First report of Asia II 7 and China 3 cryptic species of Bemisia tabaci (Gennadius) and identification of its associated endosymbionts in Bihar

doi:10.21203/rs.3.rs-1540624/v1

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First report of Asia II 7 and China 3 cryptic species of Bemisia tabaci (Gennadius) and identification of its associated endosymbionts in Bihar

https://doi.org/10.21203/rs.3.rs-1540624/v1

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Bemisia tabaci (Hemiptera: Aleyrodidae) is a polyphagous invasive insect pest in the world that infests over 1000 plant species, spreads 300 plant viruses and associated with different endosymbiotic bacteria. In the current study, we looked at the genetic variation of B. tabaci populations and their association with endosymbiotic bacteria in Bihar (29 populations: 16 interhost and 13 interlocations). For this purpose, we conducted an inter-host survey and discovered that Brinjal was highly infested by B. tabaci, so, an interlocation survey was planned. The genetic variability among 29 populations were explored using nuclear markers i.e., Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR) along with extra-nuclear marker (Cytochrome c oxidase subunit I -COI) for their molecular characterization. Interestingly, Bayesian phylogenetic analysis of 657 bp mtCOI sequences showed the existence of four cryptic species belonging to two genetic groups (Asia and China) viz., Asia I, Asia II 1, Asia II 7 and China 3. In this study we have reported different bacterial endosymbionts associated with 29 B. tabaci populations in Bihar. By amplifying of 16S rRNA of specific bacteria we were able to identify Portiera sp, the primary endosymbiont of B. tabaci, and other secondary endosymbionts like Arsenophonus sp, Wolbachia sp, Rickettsia sp, Hamiltonella sp and Cardinium sp. Overall, this study found that Asia II 7 and China 3 cryptic species were reported for the first time in Bihar region and Asia I was the dominant cryptic species and that secondary endosymbionts were not uniformly distributed in all the populations. Furthermore, our findings in mapping the distribution of B. tabaci populations and their associated endosymbionts in various ecosystems paved the way for future research and the development of area-wide management tactics for B. tabaci cryptic species in Bihar.

B. tabaci

interlocation

interhost

RAPD

SSR

mtCOI

cryptic species

16S rRNA

Bemisia tabaci (Gennadius) is a common insect pest which infests a wide variety of host plants and causes significant crop losses under adverse conditions. In 1889, it was first identified in Cotton fields in Greece (Cock, 1993) whereas in India, in 1905 it was first reported on Cotton in Pusa (Bihar) (Misra and Lamba, 1929). It is regarded as one of the world's 100 vilest invasive alien species due to its remarkable competency to move, develop, adapt, and monopolise in new environments (Ramos et al., 2018). There are no morphological characteristics that would be used to distinguish different members of the B. tabaci species complex (Boykin et al., 2018). However, research has made known the occurrence of B. tabaci cryptic species complex which is morphologically indefinite but they do possess characteristic biological, physiological, genetic variations owing to which the prominence of B. tabaci has been changing initially from the biotypes (Costa et al., 1991), to races (De Barro et al., 2005), to genetic groups (Boykin et al., 2007) and species (Tay et al., 2013).

Globally, 46 cryptic species with up to 4 per cent genetic divergence have been identified under 11 genetic groups (Mugerwa et al., 2018; Lestari et al., 2021; Rehman et al., 2021), amongst Asia I, Asia II 1, Asia II 5, Asia II 7, Asia II 8, Asia II 11, Asia IV, China 3, China 7 and Middle East Asia Minor 1 (MEAM 1) cryptic species have all been reported from India. B. tabaci feeds solely on phloem sap (Dong et al., 2020), which is low in essential amino acids are provided by the obligatory primary endosymbiont, Portiera aleyrodidarum (Andreason et al., 2020). It harbours secondary endosymbionts that provide them with thermo tolerance, insecticide resistance, host fitness, pathogen defence, virus transmission and disruption of host physiology and reproduction (Shi et al., 2018; Lestari et al., 2021). Subsequently, determining endosymbiont associations is therefore crucial to understand their relationship with the B. tabaci populations. With the given climate change, increasing international transportation of agricultural products, and intensive pest control tactics, understanding the genetic diversity and distribution of B. tabaci cryptic species and their associated endosymbionts has become extremely crucial.

