Abstract

DNA Barcoding is one of the emerging tools in molecular identification of faunal diversity, specifically insect fauna. The Surinam cockroach, Pycnoscelus surinamensis is the only known roach to be obligatorily parthenogenetic, with reported haplotypes. P. surinamensis is well established in Indomalayan, tropical and subtropical regions and substantially documented from India with a phenetic approach. Herewith we report the first set of mt DNA barcode from a vouchered collection for the species from southern Western Ghats India. Discussions are made on the identity of two sequences each of Blatteria species and Pycnoscelus species reported from USA.

Keywords: Surinam cockroach, Greenhouse cockroach, DNA barcode, Western Ghats.

Received: 10 August 2021 / Revised: 6 December 2021 / Accepted: 24 December 2021/ Published: 13 January 2022

Contribution/ Originality

This study contributes to the existing literature of Pycnoscelus surinamensis species from India with additional DNA barcode data from vouchered specimens for the species as well as the genus from the Western Ghats. Barcodes linked with the vouchered specimens forms the authenticated DNA reference library having utility in metabarcoding.

1. INTRODUCTION

In the trending era of ‘DNA barcoding’, the utility of short DNA stretches manifesting nucleotides divergence amongst species renders an intangible aid for precise identification and documentation of various life forms [1-3]. This nucleotide-based taxonomic research tool [3] have remarkably increased the pace of taxonomic discoveries facilitating access to Linnaean taxonomy for the experts and non-experts (for e.g. [4, 5]). Additionally, its usage has allowed the study of multifaceted heterogeneous questions outlining numerous paradigms [6]. Hebert's group [1] suggested the mitochondrial Cytochrome C oxidase subunit I gene (mt COI) as a barcode gene for the delimitation of animal taxa and it is widely accepted for delimitation of different invertebrate groups including Blattodea [1, 2]. Identification based on DNA barcodes solemnly depends on the DNA barcode reference library, whose caliber increases when the DNA barcodes are affiliated to a taxonomically identified vouchered specimen.
The order Blattodea is composed of roaches and termites [2, 7] of which roaches have a cosmopolitan distribution. Cockroaches are among the highly diverse insect groups with 4600 species/subspecies globally and 181 species in India [8] of which only 33 species (~18%) have DNA barcode data. The genus Pycnoscelus Scudder, 1862 is denoted by 15 species of which four species are known from India, Pycnoscelus indicus [9]; Pycnoscelus nigra [10]; Pycnoscelus surinamensis [11] and Pycnoscelus tenebrigera [12-14].

The species Pycnoscelus surinamensis belongs to the second largest cockroach family Blaberidae . It is an introduced species to India identified to have medical and economic importance [15, 16], native to the Sunda Islands of the Malaya Archipelago with a predominant range of distribution in the Indomalayan region [16, 17]. The species has recently invaded large parts of the tropical as well as subtropical regions of the world due to human intrusions [15-17]. P. surinamensis is reported to have many parthenogenetic clonal forms from different regions including Florida, Australia, Panama, Hawaii, and Indonesia [18, 19]. The Surinam cockroach is known as peridomestic species worldwide [15, 17] and acts as plants pest, biological vector, and is known for ecological associations [15].

The species P. surinamensis is thought to have evolved from its bisexual intimate progenitor P. indicus for rapid establishment [16, 18, 19]. GenBank searches suggest no DNA Barcode data is available for the members of Pycnoscelus from India. Hence, as a part of our exercise to generate mt DNA barcodes and documentation of species diversity from the Western Ghats, we report the first mt DNA barcode for the species P. surinamensis from southern Western Ghats, India with a vouchered specimen deposited at the National Zoological Collections (NZC).

2. MATERIAL AND METHODOLOGY

Three specimens were collected below the leaf litter during the daytime from the Courtallam forest (N 8.9331; E 77.2674), Tamil Nadu, India on 26.viii.2019 by K.P. Dinesh and Party. Specimens were fixed and preserved in 70% alcohol for further studies. Images were procured using DSLR camera Figure 1. For molecular studies, tissue was derived from leg and thoracic muscles. Studied examples are deposited in the National repository of Zoological Survey of India, Western Regional Centre, Pune, Maharashtra, India with the register number ZSI/WRC/Ent-12/81.

