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Wahlgreniella nervata
(Hemiptera: Aphididae), a New Pest of
Rose in India
Author(s): Sunil Joshi, D. Lokeshwari, N. K. Krishna Kumar, H. Manjunatha,
Abraham Verghese and S. K. Jalali
Source: Florida Entomologist, 97(1):162-167.
Published By: Florida Entomological Society
DOI: http://dx.doi.org/10.1653/024.097.0122
URL: http://www.bioone.org/doi/full/10.1653/024.097.0122
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162 Florida Entomologist 97(1) March 2014
WAHLGRENIELLA NERVATA (HEMIPTERA: APHIDIDAE),
A NEW PEST OF ROSE IN INDIA
Sunil JoShi
1
, D. lokeShwari
2,*
, n. k. kriShna kumar
3
, h. manJunatha
4
, abraham VergheSe
1
anD S. k. Jalali
1
1
National Bureau of Agriculturally Important Insects, Hebbal, Bengaluru–560024, India
2
Division of Entomology and Nematology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post,
Bengaluru–560089, India
3
Division of Horticulture, Indian Council of Agriculture Research, New Delhi–110012, India
4
Department of Biotechnology and Bioinformatics, Kuvempu University, Jnanasahyadri, Shankaraghatta,
Shimoga–577451, India.
*Corresponding author; E-mail: lokibiotech@gmail.com
abStract
First occurrence of the invasive aphid species, Wahlgreniella nervata (Gillette) (Hemiptera:
Aphididae) is reported from material collected in Bengaluru, India. Apterous and alate vi-
viparous females were found feeding on tender shoots, young leaves and flower buds of rose
(Rosaceae). The identity of this aphid species is supported by morphometrics (aptera and
alata) corroborated with molecular analysis based on a fragment of the mitochondrial DNA
containing the 5’ region of the cytochrome c oxidase 1 (mtCOI). Diagnostic characters to dif-
ferentiate W. nervata from other major aphid species viz., Macrosiphum rosae (Linnaeus)
and Sitobion rosaeiformis (Das), on Rosa spp., its host plants, distribution and natural en-
emies are provided. This is a new distributional record of W. nervata. The invasive species
compendium developed by CAB International, 2013 has listed W. nervata as invasive in
nature.
Key Words: Wahlgreniella nervata, India, rose, mtCOI, phylogenetic tree
reSumen
Se re-describe la especie el áfido, Wahlgreniella nervata (Gillette) (Hemiptera: Aphididae)
de material recolectado en Bengaluru, India. Se encontraron hembras vivíparas ápteros y
alados alimentándose de brotes tiernos, hojas jóvenes y capullos de rosa (Rosaceae). La iden-
tidad de esta especie de áfido es apoyado por morfometría (aptera y alata) corroborada con
el análisis molecular basado en un fragmento del ADN mitocondrial que contiene la región
5 ‘del citocromo c oxidasa 1 (mtCOI). Se proporcionan claves diagnósticas, descripciones
morfológicas, plantas hospederas, distribuciones y enemigos naturales de este áfido. Es un
nuevo registro para la fauna de áfidos. El compendio de especies invasoras elaborado por
CAB International en 2013 lista W. nervata como un invasor potencial natural.
Palabras Clave: Wahlgreniella nervata, India, rosa, mtCOI, árbol filogenético
Rose (Rosa spp. L; Rosales: Rosaceae), is in-
fested by 55 aphid species (Blackman & Eastop
2000; http://www.aphidsonworldsplants.info/C_
HOSTS_Ros_Ryt.htm#Rosa) of which 39 are
previously recognized from India (Raychaudhuri
1983; Chakrabarti & Sarkar 2001). Wahlgreni-
ella nervata (Gillette 1908) is a spindle-shaped,
pale green aphid characterized by elongate swol-
len siphunculi, apterae without sensoria on third
antennal segment, cauda bearing five hairs and
well developed lateral frontal tubercles (Heie
1986). It is associated with Rosa spp. and other
plants, e.g., Ericales: Ericaceae. It can be con-
sidered a monotypic species, or divided into 2
subspecies, which present very similar morpho-
logical features: Wahlgreniella nervata nervata
and W. nervata arbuti (Davidson, 1910). The
nominotypical subspecies is heteroecious holocy-
clic between Rosa spp. and species of Ericaceae
(Stroyan 1979); or paramonoecious anholocyclic
on Rosa spp. (Blackman & Eastop 2006) and it
is primarily North American. Wahlgreniella n.
arbuti is monoecious anholocyclic on Ericaceae
(Arbutus, Arctostaphylos and Pieris) as well as
Empetraceae (Empetrum) species and it is pri-
marily European.