Therefore in the current study we conducted an interhost survey and discovered that Brinjal was the most infested by B. tabaci. As a result, an interlocation survey was conducted in the Brinjal crop. Subsequently, molecular markers such as Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSRs) were used to study genetic variability, mitochondrial cytochrome oxidase I (mtCOI) for characterizing the cryptic species of B. tabaci (Mugerwa et al., 2018) and 16S rRNA for the identification of distinct strains of endosymbionts.

Sample collection

A survey was conducted during 2020–2021 and the populations were collected from Brinjal fields of interlocation (Bihar-Kothia, Pusa, Dhrubgama, Mandai Dih, Mirapur, Madhurapur, Jhakra, Alipur Bihta, Faridpur, Charuipar and Dariapur; Telangana and Andhra Pradesh states as check to explore the diversity) and interhost at Dr. Rajendra Prasad Central Agricultural University, Pusa, Bihar (Table 1 and Fig. 1). The collected samples were preserved in 99.0 per cent ethanol and stored at -20^oC until further studies. Each sample from different locations (Brinjal) and hosts was identified for confirmation of B. tabaci using morphological keys (Calvert et al., 2001; Baig et al., 2015).

Table 1

Details of interhost and interlocation survey conducted during the year 2020–2021
Sl. No	Accession number	Host plant/Location name	Survey location	District	State	Date of survey	Latitude	Longitude	Altitude/m
1	MZ148550	Congress grass	Pusa	Samastipur	Bihar	05-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
2	MZ148551	Indian jujube	Pusa	Samastipur	Bihar	05-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
3	MZ148552	French bean	Pusa	Samastipur	Bihar	07-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
4	MZ148553	Tomato	Pusa	Samastipur	Bihar	07-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
5	MZ148554	Potato	Pusa	Samastipur	Bihar	07-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
6	MZ148555	White fig	Pusa	Samastipur	Bihar	12-11-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
7	MZ148556	Chinese hibiscus	Pusa	Samastipur	Bihar	12-11-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
8	MZ148557	Common jasmine	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
9	MZ148558	Black nightshade	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
10	MZ148559	Barberton daisy	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
11	MZ148560	Mexican marigold	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
12	MZ148561	Cluster fig	Pusa	Samastipur	Bihar	12-11-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
13	MZ148562	Pointed gourd	Pusa	Samastipur	Bihar	19-02-2021	25°59'05.4"N	85°40'50.5"E	53.04 m
14	MZ148563	Cucumber	Pusa	Samastipur	Bihar	19-02-2021	25°59'05.4"N	85°40'50.5"E	53.04 m
15	MZ148564	Dolichos bean	Pusa	Samastipur	Bihar	19-02-2021	25°59'05.4"N	85°40'50.5"E	53.04 m
16	MZ148565	Okra	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
17	MZ148566	Brinjal	Kothia	Samastipur	Bihar	01-10-2020	25°51'17.7"N	85°37'03.5"E	20.42 m
18	MZ148567	Brinjal	Madhurapur	Samastipur	Bihar	05-10-2020	25°58'37.6"N	85°44'09.1"E	26.74 m
19	MZ148568	Brinjal	Dhrubgama	Samastipur	Bihar	05-10-2020	25°58'11.4"N	85°47'41.1"E	44.63 m
20	MZ148569	Brinjal	Pusa	Samastipur	Bihar	01-10-2020	25°59'05.4"N	85°40'50.5"E	53.04 m
21	MZ148570	Brinjal	Mirapur	Muzaffarpur	Bihar	04-10-2020	26°01'42.8"N	85°33'57.6"E	54.16 m
22	MZ148571	Brinjal	Mandai Dih	Vaishali	Bihar	01-10-2020	25°50'16.2"N	85°30'04.6"E	51.53 m
23	MZ148572	Brinjal	Jhakra	East Champaran	Bihar	04-10-2020	26°33'27.9"N	84°57'51.6"E	64.09 m
24	MZ148573	Brinjal	Alipur Bihta	Patna	Bihar	03-01-2021	25°27'19.1"N	85°27'43.4"E	41.11 m
25	MZ148574	Brinjal	Faridpur	Sheikhpura	Bihar	02-10-2020	25°10'58.5"N	85°46'32.1"E	45.11 m
26	MZ148575	Brinjal	Charuipar	Nalanda	Bihar	03-01-2021	25°17'10.1"N	85°27'30.6"E	54.41 m
27	MZ148576	Brinjal	Dariapur	Lakhisarai	Bihar	02-10-2020	25°13'01.3"N	86°04'19.7"E	26.42 m
28	MZ148577	Brinjal	Tadikonda	Mahbubnagar	Telangana	23-11-2020	16°38'59.9"N	78°00'36.7"E	425.7 m
29	MZ148578	Brinjal	Bapatla	Guntur	Andhra Pradesh	23-01-2021	15°56'03.7"N	80°29'42.2"E	121.21 m