Figure 1. Pycnoscelus surinamensis from Courtallam forest, Tamil Nadu, India (A: Dorsal and 1 B: Ventral side of ZSI/WRC/Ent-12/81a; C: Dorsal and D: Ventral side of ZSI/WRC/Ent-2 12/81b; E: Dorsal and F: Ventral side of ZSI/WRC/Ent-12/81c).

Genomic DNA extraction and purification were completed by the DNeasy Blood and Tissue Kit (Qiagen) as per the manufacturer's protocol from the alcohol preserved tissue samples. The eluted DNA was quantitated by Qubit 2.0 fluorometer ds DNA HS Assay. PCR amplification was performed using universal COI primers, LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' [20] in 25µL reaction volume consisting of 12.5µL of Go Taq Hot Start Green Master mix (Promega) 2µL (100ng) of genomic DNA, 1µL (10pmol) of each primer and nuclease-free water up to Q.S. The thermal cycling profile was as per [1]. The amplified PCR product was verified on 1% agarose gel by electrophoresis stained by EtBr dye and visualized below UV light through Gelstain gel documentation system. Amplified PCR product was purified using Invitrogen’s Pure Link PCR Purification Kit. Purified PCR product was sequenced bidirectionally by Sanger’s method on ABI 377 (Applied Biosciences) sequencer.

3. RESULT AND DISCUSSION

The obtained sequences were manually verified for corrections and the mt COI gene sequences available for the species of Pycnoscelus were downloaded from the GenBank Table 1 and were aligned by Clustal W in MEGA X software [21]. For phylogenetic reconstruction, the Maximum Likelihood tree was built with RaxML [22] under the GTR+GAMMA+I model, with 1000 bootstrap replicates and the final consensus tree was visualized by FigTree v1.4.0 treating species of the closest subfamily as outgroups.

Figure 2. Maximum Likelihood tree for the members of Pycnoscelus based on the 588 bp of mt COI DNA Table 1.

Species identity was confirmed as Pycnoscelus surinamensis based on the Basic Local Alignment Search Tool (BLAST) search of National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) with 100% match for the DNA barcodes generated in our study. In the preliminary phylogenetic tree our samples formed a monophyletic clade Figure 2 with Pycnoscelus surinamensis sequences reported from China, Indonesia, Austria, and USA Table 1 complying with parthenogenesis. The P. surinamensis clade Figure 2 had specimen representations from, India, Austria, French Polynesia, Indonesia, Costa Rica, USA, and Guyana supporting the distribution range of the species to be cosmopolitan. The two sequences (HM385633.1, KX054562.1) from the USA were making a separate sub-clade. Additionally, JN301633.1 from the USA forms a sub-clade with JN301630.1 from French Polynesia Figure 2 intimating potential haplotypic forms of Pycnoscelus surinamensis.

Table 1. mt COI DNA sequences used for building Maximum Likelihood tree.

