Gene Based Identification and Control of Invasive Stink Bug Species: a Review

© 2019 JETIR June 2019, Volume 6, Issue 6 www.jetir.org (ISSN-2349-5162) Mitochondrial Cytochrome c Oxydase Subunit I (COI) Gene based Identification and Control of Invasive Stink Bug Species: A Review

1Anant Shinde, 2Rajesh Dhakane,3 Harishchandra Kulkarni

1Associate Professor, 2Assistant Professor, 3Principal

1Department of Zoology, Yashwantrao Chavan Arts and Science Mahavidyalaya, Mangrulpir, District Washim, Maharashtra, India. 2Department of Microbiology Jaywantrao Sawant College of Science and Commerce, Hadapsar, Pune, India. 3Department of Physics, Jaywantrao Sawant College of Science and Commerce, Hadapsar, Pune, India.

Abstract: Infestation of economically important crops by invasive pests such as stink bugs has been ever increasing problem in the globe from many years and their controlling strategies are being implemented in many countries. However, their identification and relationship with hosts have been poorly understood because of insufficient platform of morphology based taxonomical science, especially in the cases of immature or damaged specimens where external characters are uneasy to detect. Putting effective pest management programs into the operation is merely impossible if the target stink bug species, harms caused by them and their hosts are poorly investigated. Moreover, target oriented pest control strategies avoiding disastrous effects on non-harmful biota require understanding of species diversity of pests and their host plants under study which are difficult if morphological database is implemented. Therefore, in this review, we assessed the COI gene based species identification methodology of potentially destructive stink bugs, economic losses caused by them, their chemical and biological controlling strategies in addition with role of DNA barcoding in biomonitoring of these pests generating awareness among global farmers to overcome the problem of losses to commercial crops and orchards.

Index Terms - Stink bugs, pest management, COI gene, host specificity, identification problems, crop loss, biota.

INTRODUCTION Many natural and anthropogenic calamities are responsible for huge agroecological losses in the world resulting into undersupply of food to growing population. This problem is more severe in agriculture based developing countries where many people are dependent on agricultural sources for their survival. The potential pests of agricultural systems are true bugs, grasshoppers, beetles, aphids, green flies, sunflower maggot flies, plant hoppers etc. that cause major losses to fruit trees and crops. Stink bug, which is one of the groups of true bug species is substantial crop pest in India and western countries. The controlling strategies of these insect species are existing and are being implemented although their success rates are restricted, particularly in developing countries owing to lack of awareness among people and methodologies used in pest control management program. Also, the species identification of such insects is one of the challenging issues as these have more than 42,000 species belonging to greater than 500 genera and 140 families (Henry TJ, 2009) suggesting their diverse speciation and distribution in the globe. Species identification of such insect fauna which is primary requirement of their controlling program is challenging task due to their vast diversity and lack of proper morphological characters of immature specimens, unavailability of expert taxonomists and underdeveloped knowledge of cryptic and sibling species. Moreover, adequate knowledge of hosts susceptible to infection plays key role in target specific pest control program avoiding harm to non-harmful biota and supporting earth’s natural cycles. In addition, the severity of crop loss due to species in question should be known in order to understand emergency of launching pest control programs in affected areas. In this review, we appraised occurrence of stink bugs along with potential harms caused by them to fruit trees and economically valuable crops along with their COI based taxonomical identification, species variation pattern and phylogenitic relationships. Furthermore, we enlightened their control measures including both chemical and biological methods with particular emphasis to biological methods. Moreover, we evaluated the possible applicability of DNA barcodes for eradication of stink bug species from commercially important agricultural crops and orchards preventing their losses due to invasions. Occurrence stink bugs: Stink bugs, especially oriental species are found in countries such as India, Bangladesh and Sri Lanka (Memon et al., 2002) of southern Asia on various crops due to availability of favorable environmental conditions. However, individuals of Nezara viridula are also distributed in other continents including not only tropical but also subtropical areas of Eurasia, Africa, Australia, and the Americas (Hoffman et al. 1987, Panizzi, 2000), whereas Nezara antennata was reported to occur in oriental and the southeastern edge of the Palaearctic region by Li M et al. (2014). Furthermore, stink bugs have various coloration and some of them have brown (brown marmorated stink bug Halyomorpha halys (Stål)) and yellow (yellow-brown stink bug-Halyomorpha halys) colors which are found in China, Japan, Korea and Taiwan (Jesus Lara et al., 2016) and East Asia (China, Korea, Japan), Allentown Pennsylvania in US JETIR1907R57 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 345