Genomic Dna Extraction And Pcr Protocol

DNA was isolated from single adult B. tabaci using modified CTAB (Cetyl Trimethyl Ammonium Bromide) procedure of Cubero et al. (1999). The intact genomic DNA was visualised in 0.8 per cent agarose gel and was diluted if requisite to obtain a workable solution of 100 ng/µl (Sambrook et al., 1989).

The RAPD, SSR, endosymbiont and mtCOI primers used in the study were given in the Supplementary 1. PCR was executed with a 20 µl reaction mixture comprising of 2 µl template DNA (100 ng), 1.5 µl of forward and reverse primers, 8 µl of Taq mixture and 8.5 µl of nuclease free water. The following conditions were used to run the thermo cycler: initial denaturation at 95^oC for 2.30 sec, 35 cycles of denaturation at 94^oC for 45 sec, annealing at time (Supplementary 1) for 30 sec and extension at 72^oC for 5 min followed by final extension of 72^oC for 10 min. PCR products were visualized in 2.2 per cent agarose gel stained with 5 µl ethidium bromide, which were then viewed using Syngene gel documentation system.

Amplicon Scoring And Data Analysis Of Rapd And Ssr Primers

Markers (11 RAPD and 9 SSR primers) (Supplementary 1) were used to amplify the genomic DNA isolated from the adults of B. tabaci populations. The amplicons were visualized under UV light and catalogued using an Alpha image analyzer (Bio-Rad). A DNA ladder of 100 bp was run alongside the samples in each gel to aid in the identification of polymorphic bands, and documented RAPD and SSR profiles were scored as one and zero for the presence and absence of bands, respectively to identify the variability among B. tabaci populations. DARwin 6 software (Perrier et al., 2003) was used to analyze the scored marker data matrix, and genetic dissimilarity was calculated using the Jaccard similarity coefficient (Jaccard, 1908). The Unweighted Pair Group Method Analysis (UPGMA) was used to create a dendrogram after clustering the dissimilarity coefficients (Sneath and Sokal, 1973). Consequently, for each RAPD and SSR marker, total amplified bands, number of polymorphic bands (PB), percentage of polymorphic bands (PPB), Polymorphism Information Content (PIC) (Anderson et al., 1993); Resolving Power (RP) (Prevost and Wilkinson, 1999); Effective multiplex ratio (EMR) (Kumar et al., 2009); Marker Index (MI) (Powell et al., 1996) were calculated to determine the informativeness of markers.

Partial mtCOI sequence alignment and phylogenetic analysis

The genetic variations between interhost and interlocation populations of B. tabaci were analyzed using RAPD and SSR primers (1st tier). All the populations found divergent in the 1st tier were promoted to the 2nd tier (universal primer: mtCOI analysis). The PCR products were purified using a QIA quick PCR purification kit (Qiagen Inc. Valencia, CA and USA) and then directly sequenced by ABI 3130XL genetic analyzer at Eurofins Genomics Bangalore, Karnataka. To obtain the final sequences, the sequences (850 bp) were aligned in MEGA X and terminally trimmed to 657 bp. Following BLASTn analysis, the consensus sequences were submitted to GenBank (Altschul et al., 1997). The accession numbers for both interhost and interlocation were given in the Table 1.