Joshi et al.: Wahlgreniella nervata a New Pest of Rose in India 163
It has been recorded from several countries
(or territories) of Europe: under W. nervata: An-
dorra, Austria, Belgium, Britain Is., Canary Is.,
Sicily and mainland Spain (http://www.faunaeur.
org/full_results.php?id=56351); under W. n. ar-
buti (a subspecies of W. nervata): Greece (Tsitsi-
pis et al. 2007), Balearic Is., Corsica, mainland
France, mainland Italy, Madeira, Portugal and
Sardinia (http://www.faunaeur.org/full_results.
php?id=56349); the Americas: Canada (Heie
1994), Colorado, USA (Gillette 1908), Mexico
(Heie 1994), Argentina (Nieto Nafria et al. 1994),
Brazil (Smith & Cermeli 1979), Chile (Eastop et
al. 1997) and Peru (Mallqui & Cobian 2011); and
other continents: Turkey (Barjadze et al. 2011),
Iran (Rezwani 2001), Israel (Halperin et al. 1988),
Burundi, Africa (Blackman & Eastop 2000), in-
cluding Quetta-Pakistan (Naumann-Etienne &
Remaudière 1995). Hitherto this aphid species
has not been reported from India.
During a regular survey, apterous and alate
viviparous females of an unknown aphid colony
were observed infesting rose at Bengaluru, India,
i.e., at the homestead garden of Sanjay Nagar,
Dec 2011 by Sunil Joshi, and in an experimen-
tal field of the Indian Institute of Horticultural
Research, Mar 2012 by D. Lokeshwari (Fig. 1).
Aphids were found on the leaves, tender shoots
and young flower buds of cultivated Rosa sp. The
aphids excreted honeydew and left their exuviae
on the leaves and flower buds. Natural enemies of
common occurrence on other species of aphids on
rose, viz., Betasyrphus linga Ghorpade (Diptera:
Syrphidae), Ischiodon scutellaris F. (Diptera:
Syrphidae) and Cheilomenes sexmaculata F. (Co-
leoptera: Coccinellidae) were observed to feed on
these aphids. No serious damage was observed as
the infestation level was kept low by the natural
enemies.
Live aphids along with the plant material
were transferred to the laboratory. Specimens
were collected and preserved in 70% ethanol at
-20 °C. Morphological identifications were carried
out prior to molecular studies (Blackman & Ea-
stop 2000); examinations showed that the aphid
characteristics matched those of W. nervata. The
viviparous apterous females were deposited as
microscopic slide-mounted specimens at National
Bureau of Agriculturally Important Insects, Ben-
galuru. Literature searches indicated that this is
the first report of the occurrence of W. nervata in
India and this species is a new inclusion for In-
dian aphid fauna.
Diagnostic characters for identifying W. ner-
vata (Gillette), Macrosiphum rosae (Linnaeus)
and Sitobion rosaeiformis (Das) are given in Fig.
2, Fig. 3 and Fig. 4 respectively. The pictured
specimen was stained during the slide mount-
ing process to facilitate illustration of important
characters.
Over the centuries, identification of insect
pests has remained a challenging task (Joshi &
Lokeshwari 2013). Accurate and rapid identifi-
cation of invasive aphid species like W. nervata
is important in order to evolve effective man-
agement practices, develop biological control
Fig. 1. Late instar nymph (left) and apterous viviparous female (right) of Wahlgreniella nervata (Gillette) on rose.
164 Florida Entomologist 97(1) March 2014
programs and detect invasive quarantine pest
species introduced into countries along with ag-
ricultural products at the port of entry. Molecu-
lar identification comes in handy at this juncture
and aids in quick identification of insect pests.
Hence, in this study morphological identification
corroborated with molecular methods to identify
W. nervata from other major aphid species on rose
are provided along with its host plants, distribu-
tion and natural enemies.
For molecular identification, total genomic
DNA was extracted from single individuals us-
ing CTAB method (Stewart et al. 1993) and PCR
was carried out in a thermal cycler (Eppendorf,
New York, USA) with the following cycling pa-
rameters; 94 °C for 3 min as initial denaturation
followed by 35 cycles of 94 °C for 30 s, 47 °C for
45 s, 72 °C for 45 s and 72 °C for 20 min as final
extension using primers specific to mtCOI, viz.,
LCO-1490F; 5’-GGT CAA CAA ATC ATA AAG
ATA TTG G-3’and HCO-2198R; 5’-TAA ACT TCA
GGG TGA CCA AAA AAT CA-3’(Hebert et al.
2004). PCR was performed in 25 μL total reaction
volume containing 10 picomoles of each primer,
1.5 mM MgCl
2
, 0.25 mM of each dNTP and 0.5
U of Taq DNA polymerase (Thermo scientific,
USA). The amplified products were resolved in
1.0% agarose gel, stained with ethidium bromide
(10 μg/mL) and visualized in a gel documentation
system (Syngene, USA).
