Molecular identification of dipteran pests (Diptera: Sciaroidea) from shiitake mushroom

Molecular identification of dipteran pests(Diptera: Sciaroidea) from shiitake mushroom

SEUNGGWAN SHIN,*†‡ SUNGHOON JUNG,*†‡ HEUNGSIK LEE§ and SEUNGHWAN LEE*†‡

*Insect Biosystematics Laboratory, Seoul National University, Seoul 151-921, Korea, †Research Institute for Agricultural and Life

Sciences, Seoul National University, Seoul 151-921, Korea, ‡Department of Agricultural Biotechnology, Seoul National University,

Seoul 151-921, Korea, §Central Post-Entry Quarantine Station, Animal Plant and Fisheries Quarantine and Inspection Agency,

Suwon 443-400, Korea

Abstract

On shiitake farms, mycophagous maggots can cause serious damage by preventing formation of the fruiting body.

Recently, these pests have significantly reduced shiitake production in Korea. However, larvae and female adults

cannot be identified due to their lack of morphological characteristics. Therefore, farmers and applied entomologists

are unable to determine which species is the primary cause of the shiitake damage. In this study, mycophagous flies

(colonized larvae) were collected from damaged shiitake farms and subsequently identified by matching identified

males with the cytochrome c oxidase subunit I (COI) sequences from the larvae. Divergences of the COI sequences

among the species discriminated the clusters clearly, and the mycophagous pests were identified as Camptomyia

corticalis and C. heterobia. Interestingly, these two species coexisted under the bark of shiitake oak bed logs.

Keywords: Camptomyia corticalis, Camptomyia heterobia, COI barcoding, Shiitake mushroom pest

Received 14 December 2011; revision received 27 November 2012; accepted 29 November 2012

Introduction

The shiitake Lentinula edodes (Berk.) Pegler 1976 (Agari-

cales: Marasmiaceae) is an edible and medicinal mush-

room grown in Far East Asia (Mimura et al. 1998;

Hirasawa et al. 2000; Brauer et al. 2002; Shimada et al.

2002). It is cultivated widely in Asia as well as in

Holland, the United States and Canada (Gold et al.

2008), as one of most-cultivated types globally, second

only to Agaricus [white button mushroom and portobel-

lo mushroom (Gold et al. 2008)]. In Asian countries, the

commercial shiitake is generally cultivated in green-

houses in which the temperature, brightness and

humidity are controlled. Recently, in many shiitake

nurseries of Korea, farmers have experienced a serious

loss of crops and have even had to abandon mushroom

farming due to severely damaged bed logs (Kim et al.

2010a; Shin et al. 2011). The damage was caused by

the propagation of a large number of mycophagous

dipteran larvae (Cecidomyiidae and/or Sciaridae) and

green fungi (green mould disease) Gliocladium viride

Matr. 1893 (Hypocreales: Hypocreaceae) (Kim et al.

2010a). Thus, these flies are believed to be an important

pest in shiitake bed logs; however, it remains uncon-

firmed due to the lack of information regarding larval

morphological characteristics (Panelius 1965; McAlpine

1981; Shin et al. 2011; Sutou et al. 2011) (Fig. 1A).

These mycophagous flies can be hardly separated by

the morphological characteristics of adult males, and

larval samples must be reared to adulthood to get the

adult male specimens (Panelius 1965; McAlpine 1981;

Gagne 2004). Consequently, DNA-based methodologies

could be used to identify the immature and female speci-

mens (Boehme et al. 2010).

Recently, ‘DNA barcoding’ has been used for pest

monitoring and quarantine issues (Armstrong & Ball

2005; Ball & Armstrong 2006; Hebert & Ratnasingham

2007). Its utility has been confirmed in several hexapod

orders; e.g. Coleoptera (L€obl & Leschen 2005), Diptera

(Scheffer et al. 2006; Smith et al. 2006; Rivera & Currie

2009), Ephemeroptera (Ball et al. 2005), Hemiptera (Foot-

tit et al. 2008; Kim et al. 2010b; Lee et al. 2010; Jung et al.