Each B. tabaci sequence was aligned in MEGA X using Clustal W to look for duplicates, gaps, indels and pseudogenes (Tamura et al., 2011). The final dataset included 657 nucleotide sequences were analysed based on mtCOI gene. To select the best model for phylogenetic tree construction, maximum likelihood fits of 24 different nucleotide substitution were performed (Felsenstein, 1981). Bayesian phylogenetic tree inference was constructed (Kumar et al., 2018). Estimates of evolutionary divergence between sequences and maximum composite likelihood of nucleotide substitution pattern were computed. The nucleotide sequences were aligned using Clustal omega online software to look for conserved, semi-conserved, and fully conserved regions (Sievers et al., 2011). The nucleotide sequences were converted into amino acid sequences using ExPASy translate (Gasteiger et al., 2003), which were then aligned using Clustal Omega software to detect conserved amino acids. Neutrality tests such as Fisher's (Fisher, 1935) and Tajima's (Tajima, 1989) were used to validate sequences that did not fit the neutral theory.

Genetic diversity among 29 B. tabaci population using RAPD primers (1st tier)

Rapd Marker Polymorphism

The genetic variability of 29 populations of B. tabaci was explored using 11 RAPD primers, which were amplified with polymorphism percentage range of 90.00 to 100.00, yielding a total of 110 bands which varied from seven (OPA-15) to 14 (F12). The mean number of total bands and polymorphic bands per primer were 10.00 and 9.09 respectively. Higher PIC (0.81) in F2 and higher EMR, MI and RP (14.00, 10.42, 8.07) in F12 primer revealed greater informativeness and low EMR, RP (7.00, 2.82) in OPA-15; low PIC, MI (0.49, 4.45) in OPA-5 revealed its lower informativeness in checking variability of B. tabaci populations (Table 2). Among all the primers OPA 11 was found to be potential genetic marker which had one monomorphic band with 90.00 per cent polymorphism, because if no monomorphic bands were found then that population would be deliberated as a distinct species (Maurya et al., 2020). Quieroz et al. (2017) recorded more than 70 per cent polymorphism in OPA-05 (70.0), OPA-10 (77.9), OPA-11 (73.8), OPA-13 (77.3), OPA-15 (70.8) and these observations are quite consistent with our findings. Likewise, Hameed et al., 2012; Hopkinson et al., 2020 have used RAPD primers for identifying polymorphism among B. tabaci populations.

Table 2

Details on amplification of RAPD region in genomic DNA of 29 *B. tabaci* populations
Sl. No	Primers	T(^oC)	TB	PB	MB	PPB (%)	PIC	EMR	MI	RP
1	OPA-02	37.3	12	12	00	100.00	0.63	12.0	7.61	5.86
2	OPA-04	37.3	09	09	00	100.00	0.67	09.0	6.06	5.79
3	OPA-05	37.3	09	09	00	100.00	0.49	09.0	4.45	4.62
4	OPA-10	37.3	09	09	00	100.00	0.50	09.0	4.52	4.90
5	OPA-11	37.3	10	09	01	90.00	0.61	08.1	4.93	3.69
6	OPA-13	37.3	11	11	00	100.00	0.72	11.0	7.87	5.02
7	OPA-15	37.3	07	07	00	100.00	0.69	07.0	4.89	2.82
8	OPA-20	37.3	09	09	00	100.00	0.61	09.0	5.41	5.59
9	OPR-07	37.3	09	09	00	100.00	0.81	09.0	7.28	4.61
10	F2	37.3	11	11	00	100.00	0.82	11.0	9.02	4.75
11	F12	37.3	14	14	00	100.00	0.74	14.0	10.42	8.07
	Total	-	110	109	-	-	-	-	-	-
	Mean	-	10.0	9.09	-	-	0.663	108.1	6.586	5.06
Note: T (^oC): Annealing temperature, TB: Total Band, PB: Polymorphic Band, MB: Monomorphic Band, PPB (%): Percentage of Polymorphic Band, PIC: Polymorphism Information Content, EMR: Effective Multiplex Ratio, MI: Marker Index, RP: Resolving Power

