Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a devastating phloem-feeding pest of many crops (Basu et al. 2019) that transmits several plant viruses (Gilbertson et al. 2015, Pinheiro-Lima et al. 2020). Based on the mitochondrial cytochrome oxidase 1 (mtCO1) sequence, 39–42 cryptic (putative) species have been described within the species (Kanakala and Ghanim 2019). In Iran, it has been showed that the putative species of Africa/Middle East/Asia Minor group and the Middle East/Asia Minor 1 subgroup is the dominant species (Shahbazi et al. 2014). However, there is no information on the putative species within Khuzestan province of southwestern Iran. The objective of this study was to determine the putative species among B. tabaci populations from the region. Moreover, the mtCOI sequences of Iranian specimens were subjected to DNA polymorphism and phylogenetic analysis.

Materials and Methods

Bemisia tabaci adults were collected from tobacco, tomato, cucumber, and cowpea fields in six counties of Khuzestan province during 2019–2020 (Table 1). Specimens were preserved individually in microtubes containing 70% alcohol and stored at -20°C. At each sampling, 20–30 whiteflies representing 10 distinct populations were collected. Morphologic characters of pupae from each population were used to confirm species identity (Hodges and Evans 2005).

Table 1 Bemisia tabaci specimens collected from different regions in Khuzestan province, southwestern Iran.

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Total DNA was extracted from the specimens using the Animal DNA Isolation Kit (Denazist, Iran) and stored at -20°C. Polymerase chain reaction (PCR) amplification was performed according to Masood et al. (2017). PCR products were then sequenced at BIONEER Corp. (Daejeon, South Korea).

Sequences were primarily checked using Chromas (ver. 2.6.6) (Technelysium, South Brisbane, Australia) software, and a complete sequence was obtained by assembling the reads of 3′ and 5′ termini using SeqMan Pro software (DNASTAR Lasergene, ver. 8). Nuclear mitochondrial sequences (NUMTs) and PCR-derived pseudogenes were excluded according to Kunz et al. (2019). BLASTn was performed, and the confirmed sequences were then submitted to GenBank under the accession numbers presented in Table 2. DNA polymorphism and haplotype analysis were performed using DnaSP software (ver. 6). Haplotype diversity and sampling variance were conducted according to Nei (1987). Nucleotide diversity (π), mean nucleotide differences per site, sampling variance, and standard deviation of the sequences were determined. Theta (h) per site from π, S, and g and mean the number of nucleotide differences (k) were also calculated. Diallelic model was applied to analyze insertion/deletion (InDel) polymorphism among the sequences, and possible InDel haplotypes were investigated.

Table 2 Mitochondrial cytochrome oxidase 1 sequences of B. tabaci specimens from different provinces in Iran.

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Fifty-six sequences of mtCOI, including 4 sequences obtained here and 52 sequences from GenBank, were used to perform a phylogenetic analysis. The sequences were aligned by CLC Main Workbench software (ver. 7.6.2), and the maximum likelihood algorithm using the general time reversible model test with 100 bootstrap replicates was used to construct nucleotide-based phylogenetic trees. A corresponding sequence from Trialeurodes abutilonea Haldeman was used as an outgroup.

Results

The examined pupal specimens from the populations were identified as B. tabaci. The partial mtCOI gene (780 bp) was amplified using the specific primers, and four haplotypes (H) were identified among the sequences (Table 3). The sequences from Khuzestan province exhibited the least values of polymorphism parameters including the number of polymorphic sites (S), haplotype diversity (HD), Pi, Theta (per site) from Eta (Theta_eta), Theta (per site) from S (Theta-W), and K. In contrast, the highest values of S, HD, Pi, Theta_eta, Theta-W, and K were observed in the population from Kerman, Hormozgan, Khorasan, and Yazd provinces. The sequences from Fars province were placed in the second rank relative to the values. No InDel polymorphic event was observed among the sequences.

Table 3 DNA polymorphism parameters of B. tabaci populations examined in this study using DNASP software (ver. 6).

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All sequences obtained herein were highly identical (99.88%) to a sample from Pakistan (LN835385). The second highest identity (99.84%) was found with an isolate of MEAM1-subcladeB from the UAE (DQ133382). Additionally, a 99.69% identity was observed against three Iraqi isolates (HM070413, KX679575, and KX679577) and one isolate from the Kingdom of Saudi Arabia (GU086344). Two Syrian isolates (KP342512 and AB473559) exhibited high levels of identity (99.74 and 99.61%, respectively) to the sequences determined here. Isolates from Yemen and Kuwait (GU086343 and GU086346, respectively) showed a 98.96% identity to Khuzestan-originated sequences. A putative species MEAM1-subcladeB2 from Pakistan (GU977267) differed by 21 nucleotides from the sequences obtained herein. The lowest identity (95.06%) was observed against an isolate of the putative species MEAM2 from Uganda (KX570778). The sequences obtained here were identified as putative species MEAM1-subcladeB.