(Hoebeke and Carter 2003), Switzerland, Canada (Quebec and Ontario) Wermelinger et al. 2008; Harris 2010; Fogain and Graff 2011), respectively. In addition, Litchi stink bug species is distributed in India (Kumar et al. 2008). Crop and economic losses: Nezara viridula which is actively invasive (Hoffman et al. 1987; Panizzi 2000), polymorphic and cosmopolitan pentatomid crop pest causes economic loss by infecting number of crop species (Panizzi 2000; Reid 2006). Moreover, according to Matteo Bracalini et al. (2015) and Rodney N. Nagoshi et al. (2012), crop pests have serious effects on host plants on which they thrive. For instance, the brown marmorated stink bug (BMSB), Halyomorpha halys (Hemiptera: Pentatomidae) is a pest of plants that infect ornamentals, crops, weeds, forests and fruit crops such as apples, pears, persimmon and peaches across China, Japan, Korea, and the USA (Funayama, 2004; Yu and Zhang, 2007; Nielsen and Hamilton, 2009; Son et al., 2009) and is causative agent of phytoplasma disease of Paulownia tomentosa in Asia and suspected vector of several phytoplasmas (Jones and Lambdin, 2009). Also, Halyomorpha halys (yellow-brown stink bug) is agricultural pest of tree fruits and soyabeans (Hoffman 1931; Kobayashi et al. 1972; Funayama 2004). As well, BMSB generate public nuisance during late autumn and eventually entering them to overwinter (Hamilton, 2009) generating unpleasant odor after disturbance (Dhami et al., 2016) and have affected fruit industry in the North America (Nielsen and Hamilton, 2009; Nielsen et al., 2011). In addition, Jentsch (2012), Leskey et al. (2012), Nielsen and Hamilton (2009), Pfeiffer et al. (2012), Rice et al. 2014) stated that BMSB are responsible for causing damage to crops including apples, soybeans, tomatoes, peaches, corn, grapes and caneberries in East Coast states viz. Delaware, New Jersey, Pennsylvania, New York, Maryland, West Virginia and Virginia. Additionally, it is known as fruit-piercing stink bug which causes invasion to diverse fruits including soybean and apple in Japan (Toyama M et al. 2011) along with orchard crops, vegetables, grapes, row crops, ornamentals and nursery crops (ESA, 2011). Likewise, these invasive pests caused damage and economical loss of estimated $37 million to apple farmers from Mid-Atlantic States (Leskey et al. 2012) and have increasing concern on their detections on commercial crops viz. hazelnuts, blackberries and wine grapes in Oregon (Hansen and Mullinax, 2014) as well as on crops such as peppers, apples, peaches, plums and cherries in Washington (Eddy 2015). Additionally, soyabean production is threatened by stink bugs in US (Peiffer M and Felton GW, 2014) and endemic species such as southern green stink bug (Nezara viridula), green stink bug (Chinavia hilaris or Acrosternum hilare), and brown stink bug (Euschistus servus), brown marmorated stink bug (Halyomorpha halys), the red-banded stink bug (Piezodorus guildinii), and the kudzu bug (Plataspidae) (Megacopta cribraria) are recent and serious pests in crops of U.S (Akin S et al., 2011). Besides, invasive pests resulted into >$3 billion/year economic loss to California (Metcalf, 1995) and H. halys has dominated mid-Atlantic areas (Nielsen and Hamilton 2009; Nielsen et al. 2011) and caused loss of $37 million by infecting apples in 2010 (Seetin 2011) along with not recorded losses to diverse ornamentals, vegetables and field crops (Leskey and Hamilton 2010; Kuhar et al. 2012). Need of stink bug identification: Identifying invasive agricultural pests causing potential crop loss is very important for their effective control (Jiawu Xu et al., 2014). According to Raupach