2011), Hymenoptera (Smith et al. 2008), and Lepidoptera

(Hebert et al. 2003; Hajibabaei et al. 2006). The cyto-

chrome c oxidase subunit I (COI) DNA barcodes of

Cecidomyiidae have been studied and applied to gall-

making species, which are pests of live plants (Kolesik &

Veenstra-Quah 2008; Uechi et al. 2011; Veenstra et al.

2011; Mathur et al. 2012).Correspondence: Seunghwan Lee, Fax: +82 2 873 2319; E-mail:

[email protected]

© 2013 Blackwell Publishing Ltd

Molecular Ecology Resources (2013) 13, 200–209 doi: 10.1111/1755-0998.12057

In this study, we compiled the DNA barcode

sequences of morphologically identified male specimens

of the major dipteran species found on shiitake farms,

and applied them to identify the immature and/or the

female adult specimens, which is hardly determined by

morphological characteristics.

Materials and methods

Taxon sampling

All of the examined taxa were outbreak species on

damaged shiitake farms in the Korean Peninsula from

2008 to 2010 (Figs 1 and 2). To collect adults, Malaise

traps and sweeping nets were used in the shiitake farms

(Table 1). All larval samples were collected from the

infected phloem of the shiitake bedlogs (Fig. 1; Table 1).

Larvae from the infected parts of the oak timbers were

transferred to the laboratory and placed in plastic emer-

gence cages at 25 � 3 °C (Shin et al. 2011). Samples

stored in 99% ethanol were used for genomic DNA

extraction (Table 1, Table S1). The identification of each

species was based on the exterior morphologies of the

slide-mounted specimens. All samples and voucher

specimens were preserved in the Insect Collection of the

College of Agriculture and Life Sciences at Seoul

National University, Korea. DNA sequences for the COI

barcode region were obtained from a total of 138 individ-

uals. Of these, 70 male specimens were identified at the

species level based on their morphology (Shin et al.

2011). The remaining specimens (54 adult females, 13

larvae, and one pupa) were used to challenge the DNA

barcode data set to ensure that all species of concern in

this particular habitat could be identified based on their

COI sequences.

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from single individu-

als using a DNeasy Blood and Tissue kit (QIAGEN, Inc).

To create the voucher specimens from the DNA extracted

samples, we used a nondestructive DNA extraction pro-

tocol slightly modified from the methods of Favret (2005)

and Kim et al. (2010a). Before extraction, the head, wings

and genitalia of each specimen were dissected and

mounted onto microscope slides. The thorax and abdo-

men left in lysis buffer with proteinase K solution at

56 °C for 6 h. After incubation, the cleared cuticle was

dehydrated and mounted with other mounted parts onto

a microscope slide. The primers used for the polymerase

chain reaction (PCR) amplifications were as follows:

LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′)

and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAA-

ATCA-3′) (Folmer et al. 1994) for COI. The DNA frag-

ments to be analysed were amplified using AccuPower

PCR PreMix, a total of 20 lL reaction mixture contained

(a) (b) Fig. 1 Damage by the dipteran pests in

the shiitake farms. A: Mixed colony of

two Camptomyia larvae (C. corticalis and C.

heterobia) feeding on phloem of damaged

shiitake bedlogs; B: Damaged shiitake

logs.

Fig. 2 Collection sites. BA: Jeollabuk-do, Buan-gun; CA: Chung-

cheongnam-do, Cheonan-si; GJ: Gyeongsangnam-do, Geoje-si;

HS: Gangwon-do, Hoengseong-gun; JH: Jeollanam-do,

Jangheung-gun; PC: Gangwon-do, Pyeongchang-gun; PJ:

Gyeonggi-do, Paju-si; PT: Gyeonggi-do, Pyeongtaek-si; SG: Jeju-

do, Seogwipo-si; SU: Seoul-si; SW: Gyeonggi-do, Suwon-si


BARCODING OF DIPTERAN PESTS 201

Table

1Sam

ple

list

ofsp

ecim

ens

Status

Fam

ily

Species

ID

No.

individual

Collectionlocality

Date

Collectionno.

