American Cockroach (Periplaneta americana): Genomic Analysis of Emerging New Strains

American cockroaches (Periplaneta americana) (Linnaeus), order Dictyoptera, is an important insect in medical (Akbari et al., 2015), they are the most notorious pests, found in kitchens (El-Sherbini and Gneidy, 2012). It is among the largest common cockroach species (Gary, 2015). Dictyoptera comprises about 5000 species under 398 genera in 28 families (Hanitsch, 1917). Out of 500, 30 species are considered as household pest (Chung et al., 2005). Several cockroach pests live in/or around homes and they are omnivorous scavengers (Akbari et al., 2015). They survive in warm weather with high moisture conditions as well as in unfavorable environments for humans (i.e., sewers and other human-made habitats) (Jaramillo-Ramirez et al., 2010).

P. americana can spread bacteria, fungi and other pathogenic microorganisms from infected areas (Czajka et al., 2003) and cause allergies to humans (Kinfu and Erko, 2008). They play an important role in the transmission of different diseases in mechanical and biological ways (Kim et al., 2016). P. americana spends most of its time in sewage and sewer pipe which usually contains a high density of pathogens (Basseri et al., 2016). Also, they feed on garbage and they have large opportunities to disseminate human pathogens (Pai et al., 2005). In addition, their nocturnal and filthy habits of eating their feces make them ideal carriers of numerous pathogenic microbes (Allen, 1987). Cockroaches spread pathogens through their cuticle (Mpuchane et al., 2006b), because their nymphal cuticles go through ecdysis (Mpuchane et al., 2006a). Therefore, they transfer pathogens in different ways such as vertical transmission which occurs when an infected mother passes on the pathogen or disease to her progeny (Jennifer, 2008).  All these pathogens are used as dangerous organisms targeting animal or human populations.

Much of the work regarding insecticidal efficacy has been done on B. germanica, however, very little data is available concerning P. americana. Therefore, keeping in view the work carried out by various researchers, the present work was designed to identify collected P. americana using morphological and molecular techniques and to investigate the insecticidal efficacy of two groups of insecticides (conventional and non-conventional), with different classes on P. americana and study the susceptibility of different stages to these insecticides through laboratory bioassay using feeding and contact toxicity methods. Little is known about the infection of P. americana oothecae isolated bacteria as a safety method for controlling this phase, however, the possibility of finding a new method to control P. americana oothecae can bring new perspectives of controlling, since no insecticides are effective so far when applied topically on this phase of the pest and it is important to inhibit embryogenesis of P. americana to prevent them from hatching. The present work aims to identify P. americana by using morphological and molecular techniques.

Molecular identification of P. americana
 
P. americana samples were collected from five different places in Jeddah city. All genomic DNA extraction experiments were conducted in the molecular identification experimental unit, in the Dengue Mosquito Experimental Station (DMES), belonging to the Department of Biological Sciences, Faculty of Sciences, King Abdul-Aziz University, Jeddah, Saudi Arabia. The place is intended to be a working and training unit in insect molecular identification and help university postgraduate students perform their specialized research. Equipment, tools and instruments were kindly supplied by the author of this work.
 
Genomic DNA extraction
 
Thermo Scientific GeneJET Genomic DNA Purification Kit #K0721 was used for P. americana DNA extraction, with some modifications. P. americana body (antenna, legs and wings), was cut into small pieces and collected into three tubes of 1.5 ml Eppendorf tube. Samples were resuspended in 180 μl of digestion solution and sterilized white sand. 20 μl of Proteinase-K solution was added and mixed thoroughly by vortexing to obtain a uniform suspension. Samples were incubated at 56°C for one hour until the tissue was completely lysed and no particles remained. 20 μl of RNase was added, mixed by vortexing then incubated for 10min at room temperature. Then, 200 μl of lysis solution was added and mixed thoroughly by vortexing for 15 seconds until a homogeneous mixture was obtained. After that, 400 μl of 50% ethanol was added and mixed by vortexing. The lysate solution was then transferred to a GeneJET Genomic DNA Purification column inserted in a collection tube. The column was centrifuged for 1 min at 8000 rpm. The collection tube containing the flow-through solution were then discarded. 500 μl of wash buffer I were added, centrifuged for 1 min at 10,000 xg. The flow-through were discarded and placed the purification column back into the collection tube. Then, 500 μl of wash buffer II were added to the GeneJET Genomic DNA Purification column, centrifuged for 3 min at maximum speed (12,000 xg). Exactly 200 μl of elution buffer were added to the centre of the GeneJET Genomic DNA Purification column membrane to elute genomic DNA, samples were incubated for 2 min at room temperature and centrifuged for 1 min at 10000 rpm. Finally, the purification column was discarded. The eluted DNA was measured with Nano Drop. Purified DNA was stored at -20°C.
 
