomplexa is a diverse group of unicellular eukaryotes that parasitize on the body

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cavities, tissues and guts of metazoans (Leander et al., 2003). Some Apicomplexans
such as Haemosporidians (e.g. Plasmodium, the causative agent of malaria),

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Coccidians (e.g. Toxoplasma and Eimeria) and Piroplasms (e.g. Babesia) are
pathogens of humans and domesticated vertebrates. The Apicomplexa is a large and
complex phylum, consisting of nearly 5000 described species, with as many as 60,000
yet to be named (Escalante and Ayala 1995; Beck et al., 2009). The Apicomplexans are

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all parasites, characterized by the "apical complex," a structure that inspired the
phylum's name. The apical complex is functionally linked to secretory organelles which
together play a role in the attachment to the host and in most taxa, intracellular
invasion (Black and Boothroyd, 2000).
Gregarine has not been a well-known parasite in Coleoptera. As described above,
gregarine is parasitic on insects including DLB which is one of the major pests in
organic

cabbage

cultivation.

DLB

ecological

characteristics

and

insecticide

susceptibility was not well studied. When we performed a bioassay to select efficient
insecticide, cyst formed parasite observed from excreta of DLB larvae. After the first
finding, we hypothesized that gregarine could be used as a bio-pesticide based on
some reports (Lopes and Alves 2005; Schilder and Marden 2007). We identified this
new gregarine before their application.

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Materials and methods

Insects

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The daikon leaf beetle (DLB), Phaedon brassicae Baly, (Coleoptera: Chrysomelidae)
was initially collected from Kimchi cabbage fields of the Highland Agriculture Research

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Center (HARC), Daegwallyeong, Pyeongchang, Korea in 2008. DLB has been

maintained in an insect rearing room at HARC under the conditions of 25 2C, 16L:
8D photoperiod, and 50~70% relative humidity. For DLB developmental period
comparisons between the gregarine infected strain (G+) and uninfected strain (G-)

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randomly separated in the pupa stage. During the pre-pupal stage, DLB secreted the
gut contents, containing gregarine. The newly emerged G+ adult gave the gamontocyst

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stage of gregarine with Kimchi cabbage. After confirming gregarine infection at 15 days
after emergence, eggs were collected. These eggs were designated G+ and thirty eggs
were observed daily with that of G-. All experiments were replicated three times.

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in the Daegwallyeong area.

Gregarine populations were surveyed during 2008~2012 from DLB which was collected

Microscopy

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The 3rd instar larvae of DLB were dissected in a magnetic dissecting chamber with
PBS (10mM phosphate-buffered saline pH 7.2). The whole gut was opened, mounting
the gut contents or soaked in fixing solution (4% paraformaldehyde) for 40min at room
temperature in a 0.2ml PCR tube. Subsequently, the fixed gut contents were

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permeabilized in 0.2% PBT (10mM phosphate-bufered saline pH 7.2, 0.2% triton X100) for 10min, and repeated three times. After toluidine blue staining, the specimen
was fully washed with 0.2% PBT and mounted. Unstained and toluidine blue stained
gut contents were photographed on a Nikon eclipse E600 light microscope equipped
with Leica application suite program ver. 2.8.1. The whole gut and excreta of the larva
were photographed on a Nikon SMZ1500 stereoscopic zoom microscope equipped
with a Nikon coolpix digital camera.
For scanning electron microscopy, DLB larva was dissected and the whole gut was
opened, soaked in Modified Karnovsky's fixing solution (50mM sodium cacodylate
buffer pH 7.2, 2% paraformaldehyde and 2% glutaraldehyde) for 2h at 4C. After
primary fixing, the specimen was fully washed with a 50mM sodium cacodylate buffer
(pH 7.2) and soaked in a post fixing solution (50mM sodium cacodylate buffer pH 7.2,
1% osmium tetroxide). Subsequently, fixed samples were washed with distilled water at
room temperature. After dehydration, samples were mounted on metal stubs and

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coated with gold. Samples were photographed on Jeol JSM 5410LV (Jeol, Japan).

Amplification of partial rDNA from DLB and Gregarine

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Total DNA was extracted from the 2~3 DLB larvae with an appropriate volume
(0.5~1ml) of DNAzol (MRC, cincinnati, USA) according to the manufacturers protocol.