The phylogenetic tree showed the position of the putative species MEAM1-subcladeB (MW161484, MW161487, MW161488, and MW161490) from the present study (Fig. 1). These sequences, together with the isolate from the UAE (DQ133382), formed a separate cluster on the tree. The Iranian haplotypes reported by Shahbazi et al. (2014) formed two separate clusters on the tree. One cluster containing four isolates (JN559744, JN559748, JN559749, and JN559750) were clustered together with an Iraqi MEAM1-subcladeB2 species (KX679577). The other cluster consisted of two isolates (JN559751 and JN559753), which were placed close to a MEAM1-subcladeB2 species from Syria (AB473559 and KP342512). Another Iranian haplotype reported by Shoorcheh et al. (2008) (EU547771) was clustered with a MEAM1-subcladeB2 species from Pakistan (GU977267). Similarly, one unpublished Iranian specimen (MT038431) was placed close to the MEAM1-subcladeB2 species from Pakistan (GU977267) (Fig. 1).

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Multiple alignments of the sequences showed 12 nucleotide positions in which genetic variation had occurred. The sequences from Fars province had seven nucleotide sites (positions: 139, 193, 242, 468, 493, 624, and 643) with genetic variation. The sequences from Khuzestan province demonstrated three variable nucleotide sites at positions 58, 548, and 606. One sequence from Khorasan province (EU547770) showed the highest number (four) of nucleotide positions (58, 369, 548, 606, and 692) with genetic variation, whereas another sequence from the same province (MT038431) exhibited no variation. Additionally, a specimen from Yazd province (EU547771) showed only one nucleotide position (692) in which genetic variation was observed. The remaining specimens showed no variation throughout the sequence.

Discussion

Here, we report for the first time the mtCOI sequences of B. tabaci populations from Khuzestan province, southwestern Iran. We demonstrated that these sequences belong to the putative species MEAM1-subcladeB. Additionally, the sequence analysis of Iranian specimens has led to the determination of their subclade (B and B2), which had not been previously reported.

The Africa/Middle East–Asia Minor genetic group was found to be the main putative species in Iran that also has been reported from neighboring countries including Iraq (Kareem et al. 2020) and Pakistan (Masood et al. 2017). The MEAM1 group that contains two subclades (B and B2) has been observed in tropical and subtropical regions globally. The species have been considered invasive and cosmopolitan (De Barro et al. 2011). It appears that MEAM1-subcladeB and MEAM1-subcladeB2 are the dominant putative species in Iran. The MEAM1-subcladeB presented here was identical to the previous sequence (EU547770) reported from Khorasan province (Shoorcheh et al. 2008). It had shown a genetic variation among the sequences determined by Shoorcheh et al. (2008) resulting in formation of a separate branch on the phylogenetic tree (Fig. 1). Accordingly, this sequence was the only Iranian isolate with a close phylogenetic relationship to the sequences obtained here (Fig. 1). Although Fars and Khuzestan specimens showed no diversity in their putative species (MEAM1-subcladeB2 and -subcladeB, respectively), no location-dependent distribution of the putative species was observed. Accordingly, a single putative species (MEAM1-subcladeB2) has been reported from different provinces (Yazd and Khorasan) (Shoorcheh et al. 2008). This phenomenon also has been shown in other studies (De Barro et al. 2011). More specimens are required to better conceptualize the distribution of the putative species in the country.

DNA polymorphism analysis showed the highest rate of polymorphism among the sequences reported by Shoorcheh et al. (2008) (Table 2). This might be because of a relatively wider sampling span covering four provinces. In contrast, the sequences from a single province showed a relatively low diversity (Table 2). Furthermore, no host-dependent genetic diversity was found among the specimens collected from Khuzestan as B. tabaci individuals on different hosts shared identical nucleotide sequences. These results were consistent with the results of previous studies (Kareem et al. 2020, Masood et al. 2017, Shahbazi et al. 2014, Shoorcheh et al. 2008).

In Iran, control of B. tabaci is highly dependent on chemical practices; however, resistance has developed to at least six groups of pesticides (Basij et al. 2017). The alternative control practice, biocontrol, also has been challenged by the symbiont-mediated immune responses of B. tabaci individuals to their parasitoid (Mahadav et al. 2008). Considering the presence of subcladeB2 individuals, and possibly its symbionts, this might lead to the development of resistance to chemical compounds and biocontrol agents. Further experiments for detection of the symbionts are required to estimate the risk of pesticide/parasitoid-resistance, virus transmission, and insect fitness.