Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 March 2023 | 15(3): 22859–22865
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.7664.15.3.22859-22865
#7664 | Received 17
September 2021 | Final received 16 January 2023 | Finally accepted 24 February
2023
First report of the beetle Henosepilachna nana (Kapur,
1950) (Coleoptera: Coccinellidae) from Maharashtra
with special reference to molecular phylogeny and host plants
Priyanka B. Patil
1 & Sunil M. Gaikwad 2
1,2 Department of Zoology, Shivaji University, Kolhapur, Maharashtra 416004, India.
1priyankapatil7933@gmail.com (corresponding
author), 2 smg_zoo@unishivaji.ac.in
Abstract: A ladybird beetle, collected from different
localities of Kolhapur and Satara districts
(Maharashtra) was identified as Henosepilachna
nana (Kapur, 1950). The presence of this species
in Maharashtra considerably extends its range to the north-west by about 700
km. Since this species is found on vegetables, Pumpkin (Cucurbita spp.),
Cucumber (Cucumis spp.), and Karit fruit plant (Cucumis
spp.). It is being recorded for the first time that this species is a pest of
these vegetables. Also, molecular phylogeny has been studied for the first time
in this species in which, this species is the sister taxon of Henosepilachna boisduvali.
This species has been described briefly with colour
photographs of male genitalia, female coxites and the
6th sternal plate of female, tarsi, & pronotum. Henosepilachna nana is being reported for the
first time from Maharashtra and now the molecular data of this species is
available.
Keywords: Epilachna, Epilachnini,
GenBank, genitalia, first record, ladybug, phylogenetic tree, vegetable pest.
Editor: Mandar Paingankar, Government Science College Gadchiroli,
Maharashtra, India. Date of
publication: 26 March 2023 (online & print)
Citation: Patil, P.B. & S.M.
Gaikwad (2023).
First report of the beetle Henosepilachna
nana (Kapur, 1950) (Coleoptera:
Coccinellidae) from Maharashtra with special reference to molecular phylogeny
and host plants. Journal of Threatened Taxa 15(3): 22859–22865. https://doi.org/10.11609/jott.7664.15.3.22859-22865
Copyright: © Patil & Gaikwad 2023. Creative Commons Attribution 4.0
International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: Shivaji University, Kolhapur.
Competing interests: The authors declare no competing interests.
Author details: Priyanka B. Patil (PhD): working as a assistant professor at Department of Zoology, Dahiwadi College, Dahiwadi, Satara District. She is working on diversity and taxonomy of Coccinelidae. Sunil M. Gaikwad (PhD): associate professor at Department of Zoology, Shivaji University, Kolhapur. His research interest is diversity and taxonomy of insects with special reference to Pentatomidae, Mantodea, Cerambycidae and Coccinelidae.
Author contributions: SMG involved in design of the research and write up of the manuscript. PBP collected and dissected specimens and participated in design of the research, digital photography and map preparation.
Acknowledgements: The authors would like to thank
the Head, Department of Zoology, Shivaji University,
Kolhapur for providing laboratory facilities and financial assistance under the
Golden Jubilee Research Fellowship.
introduction
The tribe Epilachnini (Mulsant 1846) comes
under the subfamily Coccinellinae (Slipinski 2013; Seago et al. 2011) but previously it was
considered as a separate subfamily Epilachninae of
the family Coccinellidae (Szawaryn & Tomaszewska 2014). The members of this tribe are
economically important and distributed throughout the world (Szawaryn et al. 2015), comprising 27 genera (Tomaszewska & Szawaryn 2016).
There are about 1,000 herbivorous species of this tribe that feeds on plant
tissues and fluids (Giorgi et al. 2009; Bustamante-Navarrete et al. 2018).
The genus Epilachna is one of the genera of the
subfamily Epilachninae in which hundreds of species
from all over the world have been described and later many species were
subsequently removed to other genera (Jadwiszczak
& Węgrzynowicz 2003). Based on basal toothed
tarsal claw and longitudinally divided sixth female abdominal sternite, Li & Cook (1961) raised a separate genus Henosepilachna from Epilachna.