MJ et al. (2014), morphological as well as molecular analyses of true bugs of different families are needed. This approach is concordant with the view proposed by Sanket Tembe et al. (2014) who stated that lower amount of DNA sequences belonging to Pentatomomorpha bugs are deposited in DNA databases despite the fact that these insects are economically important and require insistent identification. Stink bug identification problem: Many stink bugs evolved as diverse species even in the same habitat making their identification difficult and time consuming. For example, in Pakistan, Individuals of genus Halys showed considerable variation on the same host plant (A. arabica; Z. jujuba) which was the source of food for them (Memon N, 2006). Moreover, different species of these insects, in many cases, appear similar creating controversial issues in their taxonomical operations. This view was exemplified by Yang (1962), according to whom, highly closely related species Nezara antennata and N. virudula were considered as same although they were distinct Kon et al. (1993), Aldrich et al. (1993), Kon et al. (1988). Likewise, Foottit et al. (1997) proposed that morphological identification strategies may be complicated because of loss in essential characters, numerous forms and plasticity in morphology as result of effects of hosts and factors of milieu. In addition, in accord with Memon N et al. (2006), use of DNA for stink bug identification is favored because morphological studies indicate only species borders. Furthermore, Dhami MK (2016) proposed that the correct identification methods which are required for efficient pest control program are often not available for some recently emerged pests such as Halyomorpha halys (brown marmorated stink bug). From this, it is obvious that the effective identification platforms are needed to be developed for assisting control systems of invasive stink bug species. Solution of identification problem: In order to solve the species identification problem in greater extent, Paul Hebert et al. (2003) and Kevin A. Shufran and Gary J. Puterkha, (2011) proposed the term ‘DNA barcode’ in which animal species are identified efficiently using mitochondrial Cytochrome C Oxydase Subunit I (COI) gene beginning from its 5| end. Furthermore, its superiority was confirmed by Sanket Tembe et al. (2014) who stated that species level taxa can be assigned to unidentified organisms using phylogeney analysis and variation of COI gene. Mitochondrial and molecular markers are robust tools to investigate biodiversity and the original area of invading pest leading to explore processes behind invasions (Ficetola et al. 2008). Genetic variance: The cases of low interspecies distance (<3%) were found in stink bug species as reported by Sanket Tembe et al. (2014) between two species Plautia splendens and Plautia stali which was as 1.3% raising question mark on 3% threshold value set by Hebert et al. (2003) for distinguishing species of order Lepidoptera, suggesting need for re-evaluation of this cut off value for species delimitation and distinct barcode gap determination of stink bug species. However, Sanket Tembe et al., (2014) reported the variation between species as 2% in two cases. Similarly, Riptortus pedestris and Riptortus clavatus revealed genetic distance of 2.1% (Livermore et al., 2014). Nevertheless, the cases of misidentification may be diagnosed in molecular identification systems as reported by Sanket Tembe et al. (2014) who stated that a bit divergence among barcode regions of Tessaratoma javanica species and other not JETIR1907R57 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 346