The PCR amplified fragments were eluted us-
ing Nucleospin® Extract II according to the man-
ufacturer’s protocol (Machery-Nagel, Germany)
and ligated into the general purpose-cloning vec-
tor, InsT/Aclone; transformation was carried out
according to manufacture’s protocol (Fermentas,
Germany) and blue/white selection was done. All
the white colonies (with insert) were maintained
on LBA containing ampicillin (100 mg/mL), incu-
bated at 37 °C overnight and stored at 4 °C. Plas-
mids were isolated from the overnight culture of
5 randomly selected positive colonies cultured in
LB broth using GeneJET™ Plasmid Miniprep
Kit (Fermentas, Germany) according to manufac-
turer’s protocol. Sequencing was carried out in an
automated sequencer (ABI Prism® 3730 XL DNA
Analyzer; Applied Biosystems, USA) using M13
universal primers both in forward and reverse
directions.
The gene mtCOI was successfully sequenced
from an individual of W. nervata. Comparison
of the triplicate sequences showed no evidence
of mismatch, indicated there were no sequenc-
ing errors. Homology search was carried out us-
Fig. 2. Diagnostic characters for identification of W. nervata (Gillette) collected on rose.
Joshi et al.: Wahlgreniella nervata a New Pest of Rose in India 165
ing BLAST (http://www.ncbi.nlm.nih.gov). Both
the sequence and collection data of W. nervata
were entered in National Center for Biotechnol-
ogy Information (NCBI), GenBank under the ac-
cession number KF285590. Sequence generated
in the present study along with other major aphid
species on rose, viz., Ericaphis wakibae (Hottes),
Metopolophium dirhodum (Walker), Macro-
siphum impatientis (Williams), Sitobion rosivo-
rum (Zhang), Macrosiphum rosae (Linnaeus) and
Pentalonia nigronervosa (Coquerel) (as outgroup)
(Retrieved from NCBI) were aligned using Clust-
al W program in BioEdit V7. 0. Phylogenetic anal-
ysis of aligned sequences was done using MEGA.
V5. 0. (Tamura et al. 2011). The method of neigh-
bor-joining (NJ) with the Kimura two-parameter
model (Kimura 1980) was utilized to build the
phylogenetic tree. To assess the robustness of the
tree, 1000 bootstrap replicates were selected.
The phylogenetic tree demonstrated genetic
distinction of the species with bootstrap values
greater than 70% (Fig. 5). Rose aphids formed 2
major groups; W. nervata and E. wakibae clus-
tered to form Group I, while M. dirhodum, M. im-
patientis, S. rosivorum and M. rosae clustered to
form Group II. Pairwise alignment of mtCOI se-
quence of W. nervata collected from India showed
1.37-1.98% variation (mean divergence of 1.68%,
SE 0.043%) corresponding to an average of 11
base changes with those collected from USA and
Canada (available at NCBI database) indicating
it is not a single cosmopolitan species (Davidson
1910) and requires further investigation to en-
sure the same. The sequence divergence between
the genus Wahlgreniella and others, viz., Erica-
phis, Metopolophium, Macrosiphum and Sitobion
ranged 3.49-5.77% (mean divergence of 4.63%, SE
0.163%) indicating W. nervata is more closely re-
lated to E. wakibae (96.5% homology) than to M.
dirhodum (95%), M. impatientis (94.7%), S. ros-
ivorum (94.4%) and M. rosae (94.2%). In addition,
the study showed that mtCOI sequences are con-
sistent among species and is able to differentiate
the species well; thus it proves to be a useful tool
for identification of aphids.
It was also observed that B. linga, I. scutellaris
and C. sexmaculata as predators of W. nervata
manage this aphid in field sites at Bengaluru,
India. Although this species has been considered
to be of invasive nature (CAB Intl. 2013), it has
not assumed serious proportions at the locations
involved in the present study. Also the aphid is
Fig. 3. Diagnostic characters for identification of Macrosiphum rosae (Linnaeus) collected on rose.
166 Florida Entomologist 97(1) March 2014
not known to transmit any viral diseases, and
thus it is of less economic significance. However,
our study will aid in proper quarantine measures
from the view point of biosecurity.
acknowleDgmentS
The authors thank the Director, Indian In-
stitute of Horticultural Research, Bengaluru for
Fig. 4. Diagnostic characters for identification of Sitobion rosaeiformis (Das) collected on rose.
Fig 5. Neighbor joining tree of W. nervata with other major rose aphids for partial sequences of CO-I with boot-
strap support (1000 replicates). Bootstrap values greater than 60% are indicated above the branches. Pentolonia
nigronervosa was used as outgroup.
Joshi et al.: Wahlgreniella nervata a New Pest of Rose in India 167
encouragement and providing facilities to carry
out the research. Thanks to Indian Council of
Agricultural Research (ICAR), New Delhi for fi-
nancial support through the National Agricultural
Innovative Project on the National Agricultural Bio-
informatics Grid. Our sincere thanks to the anony-
mous reviewers, whose suggestions and comments
helped in improving this article. This paper is a part
of doctoral degree work of the second author.
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