To date, there are about 110 described species of the genus Henosepilachna from Asia and Australia (Szawaryn 2011).
There are 33 species of Henosepilachna
in India (Poorani 2012; Poorani
et al. 2021).
The male specimen collected from Nilgiri Hills, India has been described by Kapur (1950) as E. nana and its holotype is in the
Zoological Survey of India (No. 3426/13). In the world catalogue of
Coccinellidae, Jadwiszczak & Wegrzynowicz
(2003) placed E. nana in the genus Henosepilachna,
which is followed by Poorani (2012) in the
updated checklist of Coccinellidae from Indian subregion
and Borowski (2020) in the inventory of world Epilachninae.
In the present study, we describe the detailed morphology of the male
genitalia, 6th sternal plate, female genitalia and tarsi of H.
nana along with coloured photographs. In
addition, this species is being reported for the first time in Maharashtra
through this communication and its molecular phylogeny has been given.
MATERIAL AND METHODS
Specimens of H.
nana were collected on different vegetable crops by handpicking method from
Halondi (Hatkanangale), Kanthewadi (Radhanagari) and Parite (Karveer) of Kolhapur
district and Sajjangad and Yerad
(Patan) of Satara district
of Maharashtra, India (Image 1).
Specimens were photographed on stereo zoom microscope LYNX LM-52-3621 using TCapture software and preserved dry and wet and deposited
at the Department of Zoology, Shivaji University,
Kolhapur. Captured images stacked in Helicon Focus 7 software. Measurements
were taken in ImageJ software. The genitalia was dissected and photographed
using the same microscope and software. Species was identified and confirmed
based on the detailed taxonomic description provided by Kapur
(1950) along with genitalia description.
Molecular analysis
Genomic DNA extraction was done
by the manual CTAB method (Boopathi et al. 2020). PCR
of extracted DNA was carried out using the primers
LepF1-5’-ATTCAACCAATCATAAAGATATTGG-3’ and
LepR1-5’-TAAACTTCTGGATGTCCAAAAAATCA-3’ (Hebert et al. 2004; Wilson 2012). PCR
amplified mitochondrial COI gene was sequenced by the Sanger sequencing method.
The obtained sequence were submitted to GenBank. To construct the phylogenetic
tree, sequences for the COI gene of Henosepilachna
were downloaded from NCBI. A total of 54 sequences for eleven species of Henosepilachna and one for Epilachna were downloaded from NCBI GenBank
(Table 1) of which Epilachna
sp. is an outgroup.
Generated COI gene sequences were
edited in BioEdit Sequence Alignment Editor Software.
Multiple sequence alignment was carried out using MUSCLE (Edgar 2004) in MEGA 7
(Kumar et al. 2016). Phylogenetic analysis was done for COI sequences for 13
taxa.
Phylogenetic analysis was carried
out using Maximum Likelihood (ML) method (Felsenstein
1981). MEGA 7 was used to find out the best-fit nucleotide model for the
dataset under the corrected Akaike Information Criterion (AICc).
The best nucleotide substitution found was T92+G, which is not implemented in
the RAxML program, so we used the default model which
was GTR + G (Lanave et al. 1984; Gu et al. 1995). ML
analysis was performed on RAxML HPC2 (Stamatakis 2014) at the CIPRES portal (Miller et al. 2010).
Bootstrap support values were obtained with the rapid bootstrap algorithm with
1000 bootstrap replicates.
RESULTS
Material examined: 1 male, SUKDZLB94, Halondi, Kolhapur (16.7348˚N, 74.3016˚E)
27.ii.2018, Priyanka Patil; 2 females, SUKDZLB314,
SUKDZ317, 3 males, SUKDZLB311, SUKDZLB312, SUKDZLB313, Kanthewadi,
Kolhapur (16.4693˚N, 74.0329˚E), 26.x.2018, coll.-
Priyanka Patil; 1 female, SUKDZLB450, 1 male,
SUKDZLB453, Yerad, Satara
(18.3614˚N, 73.3502˚E) 15.ix.2019, coll. Priyanka Patil.