Collectedsite

Adult

male

Cecidomyiidae

Cam

ptom

yia

corticalis

C-C

A-M

-CC

7CN,Cheo

nan

-si,Seo

buk-gu,

Ipjang-m

yeo

n

2Sep

tember

2009

090902-SKC

Shiitakefarm

(Bed

logs)

C-H

S-M

-CC

6GW,Hoen

gseong-gun,

Jogok-ri

14October

2009

091014-SKH

phloem

ofsh

iitakebed

logs

(emerged

adult)

C-H

S-M

-CC

1GW,Hoen

gseong-gun,

Jogok-ri

13June2010

100613-SKH

Shiitakefarm

(Bed

logs)

C-H

S-M

-CC

7GW,Hoen

gseong-gun,

Jogok-ri

25May

2009

090525-SKH

Shiitakefarm

(Bed

logs)

Cam

ptom

yia

heterobia

C-BA-M

-CH

3JB,Buan

-gun,boan

-myeo

n,

sangipseok-ri

7October

2010

101007-SKB

Phloem

ofsh

iitakebed

logs

(emerged

adult)

C-C

A-M

-CH

2CN,Cheo

nan

-si,Seo

buk-gu,

Ipjang-m

yeo

n

2Sep

tember

2009

090902-SKC

Shiitakefarm

(Bed

logs)

C-H

S-M

-CH

2GW,Hoen

gseong-gun,

Jogok-ri

25May

2009

090525-SKH

Shiitakefarm

(Bed

logs)

C-H

S-M

-CH

4GW,Hoen

gseong-gun,

Jogok-ri

14October

2009

091014-SKH

Phloem

ofsh

iitakebed

logs

(emerged

adult)

Sciaridae

Bradysia

difformis

S-C

A-M

-BD

1CN,Cheo

nan

-si,Seo

buk-gu,

Ipjang-m

yeo

n

2Sep

tember

2009

090902-SKC

Shiitakefarm

(Bed

logs)

S-H

S-M

-BD

1GW,Hoen

gseong-gun,

Jogok-ri

17August

to

7Sep

tember

2009

090907-SKHM

Shiitakefarm

Malaise

trap

(Bed

logs)

S-JH-M

-BD

1JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

16May

2009

090516-SKJ

Shiitakefarm

(Bed

logs)

S-PC-M

-BD

20GW,Pyeo

ngch

ang-gun,

Jinbu-m

yon

18August

2009

090818-SKP

Shiitakefarm

(Saw

dust

bed

s)

S-PT-M

-BD

1GG,Pyeo

ngtaek

-si

14May

2009

090514-SKP

Bradysia

longimentula

S-H

S-M

-BL

5GW,Hoen

gseong-gun,

Jogok-ri

25May

2009

090525-SKH

S-PJ-M-BL

1GG,Paju-si

23July

2008

080723-SKG

Lycoriella

ingenua

S-JH-M

-LI

1JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

August

2010

100800-SKJ

Shiitakefarm

(Saw

dust

bed

s)

S-JH-M

-LI

2JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

16May

2009

090516-SKJ

Shiitakefarm

(Bed

logs)

S-SU-M

-LI

2Seo

ul,SNU

CALS

20May

2009

090520-SKS

S-SW-M

-LI

3GG,Suwon,SNU

arboretum

20May

2009

090520-SKS


202 S . SHIN ET AL .

Tab

le1

(Continued

)

Status

Fam

ily

Species

ID

No.

individual

Collectionlocality

Date

Collectionno.

Collectedsite

Adult

female

Cecidomyiidae

C-C

A-F

3CN,Cheo

nan

-si,Seo

buk-gu,

Ipjang-m

yeo

n

2Sep

tember

2009

090902-SKC

Shiitakefarm

(Bed

logs)

C-H

S-F

1GW,Hoen

gseong-gun,

Jogok-ri

25May

to16

July

2009

090716-SKHM

Shiitakefarm

Malaise

trap

(Bed

logs)

C-H

S-F

1GW,Hoen

gseong-gun,

Jogok-ri

22October

2009

091022-SKH

Phloem

ofsh

iitakebed

logs

(emerged

adult)