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Partial 18S rDNA fragments in DLB and gregarine were amplified from the total
DNA of DLB or gamontocyst of gregarine with a set of primers (Table 1) which were
designed from NCBI genbank database and our results. All PCRs were performed by
EX Taq Polymerase (Takara, Shiga, Japan) and DNA Engine Dyad Peltier Thermal

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Cycler (Biorad, Hercules, USA) with the following program: 94C for 5 min, 35 cycles of
94C for 30 sec, 60C for 30 sec, 72C for 90 sec, followed by final extension at 72C

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for 5 min. The PCR product was purified from the gel or from directly using the
Qiaquick gel extraction kit and Qiaquick PCR purification kit (Qiagen, Valencia, USA),
respectively. Purified samples were directly sequenced (Macrogen sequencing center,
Seoul, Korea) and cloned into pGEM-T easy vector (Promega, Madison, USA) and

sequenced. Contig assembly, nucleotide sequence alignment and phylogenetic tree

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construction (Clustral W method) were conducted by using the Lasergene software

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(DNASTAR, Madison, USA) etc.

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Results and discussion

Kimchi cabbage is one of the major vegetables in Korea and the daikon leaf beetle

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(DLB), Phaedon brassicae is also an important insect pest in Kimchi cabbage. The
Gregarina sp. was observed through the seasonal occurrences survey of insect pests

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in cruciferous vegetables and the insecticides resistance monitoring project. The

Gregarina sp. was observed in DLB which were collected from Kimchi cabbage,
cabbage and broccoli fields, Daegwallyeong, Pyeongchang during 2008~2012. We
speculated that this species is a novel Gregarina sp. based on previously reported

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taxonomic papers (Simdianov 2004; Larsson and Koie 2006; Clopton 2009; Simdianov
2009). Although, the striped cabbage flea-beetle, Phyllotreta striolata (Fabricius) is

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generally only distributed in Kimchi cabbage, cabbage and broccoli fields in Korea.
However, the Gregarina sp. was only observed in DLB, not in P. striolata even in the
same fields. Furthermore, another gregarine species was observed in P. striolata and it
cannot infect in DLB. These results confirmed that Gregarina sp. like other gregarines

are well known species-specific parasites of various Arthropoda. The infection ratio of

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the Gregarina sp. was almost 100% in fields (n = over 30 larvae per year).
The morphological characteristics and interaction between Gregarina sp. and DLB

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were identified using a stereomicroscope and SEM (Fig. 1, 2). Tropozoite and gamonto
stages were generally observed in the midgut to the hindgut, and gamonotocyst was in
the hindgut and excreta of the host, respectively. We hypothesized that this parasite
can influx the host gut when the host uptakes cabbage at the sporozoite stage.

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Parasites grow in the host gut (tropozoite to gamonto) and gamontocyst efflux with
excreta of the host. Previous study in attachment from the host cell, especially the gut,
was focused on the trophozoite stage(Valigurova and Koudela 2008). However, we
observed that the gamonto stage also attached to the host epithelium (Fig. 2). The
individual gregarine is divided into two parts, the protomerite, and deutomerite and
epimerite from protomerite (Fig. 2). Protomerite was important to connect in the host
epithelium (Fig.2).
Molecular identification was performed by 18S rDNA sequence comparisons which
were reported in Genbank. The 18S rDNA, also known as small-subunit rDNA, is one
of the most popular molecular markers used to identify and study the phylogeny in
micro-organisms including gregarine (Leander et al., 2003a; Leander et al., 2003b;
Richards and Bass, 2005). These results confirmed that the DLB parasite, Gregarina
sp., is a member of the Eugregarinida, Gregarina genus and also this Gregarina sp. is
a newly recorded species in genbank (Fig. 3).

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After molecular identification of Gregarina sp. we focused on the interaction

between Gregarina sp. and their host, DLB, because some researchers reported that

gregarine could have negative effects against the host insect. For example, gregarine

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infected insects were more susceptible than healthy cockroaches when treated with
control agents (Lopes and Alves, 2005). In another instance, gregarine reduced flight-

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muscle performance, an inability to metabolize lipids in their host (Libertia pulchella)

muscles (Schilder and Marden 2006). However, there were no significant differences
between gregarine infected and uninfected DLB strains during whole life cycles
especially in the larval stage (Fig. 4). The adult stage was not shown here, because the

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life span of the adult stage was too long to record individually (more than two month).
That means individual biases were easily interrupted. From these results, Gregarina sp.