© 2019 JETIR June 2019, Volume 6, Issue 6 www.jetir.org (ISSN-2349-5162) identified species of the same genus were observed and according to authors, it may be due to misleading taxonomic identifications or poor sequence variation. However, this view was inconsistent with our approach according to which, this error may be due to unavailability of sufficient reference sequence library in DNA databases. It becomes easy to identify unknown specimen if its reference COI sequence is available in the DNA data banks. Hebert et al. (2003) proposed that while performing species delimitation, the genetic variation within species is generally less than among the species. This concept was consistent with the finding reported by Sanket Tembe et al. (2014) who stated that intraspecific variation among true bug species collected from India was as <1% in 97% analyzed taxa. On the other hand, authors reported that the genetic divergence among individuals of different species of the same dataset was >3% with exception 3% taxa. Although authors concluded that standard DNA barcode regions can be used for taxonomic assignments of true bug species, their opinion is lacking in certain areas since they have not mentioned about identification set up for left over less reliably identified taxa. Therefore, we propose that such taxa can be precisely identified by multi-loci approach using Next Generation Sequencing technology in which multiple gene sequences can provide more robust identification data than single gene approach. In addition, Memon N et al. (2006) proposed the use of COI marker for identification of H. sulcatus (one specimen) and 10 samples from putatively new species showed intraspecific pairwise distances within this population in the range of 0-0.16% which was slightly more than 0–2.1% reported by Sanket Tembe et al. (2014) for Indian pentatomorpha bug species and according to authors, pairwise distances between H.sulcatus and putatively new species were in the range of 2.87-3.28% that was greater than 0-2.%. Furthermore, the authors reported hierarchical raise in mean Kimura 2 parameter divergence across diverse taxa of Indian Pentatomorpha bugs supporting higher strength of COI sequences for stink bug identification. Besides, Li M et al. (2014) proposed that COI sequences can be considered as successful identifiers of stink bug species N. viridula of China as they grouped in conspecific clusters although the threshold values of intraspecific variation was found to be 5% suggesting deep intraspecific divergence in contrast to the threshold value of 3% established by Hebert (2003) for identification of lepidopteran species. NCBI for species identification: Sanket Tembe et al. (2014) reported that COI gene sequence of not identified genus Tessaratoma was having 99% similarity with DNA barcode region of Tessaratoma javanica clearly indicating that available NCBI data bank is able for species identification of unknown taxa. Also, authors observed that many COI sequences of Pentatomidae family showed matching with species of same genus indicating the successful identification of genera with sufficient and reliable reference sequence database. Interestingly, authors found that in the cases in which reference sequence is not available, identification up to only family level can be obtained indicating that the query sequences are new and can be valuable addition in the DNA sequence databases. Additionally, the obtained COI sequences of specimens of same family may not show variation in some cases although they may show slight variation when aligned with NCBI reference sequence. For example, Sanket Tembe et al. (2014) found no variation among studied haplotypes of Pentatomorpha bugs belonging to Western Ghat region Maharashtra state of India, however, 2.1% sequence diversion was observed by authors when they used COI sequence from GenBank (Accession number JX548495; source of collection-USA) in their analysis revealing DNA based distinction among population. The reported variance may be due to different rates of genetic evolution in COI sequences of Nezara viridula species belonging to USA and India. Bioinformatics tools for sequence analysis: Various bioinformatics off line and online tools are used for COI gene sequence analysis of diverse stink bug species. For example, sequences obtained from N. viridula and N. antennata collected from China were analyzed through Multiple Sequence Alignment (MSA) by Clustal W (Thompson et al. 1994) by Li M et al. (2014) and their haplotypes were differentiated by Mesquite version 2.74 (Maddison WP and Maddison DR, 2010) whereas Taxon DNA 1.0 (Meier et al. 2006) was used for calculating intraspecific genetic distances among COI sequences of N. viridula and the interspecific distance between N. viridula and N. antennata. Phylogenetic analysis: In order to study pairwise distances, the Kimura 2 parameter model of base substitution (Kimura, 1980) and to investigate variation in the specimens, neighbor-joining method (Saitu and Nei, 1987) are generally preferred for stink bug species. Efficacy of this method for phylogeny analysis was supported by Sanket Tembe et al. (2014) who stated that species and genera specific clusters were formed when Pentatomorpha bugs were analyzed. In contrast, authors argued that COI sequence of Eocanthecona furcellata failed to cluster with Eocanthecona japanicola from Korea (GQ292274) which was belonging to the same genus. This suggests either identification error or sequence alignment ambiguity or inability of Neighbour Joining method for investigating evolutionary relationship among stink bug species. Similarly, John F. Reinert et al. (2009) stated that NJ tree is phenetic method and its clades are formed by considering as a whole similarity platform and is not based on synapomorphy. In contrast, according to Li M et al. (2014), COI sequences were grouped in the cluster of conspecific sequnecs leading to give successful identification of N. viridula and N. antennata species. This explicitly suggests that the used molecular marker is the most robust and reliable for species identification and phylogeny analysis of Chinese stink bugs. Sanket Tembe et al. (2014) observed that the phylogentic tree constructed by NJ method showed that individuals of Pentatomorpha bugs belonging to the same genera were clustered in their groups. This finding clearly indicated the reliability of phylogeny analysis of the bug species by NJ method. Further, the authors stated that the phylogenetic trees constructed with COI sequences are unable to give up to the mark performance for enlightening linkage among higher taxa. More to the point, the authors claimed that Maximum Likelihood and Maximum Parsimony based trees were unable to explain monophyletic clades of Pentatomidae family. Also, Dinidoridae showed erroneous relationship since dinidorid Cyclopelta which is superfamily of Pentatomoidea grouped into the distinct group irrespective of remaining family members in both types of trees. As well, Park et al. (2011) observed polyphyly in both Superfamily Coreoidea and family Coreidae. Finally, Sanket Tembe et al. (2014) claimed that DNA barcodes are useful for species identification of true bugs and their many unseen species generating suitable system for detecting potential pest as well as invasive species on the basis of clear barcode gap and Neighbour Joining analysis. JETIR1907R57 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 347