Sr no.	Gen Bank Acc. No	Locality	Species name as per Gen Bank	Publication details as per NCBI
1	MW051034.1	Tamil Nadu, India	Pycnoscelus surinamensis	This study
2	MK074952.1	Austria: Vienna	Pycnoscelus surinamensis	Zangl, et al. [23]
3	KX054560.1	French Polynesia	Pycnoscelus surinamensis	Ramage, et al. [24]
4	KX054561.1	French Polynesia	Pycnoscelus surinamensis	Ramage, et al. [24]
5	MK074949.1	Austria: Styria	Pycnoscelus surinamensis	Zangl, et al. [23]
6	KY497675.1	Indonesia: Sumba island	Pycnoscelus sps.	Unpublished
7	MK074947.1	Austria: Styria	Pycnoscelus surinamensis	Zangl, et al. [23]
8	HM385667.1	Costa Rica	Pycnoscelus surinamensis	Unpublished
9	MK074950.1	Austria: Vienna	Pycnoscelus surinamensis	Zangl, et al. [23]
10	JN301634.1	USA	Pycnoscelus surinamensis	Unpublished
11	MW051035.1	Tamil Nadu, India	Pycnoscelus surinamensis	This study
12	KF155021.1	Guyana	Pycnoscelus sps.	Unpublished
13	KF155020.1	Guyana	Pycnoscelus sps.	Unpublished
14	KX054563.1	French Polynesia	Pycnoscelus. surinamensis	Ramage, et al. [24]
15	MK074951.1	Austria: Vienna	Pycnoscelus surinamensis	Zangl, et al. [23]
16	MK074948.1	Austria: Styria	Pycnoscelus surinamensis	Zangl, et al. [23]
17	MK074953.1	Austria: Vienna	Pycnoscelus surinamensis	Zangl, et al. [23]
18	JN301633.1	USA	Pycnoscelus surinamensis	Unpublished
19	JN301630.1	USA	Pycnoscelus surinamensis	Unpublished
20	HM385633.1	USA	Pycnoscelus surinamensis	Unpublished
21	KX054562.1	French Polynesia	Pycnoscelus surinamensis	Ramage, et al. [24]
22	MF149708.1	China	Pycnoscelus indicus	Ma, et al. [25]
23	MF149709.1	China	Pycnoscelus indicus	Ma, et al. [25]
24	MF149711.1	China	Pycnoscelus indicus	Ma, et al. [25]
25	MF149710.1	China	Pycnoscelus indicus	Ma, et al. [25]
26	MG882159.1	Australia	Pycnoscelus nigra	Bourguignon, et al. [26]
27	MG882157.1	Australia	Pycnoscelus femapterus	Bourguignon, et al. [26]
28	KF372522.1	France	Lanxoblatta emarginata	Legendre, et al. [27]
29	KF372540.1	France	Zetobora sps.	Legendre, et al. [27]
30	MG882225.1	Australia	Paranauphoeta circumdata	Bourguignon, et al. [26]
31	KF372533.1	France	Phortioeca nimbata	Legendre, et al. [27]
32	MG882194.1	Australia	Epilampra maya	Bourguignon, et al. [26]
33	MG882232.1	Australia	Galiblatta cribrosa	Bourguignon, et al. [26]
34	KY497669.1	France	Galiblatta cribrosa	Unpublished
35	EU253832.1	France	Parasphaeria boleiriana	Legendre, et al. [28]
36	KF372538.1	France	Thanatophyllum akinetum	Legendre, et al. [27]
37	KP986436.1	Brazil	Tribonium sps.	Legendre, et al. [29]

The species Pycnoscelus surinamensis is sufficiently documented in India from the states Andhra Pradesh, Arunachal Pradesh, Chhattisgarh, Delhi, Manipur, Karnataka, Maharashtra, Meghalaya, Orissa, Rajasthan, Sikkim, Tamil Nadu, Tripura, and West Bengal [8, 12, 13] based on morphological studies and checklists without any genetic or DNA barcode data. Therefore, the occurrence of any potential haplotype or genetic variant for the parthenogenetic species is yet unexamined. As the evolution of thelytokous parthenogenenis is understudied the Pycnoscelus taxon is complicated to understand [18]. Therefore, the contribution of molecular data in this particular taxon is of immense value for delimiting the species complex and their evolutionary history.

The present report of Pycnoscelus surinamensis Figure 1 from Courtallam forest (Tamil Nadu) forms the first vouchered based DNA barcode record for the species as well as for the genus from India. To precisely validate the occurrence of any potential haplotype or genetic variant, large-scale DNA-based molecular studies are warranted. The current DNA barcode data generated for Pycnoscelus surinamensis from India forms baseline data for the DNA barcoding archives of the species and is expected to have utility in taxonomy, systematics, and metabarcoding other than biodiversity documentation.

Funding: This study received no specific financial support.

Competing Interests: The authors declare that they have no competing interests.

Acknowledgement: The authors are grateful to the Director, Zoological Survey of India, Kolkata; the Officer-in-Charge, Zoological Survey of India, Western Regional Centre, Pune for the research facilities and support. We thank Dr. Sameer Kumar Pati, Zoological Survey of India, Western Regional Centre, Pune for help in photography. We acknowledge the help of the staff of ZSI, WRC, Pune for the lab and field support. We thank Ms. Sangetha Devi and Ms. Sayali Aspate, Modern College of Arts, Commerce and Science, Shivajinagar, Pune, for their help in the wet lab.

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