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TETRANEURA (TETRANEURELLA)NIGRIABDOMINALIS (SASAKI),
GALL-FORMING APHID FOUND ON MAIZE ROOTS IN SLOVENIA
Øpela MODIC, Jaka RAZINGER, Gregor UREK
Agricultural Institute of Slovenia, Plant Protection Department
Hacquetova ulica 17, SI-1000 Ljubljana, email: spela.modic@kis.si
Abstract - The root aphid Tetraneura (Tetraneurella) nigriabdominalis (Sasaki),
(Pemphiginae: Eriosomatini) was recorded on corn roots (Zea mays L.) in August 2011
in northeast Slovenia in the village Benica. The species was classified morphologically
and by molecular methods.
KEY WORDS: Pemphiginae, root aphids, fauna, Slovenia, DNA barcoding.
Izvleœek – TETRANEURA (TETRANEURELLA)NIGRIABDOMINALIS (SASAKI),
KORENINSKA UØ NAJDENA NA KORENINAH KORUZE V SLOVENIJI
Koreninsko uø Tetraneura (Tetraneurella) nigriabdominalis (Sasaki), (Pemphigi-
nae: Eriosomatini) smo ugotovili na koreninah koruze (Zea mays L.) avgusta 2011 v
severovzhodni Sloveniji, v vasici Benica. Vrsto smo doloœili morfoloøko in na pod-
lagi molekularno bioloøke analize.
KLJUŒNE BESEDE: Pemphiginae, koreninske uøi, favna, Slovenija, œrtne kode DNA.
Introduction
The root aphid Tetraneura nigriabdominalis (Sasaki) has host alternation between leaf
galls on trees from family Ulmaceae and roots of numerous species of Graminae (Poaceae)
from the genera Agropyron, Cenchrus, Chloris, Cynodon, Digitaria, Echinochloa, Eleu-
sine, Eragrostis, Oryza, Panicum, Paspalum, Saccharum, Setaria, and others. Species is
distributed in Africa, India, Nepal, Bangladesh, Pakistan, Sri Lanka, Japan, China, Ko-
rea, Indonesia, Malaysia, the Philippines, Australia, New Zealand, Fiji, Tonga, Central
America, Caribbean, the U.S.A. (Blackman and Eastop, 1984 & 1994 & 2006). It is also
presented in Fauna Europaea (2011). Species is considered as a major pest of upland rice
plants (Pathak and Khan, 1994). Ulmus spp. trees are its primary host where it forms galls
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LJUBLJANA, DECEMBER 2012 Vol. 20, øt. 2: 147–152
on upper sides of leaves. Galls are stalked, hairy, elongate; pouch or spindle shaped, usu-
ally with a pointed apex and differ from galls formed by T. ulmi L. In South and Eastern
Europe T. nigriabdominalis is heteroecious holocyclic with Ulmus spp. as primary host;
and probably anholocyclic where Ulmus is unavailable (Blackman & Eastop, 2006). Alate
leave the gall through lateral slits in May and July to establish colonies on roots of
Gramineae (Tanaka, 1961). Apterous are greenish or brownish white, plump, body
length 1.5-2.5 mm. Alate have a shiny black head and thoracic lobes and a brown ab-
domen, body length 1.5-2.3 mm. Its presence on the roots of some hosts is indicated by
a reddish-purple discoloration of the leaves (Blackmann & Eastop, 1984).
T. nigriabdominalis is often found in association with ants. The ants transport them
and construct refuges in the roots. The ants attend them for honeydew and their presence
confirms the root aphid attack (Galli and Bonvicini-Pagliai, 1998). The optimal tem-
perature for population growth of T. nigriabdominalis is 30° C (Kuo et al., 2006).
The above mentioned morphological characteristics were duly observed during mor-
phological classification of the species. Such an approach to classification can be
termed ‘classical’ taxonomy. Classical taxonomy is based on detailed morphological
analysis of morphometric characteristics of biological specimens. However, as DNA
sequencing has become increasingly reliable and affordable more DNA sequences have
become available online. This data can be used to classify an unknown specimen in a
process designated DNA barcoding. The objective of DNA barcoding is to use large-
scale screening of one or a few reference genes in order to, a), assign unknown indi-
viduals to species, and b), enhance discovery of new species (Moritz and Cicero, 2004).
DNA barcoding is based on the premise that a short standardized sequence can dis-
tinguish individuals of a species because genetic variation between species exceeds that
within species (Hajibabaei et al., 2007).
The purpose of this research was a), to confirm the occurrence of T. nigriabdominalis
on secondary host in Slovenia and b), to provide the research community with additional
DNA barcodes for this species.
Systematics – based on Remaudiere and Remaudiere, 1997
The species Tetraneura nigriabdominalis (Sasaki, 1899) has several synonyms:
Dryopeia hirsuta Baker, 1921, Tetraneura argrimoniae Shinji, 1924, Tetraneura
oryzae van der Goot ex van Herun, 1923, Tetraneura akinire Sasaki, 1904.