Primer sequence
 
For molecular identification of P. americana, Universal primers (LCO1490 and HCO2198) were used in this study (Table 1, 2 and 3).






 
Agarose gel electrophoresis
 
For estimation size estimation, 10 µl of molecular weight marker were loaded in the first well. The gel was run at 127 V for one hour, which was attached firmly and connected to the power supply (MOLECULE-ON PS-M-300V Electrophoresis Power Supply, India). 1% of agarose powder was weighed and dissolved in 1X TAE buffer by placing the suspension in a microwave oven for heating. A final concentration of 0.1 µg/ml ethidium bromide (EtBr) from 10mg/ml stock in distilled water was added to the agarose, then agarose was poured into the gel tray and the comb was placed at one end. The gel was left for one hour to solidify before the comb was removed. The gel was then placed into an electrophoresis tank and submerged with 1X TAE buffer as a running buffer. The 5X loading dye was added to the samples to be loaded according to the volume ratio 5:1 of sample to dye. The gel was run in a horizontal gel apparatus at 127 volts for 60-90 min. DNA fragments were visualized on a UV transilluminator and photographed by using a Viber Lourmat Gel Imaging System. DNA ladder was used to determine the molecular weights of DNA fragments (Sharawi, 2023).
 
Sequencing reaction
 
DNA sequencing was carried out in Macrogen Company, Korea. Deduced nucleotide and amino acid sequence data were compared with the National Centre for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST).

Molecular identification of P. americana
 
P. americana DNA extraction was prepared using Thermo Scientific GeneJET Genomic DNA Purification Kit #K0721 and examined on 1% agarose gel. PCR amplification was then carried out using universal primers, as shown in the materials and methods. The resulted 700-800 bp was examined on 1% agarose gel electrophoresis as in Fig (1). The PCR products were purified and send to Macrogen for sequencing.  The phylogenetic relationships of P. americana isolate and closely related species were analyzed using the Multisequence Alignment Program (MEGA7) and the results are presented in phylogenetic tree, as in Table 4 and Fig (2). DNA barcoding promises to be a useful tool to identify pest species assuming adequate representation of genetic variants in a reference library (Beeren et al., 2015). In our finding, we used barcode region of mt-DNA (LCO 1490 and HCO2198) gene of DNA extracted from 30 cockroach specimens (Table 5), along with the development of a PCR method. The PCR generates a single band between 700-800bp-sized in all cockroach specimens, followed by direct sequencing. Two accession numbers were found from DNA sequencing (JQ267485.1 and KM576926.1) with 96-100% and 92-96%, respectively of the similarities were related to P. americana. Anyway, the molecular identification studies of P. americana are not much studied in Saudi Arabia in general and Jeddah governorate in particular. Many researchers have studied the molecular identifications of different cockroaches. Beeren et al. (2015) found that three deeply-divergent, widely-distributed P. americana COI haplogroups. Xiao et al., (2012) sequenced the complete mitogenomes of two cockroaches, reconstructed the molecular phylogeny and attempted to infer the phylogenetic position of termites in Blattaria more reliably and he found that complete mt-DNA nucleotide sequences of P. americana is 15,025 bp in size and they found that P. americana shares only 75% sequence identity with B. germanica, which is lower than that with the two genome-sequenced termites, i.e., 79% to Z. nevadensis and 80% to M. natalensis. Ma et al., (2017) determined the complete mitochondrial genomes of two cockroach species, Periplaneta australasiae and Neostylopyga rhombifolia, 15,605 bp and 15,711 bp in length, respectively. So, this study first time confirm the molecular characters of P. americana from Jeddah region of Saudi Arabia.