C-H

S-F

1GW,Hoen

gseong-gun,

Jogok-ri

13August

2010

100813-SKH

Shiitakefarm

(Bed

logs)

C-H

S-F

15GW,Hoen

gseong-gun,

Jogok-ri

25May

2009

090525-SKH

Shiitakefarm

(Bed

logs)

C-H

S-F

8GW,Hoen

gseong-gun,

Jogok-ri

14October

2009

091014-SKH

Phloem

ofsh

iitakebed

logs

(emerged

adult)

C-H

S-F

5GW,Hoen

gseong-gun,

Jogok-ri

29Sep

tember

2008

080929-SKH

Phloem

ofsh

iitakebed

logs

(emerged

adult)

C-BA-F

8JB,Buan

-gun,boan

-myeo

n,

sangipseok-ri

7October

2010

101007-SKB

Phloem

ofsh

iitakebed

logs

(emerged

adult)

C-JH-F

1JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

16May

2009

090516-SKJ

Shiitakefarm

(Bed

logs)

Sciaridae

S-JH-F

3JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

August

2010

100800-SKJ

Shiitakefarm

(Saw

dust

bed

s)

S-JH-F

1JN

,Jangheu

ng-gun,

Gwan

gpyeo

ng-ri

16May

2009

090516-SKJ

Shiitakefarm

(Bed

logs)

S-H

S-F

1GW,Hoen

gseong-gun,

Jogok-ri

17April2008

080417-SKH

Shiitakefarm

(Bed

logs)

S-C

A-F

1CN,Cheo

nan

-si,Seo

buk-gu,

Ipjang-m

yeo

n

2Sep

tember

2009

090902-SKC

Shiitakefarm

(Bed

logs)

S-H

S-F

2GW,Hoen

gseong-gun,

Jogok-ri

17August

to

7Sep

tember

2009

090907-SKHM

Shiitakefarm

Malaise

trap

(Bed

logs)

S-SG-F

1JJ,Seo

gwipo-si,

Gan

gjung-dong

14May

2008

080514-SKJ

S-G

J-F

2GN,Geo

je-si,

Dongbu-m

yeo

n,Guch

on-ri

25August

2008

080825-SKG

Larva

Cecidomyiidae

C-H

S-L

1GW,Hoen

gseong-gun,

Jogok-ri

3October

2010

101003-SKH

Phloem

ofsh

iitakebed

logs

C-H

S-L

10GW,Hoen

gseong-gun,

Jogok-ri

3October

2009

091003-SKH

Phloem

ofsh

iitakebed

logs



1 unit of Top DNA polymerase (BIONEER, Corp.,),

250 lM of dNTP, 10 mM of Tris-HCl, 30 mM of KCl and

1.5 mM of MgCl2, 1 lL of each primers (10 pM) and 5–

20 ng of template DNA. PCR was performed using a

PTC-100 thermocycler (MJ Research Inc.,). We used the

following thermal cycle parameters for the 20 lL amplifi-

cation reactions: initial denaturation at 95 °C for 5 min,

followed by 35 cycles of 95 °C for 30 s, annealing temper-

ature of 45 °C for 40 s and extension at 72 °C for 50 s,

with a final extension at 72 °C for 10 min. The PCR prod-

ucts were visualized by electrophoresis on a 2% agarose

gel and stained with ethidium bromide, and purified

using a QIAquick PCR purification kit (QIAGEN, Inc.)

and then sequenced directly on an automated sequencer

(ABI Prism_3730 XL DNA Analyzer; Applied Biosys-

tems) at the National Instrumentation Center for Envi-

ronment Management, Seoul National University (SNU),

(Seoul, Korea). The sequences generated in this study

were all deposited to the National Center for Biotechnol-

ogy Information (NCBI) GenBank as the following acces-

sions: JN378570-JN378707. Only two species of

Camptomyia recorded in Korea (Shin et al. 2011); further-

more, this study provides first DNA data within the sub-

family Porricondylinae. Thus, Dasineura rosae (Bremi

1847) (Diptera: Cecidomyiidae: Cecidomyiinae) (Gen-

Bank accession no. AB505981) was employed as an out-

group taxon for two Camptomyia (Diptera: Cecidomyii-

dae: Porricondylinae) species.

