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Journal of Stored Products Research 48 (2012) 120e125

Contents lists available at SciVerse ScienceDirect

Journal of Stored Products Research

journal homepage: www.elsevier.com/locate/jspr

Diagnosis of Liposcelis entomophila (Insecta: Psocodea: Liposcelididae) based on

morphological characteristics and DNA barcodes
Qianqian Yang a, Zuzana Ku

cerová b, *, Zhihong Li a, *, Irma Kalinovi
c c, Václav Stejskal b,
George Opit d, Yang Cao e
a
Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, PR China
b
Crop Research Institute, Drnovská 507, 161 06 Prague 6, Czech Republic
c
Faculty of Agriculture in Osijek, Department for Plant Protection, University of Josip Juraj Strossmayer in Osijek, Trg Sv. Trojstva 3, 31000 Osijek, Croatia
d
Department of Entomology and Plant Pathology, Oklahoma State University, 127 Noble Research Center, Stillwater, OK, USA
e
Academy of State Administration of Grain, No. 11 Baiwanzhuang Street, Beijing 100037, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Liposcelis entomophila infests stored grain and is one of the most important psocid species worldwide.
Accepted 25 October 2011 Six geographically isolated strains of L. entomophila from Asia, Europe, and United States of America
(USA) were compared based on morphological attributes and by molecular methods. Decisive
Keywords: characters of morphological diagnosis were studied using body size measurements and Scanning
Storage pests Electron Microscopy (SEM). Molecular identification of the six strains was performed via identification
Electron microscopy
of DNA sequence similarities and phylogenetic analyses based on a 655-bp fragment from the 50
mtDNA COI
end of the standard mitochondrial gene cytochrome c oxidase I (COI) barcode region. The results
Diagnosis
Psocoptera
showed that both morphological and molecular approaches were able to accurately identify this species.
Kimura-2-Parameter (K2P) divergence between geographically isolated strains was on average 1.75% for
the COI sequence. Phylogenetic analyses revealed that sequences of L. entomophila strains’ COI barcodes
formed clusters with tight cohesion that were clearly distinct from those of allied species.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction (Kalinovic et al., 2006). In recent years, the importance of studying

psocids has been enhanced by the fact that they develop rapid
Psocids, which include a number of domestic and stored prod- resistance to insecticides, phosphine and controlled atmosphere
ucts pests, pose an increasing threat to stored products worldwide treatments (Cao et al., 2003; Ding et al., 2002; Nayak and Daglish,
(Rees, 2004) and are commonly found in a wide range of synan- 2007).
thropic habitats, such as processed and unprocessed dry foods in Traditional diagnosis of psocids from the genus Liposcelis
households, granaries, and product warehouses (Rees, 2004; (Liposcelididae) through identification of specific morphological
Turner, 1994). In the last two decades, psocids have become an characters is difficult, especially in the case of juvenile stages; this
increasingly serious problem not only in grain storages, warehouses method requires specialized taxonomic knowledge and microscopy
with bagged commodities but also in collection centres and export techniques (Ku
cerová et al., 2009; Lienhard, 1990). DNA-based
terminals (Opit and Throne, 2008). In addition, psocids have approaches offer an effective complement to traditional taxo-
become stored product pests of considerable economic importance, nomic methods and are currently widely employed for insect
contaminating foods and negatively affecting the quality and safety species identification. Molecular diagnostics have been applied to
of food in various parts of the world (Nayak, 2006). Additionally, the identification of the Liposcelis species; for example, PCR-RFLP
these pests are a potential cause of respiratory and dermatological (Restriction Fragment Length Polymorphism) analysis was used
allergies (Musken et al., 1998; Patil et al., 2001; Turner, 1994), for rapid discrimination of four common species of catalogued
transferring microorganisms to humans. They can transmit bacteria Liposcelis from China and the Czech Republic (Qin et al., 2008), and
and fungi both on the surfaces of their bodies and also internally 16S rDNA has been proven to be effective in defining intra-species
diversity of Liposcelis bostrychophila Badonnel (Li et al., 2011).
DNA barcoding is a DNA-based species identification system
* Corresponding authors. which offers a promising supplemental technique with standard-
E-mail addresses: [email protected] (Z. Ku

cerová), [email protected] (Z. Li). ized portions of the genome (Hebert et al., 2003). The most

0022-474X/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jspr.2011.10.007
 Author's personal copy

