�������� ����� ��

Morphological and molecular characterizations of the Gregarina sp. (Api-complexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera:Chrysomelidae)

Ju Il Kim, Jee Sun Min, Min Kwon, June-Yeol Choi, Si Hyeock Lee

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

To appear in: Journal of Asia-Pacific Entomology

Received date: 13 October 2012Revised date: 16 July 2013Accepted date: 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. (Apicom-plexa: Protozoa) parasitizing on Phaedon brassicae (Coleoptera: Chrysomelidae), Journalof Asia-Pacific Entomology (2013), doi: 10.1016/j.aspen.2013.08.008

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

1

For publication in: Send comments and proofs to:

Journal of Asia-Pacific Entomology Mr. Ju Il Kim

Draft of Oct. 6, 2012 Highland Agriculture Research Center

National Institute of Crop Science

Rural Development Administration

Pyeong-chang, Korea 232-955

Tel: +82-33-330-1991

Fax: +82-33-330-1519

Email: forweek@korea.kr

Morphological and molecular characterizations of the

Gregarina sp. (Apicomplexa: Protozoa) parasitizing on Phaedon

brassicae (Coleoptera: Chrysomelidae)

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

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

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

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

Footnotes

1 To whom correspondence should be addressed. Fax: 82-33-330-1519, E-mail:

forweek@korea.kr

2 Abbreviation used:

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

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

2

Abstract

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

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

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

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

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

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

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

regulation

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

3

Introduction

The daikon leaf beetle (DLB), Phaedon brassicae Baly (Coleoptera:

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

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

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.

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

cavities, tissues and guts of metazoans (Leander et al., 2003). Some Apicomplexans

such as Haemosporidians (e.g. Plasmodium, the causative agent of malaria),

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

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.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

4

Materials and methods

Insects

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

was initially collected from Kimchi cabbage fields of the Highland Agriculture Research

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-)

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

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.

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

in the Daegwallyeong area.

Microscopy

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

permeabilized in 0.2% PBT (10mM phosphate-bufered saline pH 7.2, 0.2% triton X-

100) 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

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

5

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

Amplification of partial rDNA from DLB and Gregarine

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 manufacturer’s protocol.

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

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

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

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

(DNASTAR, Madison, USA) etc.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

6

Results and discussion

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

(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

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

taxonomic papers (Simdianov 2004; Larsson and Koie 2006; Clopton 2009; Simdianov

2009). Although, the striped cabbage flea-beetle, Phyllotreta striolata (Fabricius) is

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

the Gregarina sp. was almost 100% in fields (n = over 30 larvae per year).

The morphological characteristics and interaction between Gregarina sp. and DLB

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.

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).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

7

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

infected insects were more susceptible than healthy cockroaches when treated with

control agents (Lopes and Alves, 2005). In another instance, gregarine reduced flight-

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

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.

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

Acknowledgments

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

code PJ006630)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

8

References

Beck, H. P., Blake, D., Darde, M. L., Felger, I., Pedraza-Diaz, S., Regidor-Cerrillo, J.,

Gomez-Bautista, M., Ortega-Mora, L. M., Putignani, L., Shiels, B., Tait, A., Weir,

W., 2009. Molecular approaches to diversity of populations of apicomplexan

parasites. Int. J. Parasitol. 39, 175-189.

Black, M. W., Boothroyd, J. C., 2000. Lytic cycle of Toxoplasma gondii. Microbiol. Mol.

Biol. Rev. 64, 607-623.

Clopton, R. E., 2009. Standard Nomenclature and Metrics of Plane Shapes for Use in

Gregarine Taxonomy.Comp. Parasitol. 76, 167-190.

Escalante, A. A., Ayala, F. J., 1995. Evolutionary origin of Plasmodium and other

Apicomplexa based on rRNA genes. Proc .Natl. Acad. Sci. U S A. 92, 5793-

5797.