What is more, the phylogenetic tree of specimens belonging to N. viridula and N. antennata was constructed by Li M et al. (2014) with neighbor joining method (Saitu and Nei, 1987), Kimura 2- parameter model (K2P) for nucleotide, and maximum parsimony methods with bootstrap support of 1000 replications, each with 10 random addition heuristic searches. These methods were testyed by PAUP 4.0 for windows (Swofford, 2003) and in the analysis, authors used sequences of two pentatomid bugs (Piezodorus lituratus (F.) and Rhaphigaster nebulosa (Poda)) as out-groups indicating that these methods can be used for phylogeny analysis of such groups supporting broad range applicability of the statistical method under consideration. Drawbacks of DNA barcoding: Despite of applicability of DNA barcoding for identification and phylogenetic investigation of broad range of animals, this technique has some demerits in the field of modern taxonomy. For instance, although Hebert et al. (2003) primarily enlightened Neighbour Joining tree for DNA barcoding and since then, it has been widely used in majority of DNA barcoding studies, Meier et al. (2006) and Virgilio et al. (2010) casted doubt on this method and stated that such trees do not work appropriately both empirically as well as theoretically. This view is consistent with the opinion of R.A. Collins and R.H Cruickshank (2012) who stated that NJ trees can be ambiguous particularly when used with incomplete reference library and problems related with NJ trees could not be solved by leftover tree inference methods, for instance, maximum likelihood or parsimony. Bootstrap support (in %) which is a method, in addition with reciprocal phylogeny, used for verification of grouped species in cluster of a tree and as a part of identification or delimitation of species is lacking in certain areas since less support would be gained by recently separated species on short branches resulting into failure in their identification although they might be morphologically separated and can be detected by unique mutations (Lowenstein et al. 2009). Moreover, implementation of cut-off for precise identification of species considerably compromises the efficiency of a reference library (Collins et al. 2012a; Zhang et al. 2012) and bootstrap resampling method does not provide examination of uncertainties of identification. In other words, specimen under testing can cluster with a reference sample with bootstrap support of 100% although it would be completely different species. In order to detect uncertainties in species identification, investigation of interspecific threshold distances should be investigated along with calculation of probabilities of group memberships (Zhang et al. 2012) and making clear caveats of sampling breath (sic) (Moritz & Cicero 2004). The threshold value (3%) for genetic distances of species delimitation has been widely controversial (Puillandre et al. 2012, Virgilio et al. 2012; Zhang et al. 2012) due to varying results of diverse species obtained by multiple researchers, for example, Hebert et al. (2003) stated that that it could be >3% in case of recently diverged species. The errors in identifications using DNA barcoding were due to misleading taxonomic identifications in GenBank databases including NCBI and BOLDsystems and singletone barcode showed to be belonged to large number of species (Virgilio et al., 2010) and latter does not show the intra-specific sequence divergence (N. Nagoshi et al., 2012), which is not expected in reliable species identification of diverse taxa. All in all, there will be impediments in identification using DNA barcoding owing to lower taxon coverage in DNA database systems and insufficient taxanomic information although N. Nagoshi et al. (2012) proposed that COI gene can be effectively used for identification of Oxycarenus species. On the other hand, Sanket Tembe et al. (2014) observed barcode sharing between Nariscus fumosus and Nariscus fasciatus revealing that DNA barcode may be problematic in cases of species with identical COI gene sequences. As well, the data obtained from mitochondrial DNA has many shortcomings such as its maternal inheritence, gain of unauthentic signals by obliterating ancestral polymorphisms as a result of lineage sorting and amplification of nuclear mitochondrial pseudogenes (numts) (Moritz & Cicero, 2004). Nonetheless, this problem can be solved by using purified mtDNA by separating it from complex of nuclear genome by density gradient centrifugation or agarose gel electrophoresis in which DNA of interest will be separated out due to their lower density and smaller size than nuclear DNA, respectively. Moreover, especially causing problems in arthropods (Hurst & Jiggins, 2005), inherited symbiontts viz. Wolbachia, Cardinium can lead to dramatic changes in mtDNA haplotypes of species. Nevertheless, Memon N. (2006) casted a doubt on this view and stated that the species boundaries of stink bugs obtained from mtDNA data were similar to their morphological data, hence mitochondrial evidence for stink bug species identification is reliable. Owing to such controversies, there is a need to expand the horizons of molecular taxonomical science by investigating multiple target genes of mtDNA for species identification (Raupach et al., 2010, Oliveira et al., 2011). COI gene sequences are easily used for barcode gap among and within species divergence (Sanket Tembe et al., 2014) due to their variability among the species and conservative nature in case of individuals of the same species. Nevertheless, overlapping sequence variation was found in Pentatomotpha species by authors when higher taxa were taken into consideration. Surprisingly, the authors failed to differentiate among available statistical methods for phylogenetic analysis such as Maximum Likelihood, Maximum Parsimony along with Neighbor Joining with 100% success since these trees showed no concordance with each other since species of Pentatomidae, Lygaeidae and Pyrrhocoridae families indicated monophyletic clades while others revealed controversial relationships. Consequently, there is need to develop statistical method of phylogeny investigation which can give supreme success in species identification of diverse taxa of stink bug species. In addition, some taxonomists use only DNA for identification of species which is a point of question (Donoghue et al., 1989). Similarly, Dunn (2003) stated that DNA sequences are utilized in the case where morphological identification is insufficient or with its combination. This approach is consistent with Ferguson (2002) and Blaxter & Floyd, (2003) who advocated that DNA based data is full with controversies and extra proof is essential before using DNA sequences for taxonomy. Moreover, the artifacts of DNA barcoding for species identification such as broad overlap between intra and interspecific COI sequence divergence (Meyer and Paulay 2005), for instance, unavailability of resolving power of COI sequences (Li M et al., 2014), use of additional markers, for instances, 16S rDNA (Vences et al. 2004; Steinke et al. 2005), Cyt b (Bradley and Baker 2001; Pfunder et al. 2004) in the case where DNA barcoding fails in complete identification of species (Hebert PDN and Gregory TR., 2005) were tried to be resolved by Li M et al. (2014) by investigating capability of proposed DNA barcode markers for identification of morphologically closely related southern green stink bug species Nezara virudula and orientel green stink bug species Nezara antennata from China in relation with their close relatedness and geographical variation pattern suggesting that the COI gene can be used for identification of closely related stink bug JETIR1907R57 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 348