Upgma Clustering And Dendrogram

An UPGMA dendrogram based on dissimilarity coefficient was constructed for the 29 populations studied. Ten clusters were noticeable in which interlocation: Madhurapur and Mirapur are most closely related sharing a dissimilarity percentage of 30.0 (cluster I) and Bapatla was found to share higher dissimilarity of 46.0 percentage (cluster IV). Whereas in interhost: okra and dolichos bean are most closely related sharing a dissimilarity percentage of 36.0 (cluster VI) and common Jasmine was found to share higher dissimilarity of 57.0 percentage (cluster X). The cluster I contained interlocation viz., Madhurapur, Mandai Dih, Mirapur, Dhrubgama, Jhakra and Kothia belonging to Northern Bihar (Fig. 2). Similarly, cluster II enclosed Charuipar, Faridpur and Dariapur belonging to Southern Bihar. This result demonstrates that populations were differentiated based on their geographical locations. Potato and tomato (cluster V) belonging to the same family, Solanaceae shared a less dissimilarity percentage of 37.0. The interlocation populations were less diverged than the interhost populations because they were mostly collected from the same host, Brinjal. Similar pattern of differentiation studies were conducted using RAPD primers in Bemisia tabaci, Myzus persicae, Helicoverpa armigera, Leucinodes orbonalis (De Barro & Driver, 1997; Zitoudi et al., 2001; Lopes et al., 2017; Murali et al., 2021).

Genetic diversity among 29 B. tabaci population using SSR primers (1st tier)

Ssr Marker Polymorphism

The genetic variability of 29 populations of B. tabaci was studied using nine SSR primers which amplified with 100 per cent polymorphism, yielding a total of 60 bands which varied from four (Btls1-6) to nine bands (Btls-2, Bta1). The mean number of total bands and polymorphic bands per primer were 6.66. Higher PIC, EMR, MI and RP (0.878, 9.0, 7.902, 5. 79 respectively) in Btls1-2 revealed the greater informativeness and low PIC (0.664) in Bta 1, EMR and MI (4.00; 3.196) in Btls 1–6 and RP (1.238) in Bta 11 revealed less informativeness in checking variability of B. tabaci populations (Table 3). Similarly, De Barro et al. (2003); Simón et al. (2007); Gauthier et al. (2008); Ben Abdelkrim et al. (2017) used these primers for identifying variability among B. tabaci populations. Contrarily, Valle et al. (2012) observed lowest per cent polymorphism in Bta11 primer, which showed 100 per cent in our study.

Table 3

Details on amplification of SSR region in genomic DNA of 29 *B. tabaci* populations
Sl. No	Primers	T(^oC)	TB	PB	MB	PPB (%)	PIC	EMR	MI	RP
1	Btls1-2	50.0	09	09	00	100.00	0.88	09	7.90	5.79
2	Btls1-6	50.0	04	04	00	100.00	0.79	04	3.19	1.38
3	Bta1	51.0	09	09	00	100.00	0.66	09	5.98	4.38
4	Bta4	51.0	06	06	00	100.00	0.69	06	4.19	3.45
5	Bta11	50.0	05	05	00	100.00	0.82	05	4.08	1.24
6	Bta12	51.0	06	06	00	100.00	0.68	06	4.11	3.73
7	BEM 12	53.0	06	06	00	100.00	0.84	06	5.03	2.27
8	BEM 23	53.0	07	07	00	100.00	0.84	07	5.91	1.58
9	BEM 37	51.0	08	08	00	100.00	0.86	08	6.86	4.41
	Total	-	60	60	-	-	-	-	-	-
	Mean	-	6.66	6.66	-	-	0.79	6.66	5.25	3.13
Note: T (^oC): Annealing temperature, TB: Total Band, PB: Polymorphic Band, MB: Monomorphic Band, PPB (%): Percentage of Polymorphic Band, PIC: Polymorphism Information Content, EMR: Effective Multiplex Ratio, MI: Marker Index, RP: Resolving Power

Upgma Clustering And Dendrogram

An UPGMA dendrogram was constructed based on the dissimilarity coefficient of 29 populations studied. Ten clusters were noticeable in which interlocation: Alipur Bihta, Dhrubgama and Mirapur (cluster V) are the most closely related sharing a dissimilarity percentage of 30.0 and Pusa was found to share higher dissimilarity of 42.0 percentage (cluster VIII). Whereas in interhost: cucumber and potato (cluster I) are the most closely related sharing a dissimilarity percentage of 38.0 and common jasmine was the most divergent sharing higher dissimilarity percentage of 56.0 (Fig. 3). According to Fakrudin et al., 2004, this increased genetic variability might aid species in evolving and adapting to new environment more quickly. The lower dissimilarity observed between B. tabaci populations (interlocation) can be enlightened by the certainty that they were all collected from the same host (Brinjal), whereas the higher dissimilarity observed among B. tabaci populations (interhost) could be due to their collection from different hosts. Similar pattern of differentiation studies were conducted using SSR primers in case of Bemisia tabaci, Helicoverpa armigera, Sesamia inferens (Valle et al., 2012; Reetha and Mohan, 2018; Reddy et al., 2022).