Acknowledgments

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was a kind of facultative parasite from DLB and it cannot be used as a bio-pesticide.

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This work was supported by a grant from the Rural Development Administration (ATIS

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code PJ006630)

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Simdianov, T. G. 2009. Difficilina cerebratuli gen. et sp. n. (Eugregarinida: Lecudinidae)a new gregarine species from the nemertean Cerebratulus barentsi (Nemertini:

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Valigurova, A., Koudela, B., 2008. Morphological analysis of the cellular interactions
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Figure captions

Fig. 1. Various features of the protozoan parasite, Gregaria sp. were observed inside of

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the gut from their insect host, Phaedon brassicae. Excreta of the host and cyst formed
parasites were showed in of P. brassicae (A). Cyst formed parasites were hatched at

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3~5 days after defecation (B, C). When the cysts hatch, numerous small sporozoites
were excreted (D). Dorsal view of the internal organs (gut, malpighian tubules, fat
bodies; E) or gut only (F) form3rd instar larva of P. brassicae. Numerous tropozoites
existed inside of the midgut (G) and tropozoites were mainly lived in midgut to hindgut

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(H). Gamontos and gamontocysts were existed in the gut lumen of hindgut (I). Nucleus

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were observed after toluidine blue staining in tropozoites (J).

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Fig . 2. Scanning electron micrographs showing the morphological features of different

stages of the Gregarina sp. and Interaction between gregarine and their insect host,

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Phaedon brassicae. Tropozoite divided three parts, epimerite, protomerite and

deutomerite (A). Early gamonto looks like fused form of two tropozoite (B). Gamonto
(C) changed to ball shape of gamontocyst (D). Tropozoites and gamontos attach the
inside of the host gut (E) and protomerite directly interact the host midgut epithelium (F).

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Fine structure of epimerite which direct interaction part of tropozoite and gamonto
between host midgut epithelium (G) and top showing more detailed view of the
epimerite which pore-like structure (H).

Fig 3. Phylogenetic analysis of various organisms based on 18S rDNA sequences by

Clustal W Method using Lasergene software (DNASTAR). Ten sequences from
Apicomplexan species, collected from the Genbank database, were aligned with that of
daikon leaf beetle, Phaedon brassicae and its parasite (1.727kb). Dotted line indicates
a negative branch length, which may results from averaging.

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Fig. 4. Developmental period comparisons between Gregarina sp. infected and

uninfected strains of the Phaedon brassicae. Gregarine infected strain (G+) and

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uninfected strain (G-) randomly separated in pupa stage. Because gregarine secret in
pupa stage. Newly emerged G+ adult gave the gamontocyst stage of gregarine with

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Kimchi cabbage. After confirming gregarine infection at 15 days after emergence, eggs
were collected. These eggs were designated G+ and thirty eggs were observed daily
with that of G-. DLB has been maintained under the conditions of 25 2C, 16L: 8D
photoperiod, and 50~70% relative humidity. All experiments were replicated for three

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times. No letter indicate that the mean is not significantly different (p=0.05, T-test, SAS

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institute 1998).

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

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Table 1. Primers used for partial 18S rDNA or gregarine rDNA replicon amplifications,

and primer walking of rDNA replicon.

Sequence

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Name

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For partial 18S rDNA amplification

GGCAAGTCTGGTGCCAGCA

18S rDNA R

GGTGTGTACAAAGGGCAGGGAC

For gregarine rDNA replicon amplification

GrDNAF

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18S rDNA F

GTCGCTTCAACCGATTGGATGATT
CCCCGACTGTCCTTATCAATCATCA

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GrDNAR

For primer walking of rDNA replicon

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GrDNAF3

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GrDNAR2

GrDNAF2

CGTCTCCAACCGTTAGTACTACA
GTGCCAAAGAGTTGCTGATCAG
GGCCATGCTTTCGCAGTTC

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GrDNAR3

CCCAAATGACGTCAGATTCTAAG

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Graphical abstract

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Highlights

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We find a gregarine parasite in the population of daikon leaf beetle, Phaedon brassicae.

Based on 18S rDNA sequence, this gregarine species was grouped in eugregarine.

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We also observed interaction of tropozoite or gamonto of gregarine and epithelium of

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host midgut.

There was no significant difference in the development of daikon leaf beetle whether it is

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infected or not.

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This is the first report of gregarine parasite in P. brassicae.

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