© 2019 JETIR June 2019, Volume 6, Issue 6 www.jetir.org (ISSN-2349-5162) species. Conversely, Nelson et al. (2007) proposed that shared DNA barcodes are not able to distinguish species that are close to each other. Thus, question about ability of COI based identification system for differentiating recently evolved species remained unanswerable and this problem can be solved by increasing accurate DNA database with molecular investigation of wide range of species belonging to diverse animal taxa.

Control programs: Although Park D-S et al. (2011) proposed that taxonomic identifications of Heteroptera would assist in management of pests as well as regulatory and environmental applications, few pesticides used for control of Brown Marmorated Stink Bugs (BMSB) such as pyrethroids, neonicotinoids have broad-spectrum functions (Leskey et al. 2012; NIPMC 2014; Rice et al. 2014). However, pesticides may have non-targeted effect on beneficial biological species causing imbalance of earth’s natural cycles. Therefore, biological control of pests is gaining importance in agricultural systems and since higher use of insecticide may cause problems of food safety and environmental pollution, optional management methods, for example, biological control are required urgently although their implementation is lacking in certain areas (Jiawu Xu et al., 2014). This view was further exemplified by Jesus Lara et al. (2016), who stated that there are many biological predators that feed on BMSB including lacewings, mantids, earwigs, lady beetles, assassin bugs, minute pirate bugs, big-eyed bugs and spiders and 12 North American species of egg parasitoids viz. Anastatus spp., Gryon sp., Ooencyrtus sp., Telenomus spp. and Trissolcus spp. in at least three families (Encyrtidae, Eupelmidae, Platygastridae) that parasitize sentinel BMSB egg masses. Also, authors commented that Laemostenus complanatus (Dejean) (Coleoptera: Carabidae) can use sentinel BMSB as a food and Euclytia flava (Townsend) (Diptera: Tachinidae) and Astata spp. (Hymenoptera:Crabronidae) act as parasite of motile stink bugs and A. unicolor Say also attack on BMSB (Jesus Lara et al., 2016) which can be controlled by Astata spp. and another parasitic fly, Trichopoda pennipes (Fabricius) (Diptera:Tachinidae)(Rice et al. 2014). In the same way, Haye (2015) and Talamas et al. (2015) highlighted the biological control of BMSB and stated that these can be controlled by parasite species T. japonicas and T. cultratus in Asia. What is more, A. pearsalli (with parasitism level 4%) and a generalist parasitoid, A. reduvii (Howard) (Burks, 1967) (with parasitism level 79%) are parasites of BMSB (Jones AL, 2013). Furthermore, Anastatus sp. and other platygastrid parasitoids are found to infect these stink bug species (Jesus Lara et al., 2016) and according to Hoffmann et al. (1991), T.basalis plays the role of classical biological control agent of Nezara viridula (Linnaeus). Finally, according to Yang et al. (2009), members of Trissolcus halyomorphae are the superior egg parasitoid of H. halys having 50 % parasitism rate. In this way, biological agents can be effectively employed for eradication of substantially invasive stink bug species.