Larsson, J. I., Koie, M., 2006. The ultrastructure and reproduction of Amphiamblys

capitellides (Microspora, Metchnikovellidae), a parasite of the gregarine Ancora

sagittata (Apicomplexa, Lecudinidae), with redescription of the species and

comments on the taxonomy. Eur. J. Protistol. 42, 233-248.

Leander, B. S., Clopton, R. E., Keeling, P. J., 2003a. Phylogeny of gregarines

(Apicomplexa) as inferred from small-subunit rDNA and beta-tubulin. Int. J. Syst.

Evol. Microbiol. 53, 345-354.

Leander, B. S., Harper, J. T., Keeling, P. J., 2003b. Molecular phylogeny and surface

morphology of marine aseptate gregarines (Apicomplexa): Selenidium spp. and

Lecudina spp. J. Parasitol. 89, 1191-1205.

Lopes, R. B., Alves, S. B., 2005. Effect of Gregarina sp. parasitism on the susceptibility

of Blattella germanica to some control agents. J. Invertebr. Pathol . 88, 261-264.

Richards, T. A., Bass, D., 2005. Molecular screening of free-living microbial eukaryotes:

diversity and distribution using a meta-analysis. Curr. Opin. Microbiol. 8, 240-

252.

Schilder, R. J., Marden, J. H., 2006. Metabolic syndrome and obesity in an insect. Proc.

Natl. Acad. Sci. U S A. 103, 18805-18809.

Schilder, R. J., Marden, J. H., 2007. Parasites, proteomics and performance: effects

of gregarine gut parasites on dragonfly flight muscle composition and function. J.

Exp. Biol. 210, 4298-4306.

Simdianov, T. G., 2004. Sphinctocystis phyllodoces gen. n., sp. n. (Eugregarinida:

Lecudinidae) a new gregarine from Phyllodoce citrina (Polychaeta:

Phyllodocidae). Parazitologiia. 38, 322-332.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

9

Simdianov, T. G. 2009. Difficilina cerebratuli gen. et sp. n. (Eugregarinida: Lecudinidae)-

a new gregarine species from the nemertean Cerebratulus barentsi (Nemertini:

Cerebratulidae). Parazitologiia. 43, 273-287.

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

between the eugregarine Gregarina garnhami (Apicomplexa) and the epithelium

of its host, the desert locust Schistocerca gregaria. Eur. J. Protistol. 44, 197-207.

Wang, X. P., Xue, F. S., Tan Y. Q., Lei C. L., 2007. The role of temperature and

photoperiod in diapause induction in the brassica leaf beetle, Phaedon

brassicae (Coleoptera: Chrysomelidae). Eur. J. Entomol. 104, 693-697.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

10

Figure captions

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

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

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

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

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

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

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

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).

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 it’s parasite (1.727kb). Dotted line indicates

a negative branch length, which may results from averaging.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

11

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

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

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

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

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

institute 1998).

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

12

Fig. 1

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

13

Fig. 2

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

14

Fig. 3

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

15

Fig. 4

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

16

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

and primer walking of rDNA replicon.

Name Sequence

For partial 18S rDNA amplification

18S rDNA F GGCAAGTCTGGTGCCAGCA

18S rDNA R GGTGTGTACAAAGGGCAGGGAC

For gregarine rDNA replicon amplification

GrDNAF GTCGCTTCAACCGATTGGATGATT

GrDNAR CCCCGACTGTCCTTATCAATCATCA

For primer walking of rDNA replicon

GrDNAF2 CCCAAATGACGTCAGATTCTAAG

GrDNAR2 CGTCTCCAACCGTTAGTACTACA

GrDNAF3 GTGCCAAAGAGTTGCTGATCAG

GrDNAR3 GGCCATGCTTTCGCAGTTC

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

Graphical abstract

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

18

Highlights

· 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.

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

host midgut.

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

infected or not.

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