Insecticidal Resistance Monitoring in mitotypes of Bemisia tabaci in South Punjab region of Pakistan

doi:10.21203/rs.3.rs-3246143/v1

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Insecticidal Resistance Monitoring in mitotypes of Bemisia tabaci in South Punjab region of Pakistan

https://doi.org/10.21203/rs.3.rs-3246143/v1

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Whitefly (Bemisia tabaci Gennadius) (Hemiptera: Aleyrodidae) is a serious pest of several summer crops in hot and dry climates with diverse cryptic species complex worldwide. Among 7 major clades of B. tabaci; Asia-II is predominant in the cotton zone of Punjab-Pakistan. Cotton is one of the favorite hosts of B. tabaci, where it feeds and spread different plant viruses. In Pakistan, the primary approach used to manage B. tabaci in the cotton environment is through the use of synthetic pesticides. A vital tool for the effective management of B. tabaci is the monitoring of insecticidal resistance. Different cotton field strains were collected from the four major cotton growing districts of South-Punjab to assess their genetic variability and resistance levels compared with the laboratory susceptible population against most commonly used insecticides during the years 2020 and 2021. Leaf-dip bioassays were performed on 2nd instar nymphal (N2) stage; while adult bioassays were conducted on G1 stage on field collected populations. Genetic analysis of mtCOI revealed that all the populations were belonged to Asia II-1 clade. A mixture of three haplotypes of Asia II-I including haplotypes-2, haplotype-3 and haplotype-4 was identified from Jampur and Bahawalpur samples. However, haplotype-4 and haplotype-3 were identified from Multan population while Vehari samples comprised of haplotype-4 and haplotype-2.

Buprofezin (RR ranged 25.75–36.71), pyriproxyfen (RR ranged 7.98–12.78) and diafenthiuron (RR ranged 20.59–28.12) were least efficient products in adult bioassays and had moderate to low level of resistance. However, spirotetramat and flonicamid both were relatively effective against adults with low to very low resistance during both the years. Pyriproxyfen demonstrated least efficacy for reducing adult emergence followed by flonicamid and buprofezin with moderate to very low resistance ratio respectively. In contrast spirotetramat (RR ranged 2.20–4.01), was highly effective against nymphs followed by diafenthiuron (RR ranged 4.63–7.68), having very low resistance ratio. However an upward trend of resistance development was observed against all the five tested insecticides during both the monitoring years. Current study concluded that different mitotypes/haplotypes of B. tabaci have various level of insecticidal resistance to the old conventional insecticides. These results establish a regional baseline that can serve as a reference for future monitoring and management of B. tabaci resistance to the tested insecticides.

Bemisia tabaci

mitotypes

resistance monitoring

insecticides

Asia-II-I

The tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), is the most devastating pest of cotton, legumes, ornamentals crops and vegetables throughout the world (Oliveira et al. 2001). It is a polyphagous pest that has wide range of host plants including more than 700 species in 86 plant families (Greathead 1986). It feeds on phloem sap and excretes honeydew on leaves and fruit, causing direct damage to the plants. The sticky sugary honeydew serves as a breeding ground for black sooty mold, which discolors the leaves and impedes photosynthesis. Agricultural crops including different ornamentals, vegetables, and cotton lose a lot of value as a result of the stickiness and discoloration. Honeydew can create fiber stickiness in cotton, which can obstruct the spinning process in textile mills and lower the product's value (Hequet et. al., 2007). Whitefly was a one of the major reasons of low production of cotton during 2019-20 in Pakistan (GOP, 2020). B. tabaci is a vector for numerous key plant virus families, among them Begomovirus are most common viruses that whitefly transmits among all (Navas-Castillo et al. 2011). Cotton leaf curl virus (CLCV) is major factor in lowering the cotton yield which accounts 20–25% production loss annually in Pakistan (Traboulsi 1994).

B. tabaci has vast genetic diversity with 34 morphologically different species, which led to the development of its mitotype complex (Brown, et. al., 1995; Perring, 2003; De Barro, et. al., 2005), now currently recognized as a complex of distinct cryptic species (Brown 2010; De Barro, et. al., 2011; Boykin, 2012). All types differ in terms of fitness cost, host preferences, insecticide resistance and ability to transmit virus (Valverde et al., 2004). B and Q types are economically important and are most devastating biotypes worldwide (Wang et al., 2010).

Previously, three cryptic groups of B. tabaci have been reported from Pakistan (Ahmed et al. 2011). The Asia I and Asia II-1 are the endemic cryptic species groups while MEAM-1(B haplotype) is exotic, originated from the Middle East-Mediterranean, North African region (Brown 2010). Currently, 6 cryptic species of B. tabaci have been identified including Asia I, Asia II-1, Asia II-5, Asia II-7, MEAM1 and a variant stated as ‘Pakistan’ (Ashfaq et al. 2014). This variant “Pakistan” was latterly identified and referred to as Asia II-8, rather than of the ‘Pakistan’ variant (Masood, et. al., 2017).

Over-dependence on insecticides in the management of insect pests led towards the development of resistance and resurgence in the field pests (Byrne and Devonshire 1993; Palumbo et al., 2003, Nauen and Denholm 2005). Subsequently, B. tabaci has been developed high level of resistance against 40 active ingredients belongs to different insecticide classes including organophosphates, carbamates and pyrethroids in Pakistan (Cahill et al. 1995; Ahmad et al. 2000, 2001, 2002, 2010; Roditakis et al. 2005). The species complex of B. tabaci is among top five arthropods with most reported cases of resistance development against the insecticides (Sparks & Nauen, 2015). Two species MEAM1 and MED are most important regarding quick insecticides resistance development studies worldwide (Roditakis et al., 2005; Fernandez et al., 2009; Castle et al., 2010; Li et al., 2012; Panini et al., 2017). However B. tabaci biotypes resistance studies got main attention in Indo-Malay and Eastern Palearctic, after the prevalence of MED species in China and India which caused huge production losses (Basit et al., 2011, Yuan et al., 2012; Basit et al., 2013; Naveen et al., 2017; Wang et al., 2017a, 2017b; Zheng et al., 2017).