Materials and methods
Specimen collection
The aphid specimens were collected on August 11th 2011 from the secondary host
– the aphids were discovered on roots of Zea mays in northeastern part of Slovenia,
near the village Benica in Prekmurje. The exact location is longitude 16.5023 latitude
Acta entomologica slovenica, 20 (2), 2012
148
46.5135. Aphid samples were preserved in 75% ethanol on 4 to 5 °C in refrigerator
for identification at a later day. Several insects were saved as vouchers at -80°C in en-
tomological collection of the Agricultural Institute of Slovenia.
Classical taxonomical identification - based on Blackman and Eastop, 2006.
Apterae of the genus Tetraneura have very short terminal process of the last an-
tennal segment. Siphunculus have slightly elevated cones and broadly rounded cau-
dae. Legs of females have only one tarsal segment. Alate females have forewings with
unbranched medial veins. Wax glands are usually present.
Øpela Modic, Jaka Razinger, Gregor Urek: Tetraneura (Tetraneurella) nigriabdominalis (Sasaki)
149
Fig. 1: Apterae clustered on roots of Zea mays.
Molecular classification
DNA purification. The specimens were preserved at -80°C until DNA extraction.
Entire aphid was used for genomic DNA purification using the NucleoMag extraction
kit (Macherey-Nagel, Germany) and MagMAX Express Magnetic Particle Processor
(Applied Biosystems). The volumes of reagents used were smaller than those used in
the NucleoMag kit’s instruction manual. The tissue was homogenized manually in a
1.5 ml Eppendorf tube using micro-pestle. The lysis buffer consisted of 10 µl of pre-
mixed proteinase K solution and 50 µl of T1 buffer. The lysate was transferred to the
first well of the MagMAX cartridge, where additionally 10 µl of magnetic particles and
110 µl of MB2 buffer were added. 150 µl of MB3, 150 µl MB4 and 200 µl of MB5 were
added to wells 2, 3 and 4, respectively. In the 5th well, 50 µl of MB6 (elution buffer)
was added.
DNA barcoding. Partial cytochrome oxidase subunit I (COI) was amplified using
forward primer LCO (5-GGTCAACAAATCATAAAGATATTGG-3) and reverse
primer HCO (5-TAAACTTCAGGCTGACCAAAAAATCA-3) according to Folmer
et al. (1994), with slight modifications. PCR was performed in Veriti thermocycler (Ap-
plied Biosystems). The 25 µl reactions contained 2.5 µl 10X PCR Buffer, 3.0 µl
MgCl2(25 mM), 1.0 µl of each primer (10 µM), 0.4 µl dNTPs (10 mM), 0.2 µl Taq
DNA polymerase (Fermentas), and 1.0 µl of DNA template. Thermocycling conditions
were 95°C for 6.5 min, followed by 40 cycles of 40 s at 95°C, 40 s at 40°C, and 1 min
at 72°C, with a final extension of 72°C for 7 min. The resulting PCR amplicon was
checked on a 1.7% agarose gel, stained with ethidium bromide and visualized under
UV light in Genegenius (Syngene). The amplicon was sequenced in Macrogen inc.,
Netherlands. The obtained sequence was deposited in Barcode of Life Database
(www.boldsystems.org).
Results & Discussion
The species was previously confirmed in former Yugoslavia. It was discovered on
an elm tree Ulmus campestris in Belgrade. It was classified as Byrsocrypta hirsuta
Backer (N. Tanasijeviœ, 1965). Until now the species has expanded considerably on
Ulmus trees in Slovenia (G. Seljak, pers. comm.). Their secondary host plants with a
high potential economic value, Zea mays L. and Sorghum bicolor (L.) Moench, do not
appear to be threatened.
This study shows benefits of combined morphological and molecular classification
approach. Although molecular barcoding is a useful aid for taxonomic workflow in
identifying specimens to a species level it is not meant as a replacement for classical
morphological taxonomic analysis. For example, when an unknown specimen does not
return a close match to existing records in the barcode library, the barcode sequence
does not qualify the unknown specimen for designation as a new species. Instead, such
specimens are marked for thorough morphological analysis (Hajibabaei et al., 2007).
Acta entomologica slovenica, 20 (2), 2012
150
This was also the case in this investigation. The aphid’s sequence was obtained and
analyzed by bioinformatics software. When it was discovered that it does not closely
match any known sequences, the aphid was thoroughly morphologically analyzed. Af-
ter our own morphological classification and confirmation by an independent foreign
laboratory, we deposited the obtained sequence in the Barcode of Life Database
(www.boldsystems.org). Thus the research community profited by one more specific
genetic fingerprint of an aphid species.
Acknowledgement
We thank Dr. Thomas Thieme (BTL, Sagerheid, Germany) for confirmation of our
taxonomic determination.
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Received / Prejeto: 2. 10. 2012
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