Sequence alignment and data analyses

Raw sequences were examined and corrected using

SeqMan-Pro version 7.1.0 (DNASTAR, Inc.,). All the

DNA sequences for each fragment were aligned using

Clustal X version 2.0.11 (Jeanmougin et al. 1997) with

default settings. Pairwise distances, number of substitu-

tions and nucleotide compositions for COI were obtained

using MEGA 4.0 (Kumar et al. 2007) based on the Kimura

two-parameter (K2P) model. Neighbour-joining (Saitou

& Nei 1987) bootstrap support analysis (1000 replicates)

were performed using MEGA 4.0 (Kumar et al. 2007).

The K2P model of nucleotide substitution (Kimura 1980),

was selected for the analyses, which is the most widely

used model for DNA barcoding analyses.

Results

Species determined from shiitake mushroom bed log

In this study, Cecidomyiidae and Sciaridae species were

collected from shiitake farms. For the five tested species

from the shiitake oak bed logs, morphological identifica-

tion was performed by examining the male genitalia

(Shin et al. 2011, 2012). Of the 70 adult male specimensTab

le1

(Continued

)

Status

Fam

ily

Species

ID

No.

individual

Collectionlocality

Date

Collectionno.

Collectedsite

C-BA-L

2JB,B

uan

-gun,boan

-myeo

n,

sangipseok-ri

7October

2010

101007-SKB

Phloem

ofsh

iitakebed

logs

C-BA-P

1JB,B

uan

-gun,boan

-myeo

n,

sangipseok-ri

7October

2010

101007-SKB

Phloem

ofsh

iitakebed

logs

BA,Jeollab

uk-do,Buan

-gun;CA,Chungch

eongnam

-do,Cheo

nan

-si;CALS,Colleg

eofAgricu

lturalan

dLifeScien

ces;CN,Chungch

eongnam

-do;GG,Gyeo

nggi-do;GJ,Gyeo

ngsang-

nam

-do,Geo

je-si;GN,Gyeo

ngsangnam

-do;GW,Gan

gwondo;HS,Gan

gwon-do,Hoen

gseong-gun;JB,Jeollab

uk-do;JH

,Jeollan

am-do,Jangheu

ng-gun;JJ,Jeju-do;JN

,Jeollan

am-do;

PC,Gan

gwon-do,Pyeo

ngch

ang-gun;SG,Jeju-do,Seo

gwipo-si;SNU,Seo

ulNational

University;SU,Seo

ul-si;SW,Gyeo

nggi-do,Suwon-si;BD,Bradysiadifformis;BL,Bradysialongi-

mentula;CCm

Cam

ptom

yiacorticalis;CH,Cam

ptom

yiaheterobia;DR,Dasineura

rosae;LI,Lycoriellaingenua;M,m

ale;F,female;L,larva;

P,pupa.



obtained, a total of five species belonging to three genera

from two families were identified based on their

morphological characteristics and sequenced for refer-

ence barcode data set [C. corticalis, (21), C. heterobia (11),

Bradysia difformis (24), B. longimentula (6), Lycoriella inge-

nua (8)]; the remaining females [54 specimens: C. cortical-

is, (23), C. heterobia (20), B. difformis (4), B. longimentula

(2), L. ingenua (5)] and larval/pupal specimens [14 speci-

mens: C. corticalis (3) and C. heterobia (11)] were identified

to the family level and also sequenced. Of these, male

specimens reared from larvae to adult were identified as

C. corticalis (6) and C. heterobia (7) based on genitalia

morphological characteristics and COI genetic diver-

gences. Also, 22 female specimens C. corticalis (11) and

C. heterobia (11) reared from larvae were identified based

on COI genetic divergences (Table 1; Table S1, Support-

ing information).

Characteristics of the COI barcoding gene fragments

The COI data set comprised 658 aligned base pairs (bp).