Role of DNA barcoding in stink bug control programs: Biological identification of pray and predators may play valuable role in agricultural pest control program. For example, the species identification of stink bugs using their COI genes can be used for estimation of infection of given agricultural landscape by them, which can assist in taking decision about the need of launching control programs in the invaded region. In turn, if the infection is sever and able to cause crop and economic losses, the decision of putting controlling measures into operation would be taken. Since chemical pesticides may be non-targeted with respect to their functions, these may be harmful for ecosystems. Moreover, many of them may be toxic to humans and as a result, these should not be preferred for eradication of stink bugs. Instead, the biological strategies of pest control should be implemented in order to approach the sustainable development of natural flora and fauna. In sequence, DNA barcoding may be very crucial in identifying varying species of stink bug predators assisting their implementation in the infected agricultural areas with approximate ratio of 30%:70% (pest: parasite) assuring effective removal of pests in question. Thus, it is clear that COI based identification of biological specimens may be helpful for reducing the invasion of pest species to agricultural crops preventing potential damage to them and resulting economic losses of farmers along with protecting natural processes of earth’s ecosystems.

CONCLUSION: Stink bug species can be reliably identified with COI gene based identification system with few exceptional cases where insufficient reference sequence library is available for identification analysis. Furthermore, the specimens under study are potential pests of commercial crops as well as fruit trees causing extensive losses to agroeconomic systems. These invasive creatures can be monitored by molecular identification and implementation of their biological predators in infected area. To end with, we advocate agricultural departments of all nations in the globe to sensitize the people for operation of suggested strategies for identification and control of stink bug species to make the public life pleasurable and joyful.

ACKNOWLEDGMENT Authors are thankful to Dr. V. R. Bhonde, Principal, Yashwantrao Chavan Arts and science Mahavidyalaya, Mangrulpir, District Washim, Maharashtra, India for his encouragement for our review work.

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