Henosepilachna nana (Kapur,
1950)
Diagnostics: Length 6.0–6.6 mm, width 4.6–5.2
mm.
The body is oval with 6 black
elytral spots over grey background (Image 2a). The black elytral spots are
surrounded by a brown ring. The dorsal body shows grey pubescence except for
black spots. Brick red coloured pronotum with four
black spots (Image 2m). In many examples, pronotal
spots on the same side join to form a quadrate spot, which is nearer to the
base than the apex and does not touch to anterolateral margins and pronotal median line (Image 2l). Scutellum brown,
triangular, pointed at the elytral side (Image 2i). Ventrally brown except dark
patches on metasternum and abdominal sternites. Tarsi are pseudotrimerous
having bifid tarsal claws (Image 2o). The tarsal claw is with a basal tooth
(Image 2p). Sixth abdominal sternite is divided in
females (Image 2h---) and emarginated in males (Image 2j).
Male genitalia: Penis is thin, with a slightly
broad penis capsule (Image 2f). Penis apex is with V-shaped notch and a small
projection at the notch base (Image 2d,e). Tegmen with paramere
tip rounded having many setae at the apex and fewer setae on the distal half.
Penis guide is uniformly curved at the distal half, curved and pointed at the
apex (Image 2b,c).
Female genitalia: Female coxites
quadrate with blunt corners, inner margin is straight except for a shallow
notch at the base and with a pear-shaped small stylus with numerous setae
(Image 2g).
Host plants: The specimens of Henosepilachna nana were found to be
associated with Cucumber (Cucumis spp.),
Pumpkin (Cucurbita spp.), and Karit fruit
plant (Cucumis spp.). On the underside
of leaves, in a bunch, approximately 20 elliptical, yellow, sparsely laid eggs
were observed. Both the larvae and adults of H. nana skeletonize the
leaves of the above vegetables by eating the chlorophyll and can act as a
serious pest of cucurbitaceous plants. Under laboratory conditions, conspecific
egg predation by this species was observed (Image 2n).
Phylogeny (Figure 1): COI gene sequenses
for Henosepilachna nana generated
during this study were first time submitted to NCBI GenBank under accession
numbers ON220741 (for SUKDZLB94) and ON220742 (for SUKDZLB311). The
phylogenetic tree is rooted with outgroup Epilachna
sp. The larger part of the tree is a nested clade that comprises four main
sub-clades. Within the first sub-clade, H. septima
and Henosepilachna sp.1 are the sister taxon
to all remaining species with high support (bootstrap (BS) 99). The second
sub-clade, H. boisduvali is sister taxa to H.
nana with a high bootstrap value (BS 88).
Within the second sub-clade, Henosepilachna
sp.2 and H. implicata are sisters to H. pusillanima
with high support (BS 98). The third sub-clade shows three different Henosepilachna sp. named H. vigintioctopunctata showing its variation among its
congeners. The sub-clade at the top of the tree shows two nodes, one for H. enneasticta and the other for H. vigintioctopunctata complex consisting of H. niponica, H. pustulosa, H. yasutomii, and H. vigintioctopunctata.
The species H. nana is genetically closer to H. boisduvali than other congeners.
DISCUSSION
Kapur (1950) wrote a note on Epilachna ocellata
with a description of three species, viz., E. nana, E. anita, and E. manipurensis.
It includes details of identification and genitalia description along with
images of all four species. After 25 years, Gordon (1975) revised Epilachninae of the Western hemisphere in which he
described one new species named E. nana. However, the species which was
described by Gordon (1975) is different from E. nana having different
characteristics than E. nana which was described by Kapur
(1950). Later, Kapur informed Gordon that the
nomenclature E. nana is already given to one ladybird species and then
Gordon (1985) replaced the E. nana name with E. minuta.
The Holotype of E. nana was collected in 1892 and paratypes were
collected in 1914 from Nilgiri Hills and Parambikulam (Kerala), respectively (Kapur
1950). The species is distributed over the Nilgiri
Hills of southern India (Kapur 1950; Poorani 2012; Borowski 2020). The localities in
Maharashtra, which are being reported in the present article, will add H.
nana to the Fauna of Maharashtra, proving that the range extension of this
species towards the north-west by about 700 km.