Q. Yang et al. / Journal of Stored Products Research 48 (2012) 120e125 121

commonly used barcode gene, mitochondrial (mt) DNA cyto- 2.3. Molecular identification
chrome c oxidase I (COI), has been shown to be a reliable, quick and
cost-effective tool for the identification of organisms of various taxa 2.3.1. DNA extraction, mtDNA COI amplification and sequencing
in all life stages (Augot et al., 2010; Cywinska et al., 2010; Hebert Total genomic DNA was isolated from the whole body of
et al., 2003; Rach et al., 2008). A threshold of 2e3% mtDNA COI L. entomophila specimens using cetyl trimethyl ammonium
sequence divergence was recommended to define separate species bromide (CTAB; Qin et al., 2008); three individuals from each
for insects and mammals (Hebert et al., 2003). In studies of geographical population were used. PCR was performed with a pair
butterflies and ants, DNA barcoding has been successful in defining of universal primers, LCO1490 (fw) 50 GGT CAA CAA ATC ATA AAG
species boundaries by genetic distance thresholds (Hebert et al., ATA TTG G 30 and HCO2198 (rev) 50 TAA ACT TCA GGG TGA CCA AAA
2004; Smith et al., 2005); however, there is no established AAT CA 30 , amplifying an approximately 710 bp fragment of the
universal distance threshold value to distinguish between taxo- standard mtDNA COI-5 barcode (Folmer et al., 1994). PCR products
nomic groups (Rach et al., 2008). were separated on a 1.0% (w/v) agarose gel (1 TAE), stained with
One of the most important stored product psocids worldwide is ethidium bromide, and visualised under UV light. The agarose gel
Liposcelis entomophila (Enderlein) (Psocodea, formerly Psocoptera; slice containing the PCR amplicon of interest was excised and
Yoshizawa and Lienhard, 2010). The objectives of research placed in a centrifuge tube. The agarose gel slice containing the PCR
presented here were the following: (1) to identify the six different amplicon of interest was excised and the DNA was gel extracted.
strains of L. entomophila from geographically different locales of Bidirectional sequencing reactions were carried out from a single
Europe, Asia and USA using morphological trait identification individual of each geographical isolate (Beijing Aoke Biotechnology
through optical and scanning electron microscopy and COI gene Co., Ltd.).
sequencing diagnostic methods and (2) to enrich the COI barcode
identification system while evaluating the proper sequence diver- 2.3.2. Genetic distance and phylogenetic analysis
gence threshold within L. entomophila. This work builds on Pairwise genetic distances for COI were calculated using the
preliminary results published in Yang et al. (2011). Kimura-2-Parameter (K2P) distance model implemented in the
software Molecular Evolutionary Genetics Analysis 5 (MEGA 5;
Tamura et al., 2011). All phylogenetic analyses were carried out using
2. Materials and methods
the program PAUP 4.0 (Swofford, 2002). Two different types of
phylogenetic trees, neighbour-joining (NJ) and maximum parsimony
2.1. Insects
(MP), were graphically displayed and compared. A heuristic search
was employed using tree bisection and reconnection (TBR) branch
The six geographical strains of L. entomophila used for the
swapping and random addition for 100 replicates, and bootstrapping
morphological and molecular study were initially collected in grain
was performed using 1000 replications (Felsenstein, 1985). In the
stores from China (Shandong and Yunnan), the Czech Republic
phylogenetic analysis, two geographical stocks were introduced: one
(Central Bohemia), Croatia (Baranja), the United Kingdom (Wales)
additional closely related Liposcelis species and one more distantly
and the United States of America (USA - Kansas). The laboratory
related Psocoptera species. Collection localities and the GenBank
cultures of these stocks (maintained by the following: Crop
accession numbers are available in Table 1.
Research Institute, Prague; China Agricultural University, Beijing;
University of Josip Juraj Strossmayer, Osijek; University of Wales
3. Results
Bangor, Bangor; Oklahoma State University, Stillwater) were reared
at 27  1  C, 75  5% relative humidity (r.h.), on a scotoperiod of
3.1. Morphological identification
24 h in glass containers (125 ml) and on a wheat based diet (Ding
et al., 2001; Opit and Throne, 2008; Ku
cerová et al., 2009).
Diagnosis of the species L. entomophila by decisive and
secondary morphological characters, as put forth by Lienhard
2.2. Morphological identification (1990); (Key e Section I, Group IA): The basic body colour is
yellowish, and abdominal terga 3e4 and 6e9 are usually marked
2.2.1. Optical microscope with a brown transverse band and often broadly interrupted
Decisive diagnostic characters and size measurements were medially. The dorsal side of the body appears as follows: the vertex
used for species identification. A minimum of 50 individuals of the head has spindle-shaped areas lacking tubercles or bear only
(females) were examined and measured for each geographic strain. indistinct tubercles of medium size; the average distance between
Head width (W) measurements were taken using a light micro-
scope equipped with an objective micrometer. Differences in size
Table 1
among specimens of the geographic strains were displayed in box
Origin of species and populations used in this study.
plots (developed using the software STATISTICA 8). Other
morphological characters examined were numbers of ommatidia Population Location collected GenBank
accession nos.
contained in the compound eye and numbers of the following
setae: pronotal setae (PNS) on the lateral lobe, and prothoracic and L. entomophila SD-P.R. China Shandong, P.R. China HQ018872
L. entomophila YN-P.R. China Yunnan, P.R. China JF910135
mesothoracic sternite setae. L. entomophila P-CZ Central Bohemia, HQ018874
Czech Republic
2.2.2. Scanning electron microscopy (SEM) L. entomophila CRO Bobota, Croatia HQ658137
Morphological details (ommatidia, vertex surface sculpturing L. entomophila UK Wales, UK JF910133
L. entomophila USA U.S.A HQ018873
and prothoracic setae) were imaged with SEM micrographs. A
L. brunnea USA U.S.A JF910139
minimum of 10 females was examined for each geographic pop- L. brunnea P-CZ Central Bohemia, JF910140
ulation. The specimens were sputter-coated with platinum in the Czech Republic
Sputter Coater SDC 050 (4 nm thick platinum layer). The head and Psocoptera sp. Arizona, U.S.A HQ582230
thorax were then studied with the JEOL JSM-6400 SEM at magni- Populations were coded by combining species names with acronyms of collection
fications of 650e2500. countries and sites.
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122 Q. Yang et al. / Journal of Stored Products Research 48 (2012) 120e125