Genetic diversity among B. tabaci population using mtCOI (2nd tier)

All the individual B. tabaci populations collected from interlocation and interhost of Bihar (Check- Bapatla and Tadikonda) produced an amplicon of 850 bp mtCOI region in PCR using C1-J-2195 and TL2-N-3014 primers (Supplementary 1). The final sequences were terminally trimmed to 657 bp. The phylogenetic tree was built based on maximum composite likelihood approach and Hasegawa- Kishino-Yano with Gamma distribution model using 29 mtCOI sequences of B. tabaci populations from Bihar, 44 reference sequences and B. atriplex, B. afer and T. vaparorium as the outgroups (Fig. 4) (Supplementary 2). It was noted that four cryptic species (out of 44 cryptic species) were found to cluster with 29 B. tabaci populations (Fig. 5). The sequences of B. tabaci from Pusa, Bapatla, Dariapur, Charuipar, Faridpur, Mirapur, Jhakra, Mandai Dih, okra, dolichos bean, pointed gourd, tomato, Dhrubgama, Alipur Bihta, Madhurapur, potato, Mexican marigold, cucumber, Kothia, French bean, Indian jujube, congress grass, white fig, cluster fig, common jasmine clustered with the cryptic species Asia I. The Asia I cryptic species was the most common, accounting for 25 of the 29 (86.20 per cent) populations sequenced, implying that Asia I has a greater potential to expand and adapt in Bihar. Similarly, Roopa et al. (2015) sequenced the 71 samples of B. tabaci, and found Asia I cryptic species to be the most predominant accounting for 44 out of the 71 (61.97 per cent), which is quite similar to our findings. Whereas, white fig and cluster fig clustered together, belong to the same genus (Ficus) and family (Moraceae). The remaining four populations, such as barberton daisy, black nightshade, Chinese hibiscus and Tadikonda clustered with the China-3, Asia II 1, Asia II 7 and Asia II 7 cryptic species respectively (Fig. 4). Among these four cryptic species, Asia II 7 and China 3 were reported for the first time in Bihar region in comparision with the previous data (Misra and Lambda, 1929; Chowda-Reddy et al., 2012; Roopa et al., 2015; Rangaswamy et al., 2019). Similarly, Roopa et al. (2015) found Asia II 7 cryptic species on hibiscus host, which supports our findings. In contrast with our study Chowda-Reddy et al. (2012) reported that Asia II 7 cryptic species was found mostly in Southern and Western India. The China 3, which is native to China and the island of Hainan (Hu et al., 2011), has also been found in the Birbhum region of West Bengal, India (Ellango et al., 2015). Climate change, overall international transportation of agricultural products, intensive pest control tactics and also with additional help from their unique bacterial endosymbionts could be the most likely causes of the emergence of these new cryptic species.

Multiple Sequence Alignment

Multiple alignment of nucleotide sequences of 29 B. tabaci samples revealed 305 fully conserved residues (Supplementary 5) and 105 Single Nucleotide Polymorphisms (SNPs). Likewise, in multiple alignment of amino acid sequences 75 fully conserved residues, 46 conserved residues and 23 semi conserved residues were obtained (Supplementary 6). This higher number of nucleotide similarity indicates that they evolved from a common origin and also SNPs can be used for differentiating the haplotypes. Similarly, Wosula et al. (2017); Kunz et al. (2019) found 7453 SNPs and 125 conserved amino acid residues with amplification of 657 bp region of mtCOI gene of B. tabaci.

Pair Wise Genetic Distance

The pair wise genetic distance of B. tabaci populations ranged from 0.00 to 0.47 (Supplementary 4) with an over mean distance of 0.08. Similarly, Dinsdale et al. (2010) reported zero to 34 per cent genetic distance among 198 B. tabaci species.