Among the data set, 259 bp were variable and 222 bp

were parsimony informative. The average of the uncor-

rected sequence divergence among all taxa for COI was

19.5%, and the average proportions of T:C:A:G were

40:15:31:14. None of the sequences from this study had

indels.

Molecular identification using COI

The COI sequences of male specimens were matched

with the morphological characteristics of the identified

specimens (Shin et al. 2011, 2012). Sequencing of the COI

gene suggested that five dipteran species were present

among the collected samples, which is consistent with

the morphological identification of male specimens.

Intra- and interspecific variations for these species are

shown in Table 2 and Fig. 3. This COI profile could pro-

vide species-level identification for all tested specimens

(Fig. S1, Supporting information). In all cases, the tested

specimens grouped most closely with their conspecific

profile sequence, confirming that the species possessed

unique arrays of COI sequences (Fig. 4). Figure 4 shows

the species names of the pest larval specimens with the

identified male specimens (Table S1, Supporting infor-

mation). For phylogenetic analysis of the COI data set

(Fig. 4; Fig. S1, Supporting information), the branch

support values were insufficient to resolve relationships

between genera and families, but major nodes subtend-

ing for each species cluster were highly supported. The

monophyly of two Camptomyia species (Fig. 4; Fig. S1,

Supporting information) and three sciarid species (Fig. 4;

Fig. S1, Supporting information) were supported by high

bootstrap values.

Interspecific divergences between C. corticalis

and C. heterobia species were typically an order of magni-

tude greater than the intraspecific divergences (Fig. 3;

Table 2). The mean interspecific divergence between the

two Camptomyia species was 10.33% (range 8.90%–

11.30%) and that between the two Bradysia species was

15.32% (range 15.30%–15.50%) (Table 2).

Table 2 Summary of interspecific and intraspecific K2P

distances of five tested species

Mean (%) Min (%) Max (%)

No.

specimens

Intraspecific K2P distances

Cecidomyiidae

Camptomyia

heterobia 0.79 0.00 2.20 42

corticalis 0.29 0.00 1.50 47

Sciaridae

Bradysia

difformis 0.00 0.00 0.00 28

longimentula 0.17 0.00 0.50 8

Lycoriella

ingenua 0.06 0.00 0.20 13

Interspecific K2P distances

Cecidomyiidae

CC-CH 10.33 8.90 11.30 47–42

DR-CC 24.19 23.60 24.60 1–47DR-CH 22.63 22.10 23.20 1–42

Sciaridae

BD-BL 15.32 15.30 15.50 28–13

BD-LI 18.40 18.40 18.40 28–13BL-LI 18.63 18.60 18.80 8–13

BD, Bradysia difformis; BL, Bradysia longimentula; CC, Camptomyia

corticalis; CH, Camptomyia heterobia; DR, Dasineura rosae; LI,

Lycoriella ingenua.

1000

1500

2000intraspecificinterspecific

0

500Num

ber o

f tax

on p

airs

Genetic distances

Fig. 3 Intraspecific and interspecific distances of the COI bar-

coding region among five Sciaroidea species.



Molecular identification of larvae

All pest larvae from the damaged shiitake bed logs were

matched with C. corticalis and C. heterobia by molecular

analysis (Fig. 4; Fig. S1, Supporting information). Twelve

male specimens of C. corticalis (C5, C9, C14, C15, C66,

C107, C108, C109, C110, C111, C112, C115) had 100%

similarity with larval specimens (C19, C103, C105). Two

groups of C. heterobia larvae matched with the male spec-

imens respectively. One male specimen (C87) had 100%

similarity with four larval specimens (C69, C70, C71,

C75), and four male specimens (C94, C97, C122, C124)

had 100% similarity with five larval and one pupal speci-

mens (C68, C67, C76, C91, C92, C88). Between the two C.

heterobia groups, a 1.4% difference was observed based

on K2P distances (see discussion). In the Hoengseong

shiitake farm, two specimens (C103, C105) of C. corticalis

and eight (C67, C68, C69, C70, C71, C75, C76, C104) of C.

heterobia were collected from the same colony (Oct 3,

2009). All Buan larval samples were identified as C. het-

erobia (Table 1). In addition, both species of adult male

and female specimens were confirmed on the Cheonan

and Hoengseong shiitake farms (Table 1, Fig. 4). There-

fore, these two Camptomyia species living in the same

habitat with forming mixed colonies and identified as

the primary pests of shiitake beg logs.