According to the illustrations
and description given by Kapur (1950), the specimens
recorded during the study are treated here as H. nana. Every specimen of
H. nana collected during the study show 12 elytral spots without any
variation in the count. Based on the number of elytral spots, H. nana differs
from the other Henoesepilachna species. The
species H. boisduvali is yellowish-red having
a median black pronotal spot and 12 elytral spots (Li
& Cook 1961; Li 1993). The species H. nana is varying from H. boisduvali in having two spots present on each
side of the median line, there is no median pronotal
spot and the median area is always spotless. In the male genitalia, the penis
tip of H. nana is similar to H. boisduvali but
differs in having a small projection at the base of the notch. Poorani et al. (2021) have also mentioned 5–7 pronotal spots in H. implicata. In H. nana, the
total number of pronotal spots is 2–4 and elytral
black spots are 12 (6 on each elytron) which agrees with the observations of Kapur (1950) and confirms the identification of H. nana.
The species H. implicata is with 26–28 black spots on the elytra,
resembles other 28-spotted species of Henosepilachna
such as H. vigintioctopunctata, H. septima, and H. pusillanima
(Poorani et al. 2021). Although the tegmen of H.
implicata and H. nana look a bit similar but it differs in their
penis structure. The penis apex in H. implicata is with a shallow notch
(Poorani et al. 2021) while the penis apex in the
species under study is with a deep notch having a central small projection at
the base of the notch which is similar to the penis illustration of H. nana given
by Kapur (1950). So far, no information is available
about the host plant of H. nana. Kapur (1950)
wrote a note on E. ocellata including H.
nana in which he mentioned that E. ocellata
is found on potato but he does not mention the host of H. nana.
According to Katoh et al. (2014), adults and larval
stages of all Epilachnini species show phytophagous
feeding nature. The record of this species on the Cucurbitaceae
plant is providing new host records for the species under study and related
species. In the present study, H. nana specimens were observed on the
leaves of pumpkin, cucumber, and karit fruit plant
which are additional host records.
A recent study on DNA sequence
data and phylogenetic tree of Henosepilachna
species includes five species (Poorani et al. 2021)
excluding species H. nana. Therefore, the COI gene sequence of H.
nana is becoming available in the NCBI database for the first time. The
present consensus tree arrangement shows the co-evolution of species from
common ancestors. When the phylogenetic tree of H. nana was constructed
using COI gene sequences of eleven species of the Henosepilachna
genus and one outgroup Epilachina sp.
from the same family from NCBI’s database. The species H. nana shows
variation among its congeners.
Table 1. Taxa used in phylogeny
analysis with their GenBank accession numbers.
|
|
Taxa |
GenBank accession numbers |
|
1 |
Henosepilachna niponica |
LC228599, LC228594, LC228597,
AB002185, LC228600 |
|
2 |
Henosepilachna pustulosa |
AB300448, LC228587, AB300446,
LC228586, AB495213, AB495212, AB495210, AB300447, AB300454, AB300457,
AB495211, LC228585, AB300452, LC228588, AB300456, AB002183, AB300451,
AB300453, AB300450, AB300449, LC228582 |
|
3 |
Henosepilachna vigintioctopunctata |
KU234209, KU234206, KU234207,
KU234208, KU234204, KU234205, KU234199, KU234210, KU234203, KU234201,
KU234200, AB002180, KU234211, KU234202 |
|
4 |
Henosepilachna yasutomii |
LC228592, AB002184 |
|
5 |
Henosepilachna enneasticta |
AB002173 |
|
6 |
Henosepilachna pusillanima |
MH395854, AB002177, MT985168 |
|
7 |
Henosepilachna implicata |
MT985166 |
|
8 |
Henosepilachna sp. 1 |
MH395855 |
|
9 |
Henosepilachna sp. 2 |
AB002174 |
|
10 |
Henosepilachna boisduvali |
AB002175 |
|
11 |
Henosepilachna septima |
KT693136, AB002176, KX503056,
MT985165 |
|
|
Epilachna sp. |
AB002179 |
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