Patterns of nucleotide substitution in B. tabaci mtCOI gene

An important characteristic of nucleic acid is their nucleotide composition. From the analysis it was found that the average largest number of nucleotide base was thiamine (T) (43.37 per cent). On the other hand, lowest number of nucleotide base was guanine (G) (19.00 per cent). Similarly, Roopa et al. (2015) observed B. tabaci nucleotide frequencies as highest in thiamine (43.10 per cent) and lowest in guanine (13.22 per cent). The base composition of the mtCOI gene fragment was biased towards Adenine (A) and Thymine (T) with an overall 67.54 per cent. A biased transition/transversion ratio was a universal feature of nucleotide diversity (Lynch, 2008).

Neutrality Tests

A negative Tajima's D = -0.726310 (Table 4) indicated excess of low frequency polymorphisms and in Fisher’s exact test, common jasmine, congress grass, Chinese hibiscus, black nightshade, cucumber, Kothia, Madhurapur, Dhrubgama, Alipur Bihta, white fig, Chinese hibiscus and Tadikonda populations having P value less than 0.05 (Supplementary 7) showed deviation from other populations and thus both tests supported the neutral theory of evolution. These findings were supported by Tocko-Marabena et al. (2017) who concluded that B. tabaci SSA1 haplotype SG1 data was found to be significant with Tajima's D (-2.45317).

Table 4

Results from Tajima's Neutrality Test
m	S	p_s	Θ	Π	D
36	402	0.611872	0.147554	0.119309	-0.726310

Note: m = number of sequences; S = Number of segregating sites; p_s = S/n; Θ = p_s/a₁; π = nucleotide diversity; D is Tajima test statistic

Association of Bemisia tabaci populations with bacterial endosymbionts

Bacterial community of B. tabaci populations (interlocation and interhost) were identified by amplification of 16S rRNA of specific bacteria (Portiera sp, Wolbachia sp, Arsenophonus sp, Rickettsia sp, Cardinium sp and Hamiltonella sp) using bacteria specific primers (Supplementary 1) (Chiel et al., 2007; Gueguen et al., 2010). The primary endosymbiont, Portiera sp detected in all samples (100.0 per cent), supports the fact that it provides nutrients and is required for whitefly survival (Jiang et al., 2012; Skaljac et al., 2017). In total, from the analysis of 16S rRNA gene of 29 B. tabaci populations we were able to detect 96.7 per cent of Arsenophonus sp followed by Wolbachia sp (93.3 per cent), Rickettsia sp (93.3 per cent), Hamiltonella sp (70.0 per cent) and Cardinium sp (60.0 per cent). It is possible that B. tabaci requirements vary depending on its environment and hosts, resulting in different combinations of secondary endosymbionts (Table 5). All five secondary endosymbionts were detected in Asia I and Asia II 7 cryptic species (Asia I: congress grass, potato, Mexican marigold, cucumber, dolichos bean, Khotia, Mirapur, Dhrubgama, Dariapur and Asia II 7: Chinese hibiscus). However, the incidence of secondary endosymbionts differed within the same cryptic species, Asia I. Asia II 1 of black nightshade was found to associated with Arsenophonus sp, Rickettsia sp, and Hamiltonella sp. Strikingly, Hamiltonella sp was found to be absent in China 3 (barberton daisy), Asia II 7 (Tadikonda) and Asia I (French bean, tomato, white fig, cluster fig, pointed gourd, okra, mustard). Arsenophonus sp and Rickettsia sp associated with all the Asia I populations except Alipur Bihta and cluster fig and French bean respectively. These observations further support the fact that endosymbionts are unevenly distributed within the B. tabaci species complex as reported in previous studies (Gueguen et al., 2010; Lestari et al., 2021). However, because of competition for space and resources in the host, this coexistence can result in uneven distribution of these endosymbionts in B. tabaci populations. According to Gottlieb et al. (2008), bacteria combinations such as Wolbachia sp and Cardinium sp, Rickettsia sp and Cardinium sp, Hamiltonella sp and Arsenophonus sp do not coexist in the B. tabaci population. On the contrary, we discovered them coexisting in the majority of the populations. This suggests that new combinations of secondary endosymbiotic bacteria may emerge under different environmental conditions and host plants.