small fine hairs on the vertex is approximately equal to their length originate behind the SI. The abdomen is compact; terga 3 and 4 lack
or slightly less. Compound eyes consist of 6e8 (most often 8, posterior delimitation by intersegmental membranes. The ventral
occasionally 7 and very rarely 6) ommatidia. The lateral lobe of the side of the body has 4e7 setae on the anterior half of the pros-
pronotum has a strong and long shoulder bristle (SI) and a trans- ternum, the posterior prosternum is without setae and the meso-
verse row of 3e5 other bristles. In addition, some small fine hairs sternal region has 6e11 setae.

Fig. 1. Liposcelis entomophila (female) e morphological comparison of the compound eye (a), vertex sculpture and setae (b) and lateral lobe of pronotum (c) in the six geographical
strains (A e China - Shandong, B e Czech Republic, C e Croatia, D e USA, E  UK, F e China - Yunnan).
 Author's personal copy

Q. Yang et al. / Journal of Stored Products Research 48 (2012) 120e125 123

All six geographic strains exhibit the decisive diagnostic Table 2

morphological characteristics of L. entomophila described above. Pairwise sequence divergence (%) based on the Kimura-2-parameter of COI of the six
population stocks of L. entomophila and two L. brunnea population stocks that were
SEM micrographs of compound eyes, the vertex surfaces and lateral investigated.
lobes of the pronotum are shown in Fig. 1. Tubercles on spindle-
shaped areas of the vertex are slightly more pronounced in 1 2 3 4 5 6 7 8