C67 HS L

C88 BA P

C92 BA L

C102 CA F

C76 HS L

C68 HS L

54

C97 BA M*

C91 BA L

C131 HS F*

C100 CA M

C101 CA M

C12 HS M*100

94

45

72

C104 HS L

C17-HS M*

C69 HS L

C70 HS L

C87 BA M*

C71 HS L

100

6374

97

51

Cam

ptom

yia

hete

robi

a

C75 HS L

C10 HS M

C6 HS M

C132 HS F*

C129 HS F*

C56 HS F7630

61

100

C4 CA F

C23 HS F

C19 HS L

C105 HS L

C5 HS M

C103 HS L

53

15

14

17

C55 HS F

C1 CA M

C2 CA M

Dasineura rosae

S9 PC M

S150 JH M

S29 SW M100

16

Cam

ptom

yia

corti

calis

Bradysia difformis

Lycoriella ingenua

S4 HS M

S5 HS M

S8 HS M82100

55

100

0.02

Bradysia longimentula

Fig. 4 Partial neighbor-joining trees based

on Kimura 2-parameter genetic distances.

Bootstrap support for nodes containing test

sequences based on 1,000 replications. The

scale bar indicates branch length (NJ tree

of total sequences are shown in Fig. S1,

Supporting information). BA: Jeollabuk-

do, Buan-gun. CA: Chungcheongnam-do,

Cheonan-si. HS: Gangwon-do, Hoengs-

eong-gun. PC: Gangwon-do, Pyeong-

chang-gun. M: Male. F: Female. L: Larva.

*: Adult specimens that emerged from

larval colonies in shiitake bedlogs.



Discussion

We confirmed that COI-based DNA barcoding can be

applied to the species determination of the mycopha-

gous dipteran pests on the shiitake mushroom, which

cannot be identified by the morphology of the adult and

larvae. Although Meier et al. (2006) suggested the

relatively low success rate of identification using DNA

barcoding in Diptera, other studies proved the useful-

ness of DNA barcoding in morphologically selective

samplings. DNA barcoding studies on Diptera showed

maximum intraspecific variation from 3.0% to 3.9%

(Cywinska et al. 2006; Smith et al. 2006; Carew et al.

2007). According to Rivera & Currie (2009), the maxi-

mum intraspecific divergence was 3.3%–3.8% for mor-

phologically identified species of Nearctic black flies.

Regarding the DNA barcode of mycophagous flies, Ma-

thur et al. (2012) suggested that the intraspecific diver-

gences of Dasineura oxycoccana (Diptera: Cecidomyiidae)

were 0%–1.3% (cranberry tipworm and blueberry gall

midge). Furthermore, the intraspecific K2P distance of

genus Sciara (Diptera: Sciaridae) was reported to be

lower than 1.6% (Sutou et al. 2011). In this study, the

maximum intraspecific divergence of morphologically

identified species was 2.2% (C. heterobia). Although the

maximum divergence was slightly higher than that in

the previous barcode studies of mycophagous flies

(Sutou et al. 2011; Mathur et al. 2012), the divergence

range was lower than reported in the DNA barcode

study of Diptera (Cywinska et al. 2006; Smith et al. 2006;

Carew et al. 2007). Consequently, larval specimens from

the damaged phloem of shiitake bed logs were matched

to C. corticalis and C. heterobia based on the COI barcod-

ing sequences of the larvae, as well as the morphological

characteristics of reared adults.

In this context, we confirm that the larvae of two

Camptomyia spp. coexist under the bark (phloem section)

of shiitake bed logs [Quercus (Fagales: Fagaceae)]. Two

species of Camptomyia larvae were collected from the

same colony (Table 1; Table S1, Supporting information),

while adult midges of the two larvae were observed in

the same shiitake farm (Table 1). Most Camptomyia

species larvae can be found in a deadwood habitat, such

as under the bark of Pinus, Ulmus, and Picea, including

C. fulva and C. spinifera larvae recorded from Quercus

(Panelius 1965) (although C. fulva and C. spinifera could

not be collected in shiitake bed logs). According to

Mamaev (1961) and Panelius (1965), C. heterobia larvae

exist under the bark of dead Ulmus (Rosales: Ulmaceae).