Table 5

Diversity of bacterial endosymbionts associated with 29 *Bemisia tabaci* populations
Sl. No	Host plant /Location name	Genetic group	Port	Wsp	Rb	Card	Ars	Ham	Total amplified	Percentage (%)
1	Congress grass	Asia I							6	100.00
2	Indian jujube	Asia I							5	83.33
3	French bean	Asia I							4	66.67
4	Tomato	Asia I							5	83.33
5	Potato	Asia I							6	100.00
6	White fig	Asia I							5	83.33
7	Common jasmine	Asia I							5	83.33
8	Mexican marigold	Asia I							6	100.00
9	Cluster fig	Asia I							3	50.00
10	Pointed gourd	Asia I							4	66.67
11	Cucumber	Asia I							6	100.00
12	Dolichos bean	Asia I							6	100.00
13	Okra	Asia I							5	83.33
14	Khotia	Asia I							6	100.00
15	Mandai Dih	Asia I							5	83.33
16	Jhakra	Asia I							5	83.33
17	Mirapur	Asia I							6	100.00
18	Madhurapur	Asia I							5	83.33
19	Dhrubgama	Asia I							6	100.00
20	Alipur Bihta	Asia I							4	66.67
21	Faridpur	Asia I							5	83.33
22	Charuipar	Asia I							5	83.33
23	Dariapur	Asia I							6	100.00
24	Pusa	Asia I							5	83.33
25	Bapatla	Asia I							5	83.33
26	Chinese hibiscus	Asia II 7							6	100.00
27	Tadikonda	Asia II 7							5	83.33
28	Black nightshade	Asia II 1							4	66.67
29	Barberton daisy	China 3							5	83.33
	Total amplified		29	27	27	17	28	21
	Percentage		100.0	93.1	93.1	58.6	96.5	72.4

Note: Empty and filled cells indicates the absence and presence of bacterial endosymbionts respectively in B. tabaci populations. Port- Portiera, Wsp-Wolbachia, Rb-Rickettsia, Card- Cardinium Ars- Arsenophonus Ham- Hamiltonella

Besides, during surveys of interlocation, farmers stated that the number of sprays they go for whitefly control in brinjal fields throughout crop period were 45 to 50, which gives an interesting clue that endosymbiont-mediated insecticide resistance could have developed in interlocation populations (Wang et al., 2010; Pan et al., 2012). Although endosymbionts are expected to be expensive for hosts in terms of resource allocation, many insects host these bacteria, implying that their presence may have functional benefits.

The findings confirmed the presence of four cryptic species of B. tabaci in Bihar (Asia I, Asia II 1, Asia II 7, and China 3). In particular, Asia II 7 and China 3 were reported for the first time in Bihar region. Association of different secondary endosymbionts among all the B. tabaci populations were not significantly linked to the geographical location or host plants. Moreover, the Asia I cryptic species and Arsenophonus sp endosymbiont dominated in all interhost and interlocation populations. Overall, this study enhance in characterizing different B. tabaci cryptic species and their association with endosymbionts for assessing ongoing changes in genetic diversity, evolutionary history, and potential spread which allow for effective pest management with avoiding the use of insecticides usage and reducing environment pressure.

Ethics approval and consent to participate

Not applicable.

Consent for Publication

All authors have consented for submission.

Availability of data and materials

All data generated or analysed during this study are included in this published article (and its supplementary information files).

Competing interests

The authors declare no conflict of interests.

Funding

The authors declare that no funds, grants, or other supports were received.

Author’s contributions

Gummudala Yashaswini: Planned and executed the research

Somala Karthik: Writing assistance, Proof reading of the article

Md. Abbas Ahmad: Proof reading of the article

Udit Kumar: Proof reading of the article

B Deepak Reddy: Data Interpretation and analysis

A Keerthana: Sample collection and DNA extraction

Gurram Mallikarjun: Sample collection and DNA extraction

S Abinaya: Sample collection and DNA extraction

M.S. Sai Reddy- Supervised and guided the researcher

Acknowledgements

Authors are grateful to the Departments of Entomology, Genetics & Plant Breeding and Biotechnology of Dr. Rajendra Prasad Central Agricultural University for their unwavering support throughout the research.

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First report of Asia II 7 and China 3 cryptic species of Bemisia tabaci (Gennadius) and identification of its associated endosymbionts in Bihar

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Materials And Methods

Sample collection

Genomic Dna Extraction And Pcr Protocol

Amplicon Scoring And Data Analysis Of Rapd And Ssr Primers

Results And Discussion

Upgma Clustering And Dendrogram

Upgma Clustering And Dendrogram

Multiple Sequence Alignment

Pair Wise Genetic Distance

Neutrality Tests

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