Croatia, USA and UK strains than in the other strains. No signifi- 1 L. entomophila
SD-P.R. China
cantly distinguishable differences among strains were found in
2 L. entomophila 0.15
other morphological characters, including the number of omma- YN-P.R. China
tidia in the compound eye, distance between setae of the vertex 3 L. entomophila P-CZ 0.77 0.92
and the number of thoracic setae. Box plots of head size 4 L. entomophila CRO 3.29 3.45 3.13
measurements (head width) for these six strains are shown in 5 L. entomophila UK 0. 61 0.77 0.61 3.13
6 L. entomophila USA 1.55 1.70 1.39 3.45 1.39
Fig. 2. Based on numerical differences among head measurements 7 L. brunnea USA 45.44 45.44 45.17 44.91 45.17 44.59
alone, the Croatia, two China, Czech, USA, and UK strains showed 8 L. brunnea P-CZ 45.44 45.44 45.17 44.91 45.17 44.59 0.00
a decreasing order of head size. The median head size of the six
strains differed, but the 25e75% confidence intervals or the
minimal and maximal values overlapped. This means that statisti-
cally the six strains do not differ in head size. six geographical isolates, along with the closely and distantly
related species, into three clades (Fig. 3). The resulting trees showed
a clear clade comprising of L. entomophila stocks, distinct from
3.2. Molecular identification
L. brunnea species and the out-group clades. There was high
bootstrap support (100%) for the terminal branches at the species
The mtDNA COI sequences of six L. entomophila strains obtained
level.
in this study have been deposited in GenBank available under
accession numbers listed in Table 1.
The sequences were all trimmed to a 655 bp core region that
could be unambiguously aligned to one another. No sequences 4. Discussion
contained indels or nonsense codons, allowing for easy alignment
and supporting their origin in the mitochondrial gene. This work shows a comprehensive diagnostic method for
All geographic isolates possessed a distinctive COI sequence, yet L. entomophila through detailed morphological observation and
most showed low intra-species divergence. Conspecific Kimura-2- molecular methods. The combination of decisive morphological
Parameter (K2P) divergence of L. entomophila averaged 1.75% characters and COI DNA barcoding analyses is a very constructive
(ranging from 0.15 to 3.45%). Liposcelis entomophila from Croatia means of species identification.
(CRO) showed the highest divergence (K2P: 3.13e3.45%) from the All geographical stocks were unambiguously diagnosed as
other conspecific stocks. All other L. entomophila strains showed L. entomophila species based on identification of decisive morpho-
less than 2% K2P divergence. In contrast, COI sequences diverged logical characteristics (Lienhard, 1990). Previous studies have
widely between the two different Liposcelis species (L. entomophila shown that certain morphological characteristics such as number
and Liposcelis brunnea), generating K2P scores of 44.59e45.44%, an of ommatidia, number of prosternal and pronotal setae, head size,
average of 45.12% (Table 2). In summary, the interspecies average colouration and surface structures (Lienhard, 1990) have some
divergence between closely related species was 25-fold higher than distinct intra-specific variability based on data from the Liposcelis
that of the intra-species divergence. strains examined. Slight variability of head size and vertex struc-
Both the neighbour-joining (NJ) and maximum parsimony (MP) tures revealed by the present study of L. entomophila geographical
phylogenetic analysis of the COI gene generated the same tree strains does not support consistent discrimination of these isolates
topology; distance value results were consistent, and grouped the based on morphological traits.
Intra-specific divergence of the mitochondrial COI gene within
L. entomophila geographical isolates from different sites all over
the world was not significant enough to be considered deep
Liposcelis entomophila
330 divergence. Inter-specific divergence between L. entomophila and
L. brunnea was 25-fold higher than the observed intra-specific
320 divergence of L. entomophila, despite the fact that L. brunnea is
a closely related species belonging to the same taxonomic group
310
(IA) (Lienhard, 1990). These results hold potential for use of DNA
barcodes in phylogenetic delineation and association studies of
300
other Liposcelis species. Very few psocid species are present in the
Head - W (µm )

290 DNA barcode databases, and our submission of Liposcelis COI

sequences will enlarge this universal and accessible identification
280 system.
Published studies show that approximately 98% of lepidopteran
270
species have been identified through molecular taxonomy within
a 3% threshold (Hebert et al., 2003), while 90% of 260 bird species
260
from North America have been identified within a 2.7% threshold
250 (Hebert et al., 2004). It is difficult to define a general threshold of
25%-75% genetic distance in distinguishing taxonomic groups, as divergence
240
CR China-1 Croatia China-2 USA UK
Min-Max rates between taxa are a dynamic process, and species boundaries
- Median
change with the time and population considered (Rubinoff et al.,
Fig. 2. Head size measurements of female L. entomophila from geographic strains from 2006). In our particular investigated stocks of L. entomophila from
China - Shandong, Czech Republic, Croatia, U.S.A, UK, and China - Yunnan. Asian, European and American populations, we determined that
 Author's personal copy

124 Q. Yang et al. / Journal of Stored Products Research 48 (2012) 120e125

Fig. 3. Neighbour-joining (NJ) and maximum parsimony (MP) phylogenetic trees developed from COI barcodes analysis. The number at each branch point is the percentage
supported by bootstrap. Psocoptera sp. (HQ582230) is the out-group.

the tested sequence threshold value might be appropriate at Acknowledgements

3e3.5%.
In phylogenetic analyses, both the NJ and MP phylogenetic trees We thank J. Hromádková (IMCH Academy of Sciences) for
yielded an identical topology, and their congruence revealed that the processing SEM micrographs and M.A. Perotti and H.R. Braig
geographical isolates from the two locations in China, the Czech (University of Wales Bangor, UK) for supplying biological material
Republic and the UK were of the closest phylogenetic relationship from the UK. This study was supported by the program of inter-
and members of the same clade. The Croatian and American strains national cooperation No. 38-46 of the ChineseeCzech cooperation
were relatively distantly related to the other four isolates and were project (China), No. 2009-1-49 of the Chinese Universities Scientific
thus placed as sister cluster relative to them. All six populations were Fund (China), No. ME 09080 e KONTAKT (Czech Republic), No.
distinct from the close related species L. brunnea and the out-group. MZOS
079-0427 (Croatia) and by the Oklahoma Agricultural
The correlation between morphological and molecular identi- Experiment Station (Project No. OKL02695).
fications in our study indicated that COI-based DNA barcode data
were useful in species discrimination for the genus Liposcelis. It
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