Molecular identification through DNA barcoding has proven to be a valuable tool for accurately identifying pest species, given an adequate reference library (Beeren et al., 2015). In our study, we utilized the barcode region of mt-DNA, specifically the LCO 1490 and HCO2198 genes, extracted from 30 P. americana specimens. The PCR method developed for this study produced a single band of 700-800 bp in all specimens, which was subsequently sequenced. The sequencing results yielded two accession numbers, JQ267485.1 and KM576926.1, with 96-100% and 92-96% similarity, respectively, to P. americana. Our findings indicate a high degree of similarity to P. americana corroborating the effectiveness of the molecular approach in identifying this species. Despite the successful identification, it is noteworthy that molecular studies on P. americana in Saudi Arabia, particularly in the Jeddah governorate, are limited. This study contributes novel molecular data from this region, filling a significant gap in the genetic documentation of P. americana. Previous research has also highlighted the genetic diversity within P. americana Beeren et al., (2015) identified three deeply-divergent, widely-distributed COI haplogroups within P. americana, demonstrating the genetic complexity of this species. Similarly, Xiao et al., (2012) sequenced the complete mitogenomes of two cockroach species and inferred the phylogenetic position of termites within Blattaria. They reported that the complete mt-DNA sequence of P. americana is 15,025 bp in size and shares only 75% sequence identity with Blattella germanica. This lower identity compared to termites (79% with Zootermopsis nevadensis and 80% with Macrotermes natalensis) suggests significant genetic divergence within the Blattaria order. Additionally, Ma et al., (2017) determined the complete mitochondrial genomes of Periplaneta australasiae and Neostylopyga rhombifolia, finding them to be 15,605 bp and 15,711 bp in length, respectively. These studies collectively underscore the extensive genetic variability and complexity within cockroach species, highlighting the importance of molecular identification in understanding their phylogeny and evolutionary relationships. Our study, for the first time, confirms the molecular characteristics of P. americana from the Jeddah region of Saudi Arabia, adding to the global genetic data on this species. The high similarity rates observed in our sequences suggest a close genetic relationship with previously identified P. americana specimens, reinforcing the utility of DNA barcoding in pest identification. Overall, the molecular identification approach used in this study provides a reliable and accurate method for identifying P. americana, which can be instrumental in pest management strategies. By contributing to the genetic database of P. americana in Saudi Arabia, this study supports future research and control measures aimed at mitigating the impact of this pervasive urban pest.

This study provides the first molecular characterization of P. americana from the Jeddah region of Saudi Arabia, contributing novel genetic data to the global database for this species. Morphological identification of various life stages, including adults, nymphs and oothecae. However, there is still a need for detailed morphological characterization of oothecae with clear photographs and identification keys for this region. Our molecular approach, utilizing the barcode region of mt-DNA (LCO 1490 and HCO2198), confirmed P. americana with high similarity rates to known sequences (JQ267485.1 and KM576926.1). These findings demonstrate the effectiveness of DNA barcoding for accurate pest identification, specifically for P. americana. By addressing a significant gap in the genetic documentation of P. americana in Saudi Arabia, this study enhances understanding of its genetic diversity, providing valuable insights that support future pest management strategies. This wording streamlines the points and emphasizes the contribution to genetic documentation and pest control. The integration of molecular and morphological methods provides a comprehensive framework for identifying and managing this urban pest.

The present study was supported by the author.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the author and do not necessarily represent the views of their affiliated institutions. The author is responsible for the accuracy and completeness of the information provided, but do not accept any liability for any
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Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The author declares that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.