In contrast, C. corticalis lives under the bark of rotten

Fagus (Fagales: Fagaceae), and cannot survive in dead

Ulmus (Mamaev 1961). However, our molecular analysis

demonstrated that two Camptomyia species could survive

under the bark of shiitake bedlogs. Therefore, in terms of

habitat selection by the genus Camptomyia, the type of

fungi could be a more important factor than the type

(family or genus) of dead wood, because the confirmed

larvae were collected mainly from the mycelium ample

part of the shiitake bed logs. Moreover, Kim et al. (2010a)

suggested shiitake green mould disease could be closely

related with Camptomyia spp. as a vector. These data sup-

port that both Camptomyia species could be pests of shii-

take mushroom cultivation.

According to previous research (Kim et al. 1999; Lee

et al. 1999; Jess & Schweizer 2009), L. ingenua has been

recorded as one of the main pests of commercial mush-

rooms. Although we collected one L. ingenua adult speci-

men from the shiitake bed logs farm, the larvae of the

species on damaged parts have not been found based on

the molecular data.

Conclusions

DNA barcoding for larval dipteran pests (two

Cecidomyiidae and three Sciaridae species) from shii-

take mushrooms was studied. Among them, two Camp-

tomyia (Cecidomyiidae) species were confirmed as the

main pests of shiitake bedlogs. The genus Camptomyia

has 73 species recorded worldwide, and belongs to sub-

family Porricondylinae (Gagne 2004). Nevertheless, the

DNA barcode data of the subfamily Porricondylinae

was reported for the first time in this study. Although

many pest species were collected at the larval stage,

they are difficult or impossible to identify at the species

level (Panelius 1965; McAlpine 1981; Shin et al. 2011; Su-

tou et al. 2011). However, COI barcoding can be useful

to identify species at all stages of insect development in

conjunction with morphological data (Armstrong & Ball

2005; Ball & Armstrong 2006). In addition, construction

of the DNA COI database could be a useful tool for the

field of applied entomology, especially for mycopha-

gous dipteran pests at all larval stages, as well as for

adults.

Acknowledgements

We thank Dr. Spungis V (Department of Zoology and Animal

Ecology, Faculty of Biology, University of Latvia), Dr. Menzel F

(Deutsches Entomologisches Institut) and Mr. Heller K (Heiken-

dorf, Germany) for checking the specimens. We are also thank-

ful to all reviewers who gave their valuable comments. This

study is a component of Project No. S120909L050000 supported

by the Korea Forest Service. Partial funding was also provided

by the Project for Export of Agricultural products by Animal,

Plant and Fisheries Quarantine and Inspection Agency, Korea,

and the Research Institute for Agriculture and Life Sciences

(RIALS), SNU.



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Data accessibility

DNA sequences: Genbank accession numbers are avail-

able in the Table S1 (Supporting information). JN378570-

JN378707.

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Fig. S1. Neighbor-joining trees based on Kimura 2-parameter

genetic distances of total sequences. Bootstrap support for nodes

containing test sequences based on 10,000 replications. Numbers

on branches represent NJ support values. The scale bar indicates

branch length. BA: Jeollabuk-do, Buan-gun. CA: Chungcheong-

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Gangwon-do, Pyeongchang-gun. M: Male. F: Female. L: Larva.

Table S1. List of specimens used in this study. SNU, Seoul

National University; CALS: College of Agricultural and Life

Sciences; CN, Chungcheongnam-do; GG, Gyeonggi-do; GN,

Gyeongsangnam-do; GW, Gangwondo; JB, Jeollabuk-do; JN,

Jeollanam-do; JJ, Jeju-do.



Molecular identification of dipteran pests (Diptera: Sciaroidea) from shiitake mushroom

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