The present study was designed to monitor the insecticidal resistance in the field populations of B. tabaci species complex prevalence in the cotton zone of the Southern Punjab-Pakistan. The aim of this study was to check the current status of different species complex of B. tabaci prevailing in South Punjab cotton zone and give the current status of insecticidal resistance among the field populations of B. tabaci cryptic species.

Insect Collection and Rearing

Whitefly populations were collected from four locations (Multan, Vehari, Bahawalpur and Rajanpur) in cotton growing region of South- Punjab during 2020 and 2021. The host plant was cotton and the locations were same in both years. To confirm the genetic diversity of Bemisia tabaci found in South Punjab we extended our collection to nearby all the districts (Khanewal, Lodhran, Rahim Yar Khan, Muzaffarghar, Dera Ghazi Khan) along-with the above mentioned resistance monitoring four main districts (Fig. 1). The adult whitefly collection was made in the early morning through battery operated and handmade aspirators and the infested leaves with whitefly nymphs were also collected from cotton fields. The collected whitefly samples were immediately shifted to the insect rearing laboratory, MNS-University of Agriculture, Multan.

Table-1 Whitefly collection sites and hosts details

Host	District	Location Address	Latitude	Longitude
Cotton	Rajanpur	Kotla Dewan, Jampur, Rajanpur-Punjab	29.6629918	70.6352471
Cotton	Multan	Marral Road, near Basti Jhok wala, Multan-Punjab	29.9402819	71.5250866
Cotton	Dera Ghazi Khan	Smena, Dera Ghazi Khan-Punjab	30.0238538	70.7511252
Cotton	Rajanpur	Wang, Kot Mithan, Rajanpur-Punjab	28.9608757	70.3381838
Cotton	Khanewal	Thata Sadiq abad, Chak No. 133/10-R, Khanewal-Punjab	29.977695	71.625291
Cotton	Rahim Yar Khan	Khanpur, R.Y. Khan-Punjab	28.6578249	70.6436912
Cotton	Muzaffargarh	Wan Patafi, Basira Qandrani, Punjab	30.0840834	71.0362035
Cotton	Lodhran	N-5, Chak No. 389/WB East Lodhran, Punjab	29.8176876	71.5473081
Cotton	Bahawalpur	Jamrani Kohna, Basti Malanhas Bahawalpur	29.3541451	71.5180957
Cotton	Vehari	Chak 58/WB Vehari, Punajb	30.1725167	72.2639717
Cotton	Multan	B Block, Near Central Library, MNS-UAM	30.146424	71.448639

Subsamples of ≈ 200 adults from each field-collected whitefly population were preserved in 90% ethanol and held at 20°C until used. Adult whitefly population and infested leaves were released in whitefly rearing cages on fifteen days old potted cotton seedlings. Standard laboratory conditions (28 ± 2°C: 65 ± 5% RH: photoperiod of 16h : 8h light:dark) for whitefly rearing were maintained in the growth chamber. Susceptible whitefly population Lab-MNSUAM was collected from Non-sprayed cotton field during 2018 in the university B-Block research area and maintained the culture without exposure of insecticide application during whole the experiment (Melamed-Madjar et al., 1984; Naveen et al., 2017).

DNA extraction protocol from single whitefly

Five adults from each field population were selected for genetic identification. Single whitefly was used for DNA extraction by using the method of Zhang et al. (1998). Individual whitefly was placed on filter paper (Ahlstrom-Munksjo Filtration LCC, USA) for several seconds until dry the excess ethanol. Then ground the individual whitefly in 600µl CTAB extraction buffer {1.4 M NaCl, 2% CTAB (hexadecyltrimethyl-ammonium bromide), 20 mM EDTA, 0.2% 2-mercaptoethanol, 100 mM Tris-HCl, pH 8.0, Distilled water (ddH₂o)} in Mini beads bead beater 96 (BioSpec Products, Fisher Scientific USA) by using 15–20 disruption beads (RPI Research Products International, USA) for 5 minutes and transferred the liquid in to new centrifuge tube. 1uL of 20 mg/mL Proteinase K (Thermo Scientific, Lithuania) was added and incubated at 55°C for 1 hour. This was incubated for 10–15 min at 65 C and mixing 3–4 times was done during the incubation. The tubes were kept at room temperature for two to three minutes then they were added with cold chloroform @ 600 µl. To avoid fumes of chloroform, this was done in the hood. The tubes were thoroughly mixwd by tilting up and down to make emulsion. Then it was centrifuge for three minutes at 12000 rpm in microcentrifuge machine. The 500 µl upper aqueous phase was transferred to a new 1.5 ml microcentrifuge tube and added 500 µl of cold isopropanol. It was then added of glycogen (Thermo Scientific, Lithuania) (2 µl) to enhance the precipitation of the DNA. The solution was thoroughly mixed and holds on ice 5–10 min. This was centrifuged for ten minutes at 12000 rpm to pellet the nucleic acid and pour off liquid supernatant. To wash the DNA pellet ethanol (70%) was added and for one min at 12000 rpm. Then the liquid was discarded. It was again centrifuged at 12000 rpm to bring down remaining ethanol which was removed with the help of pipette. Then the tubes were kept on ice for the better visualization of DNA pellet.

The DNA pellet was allowed to dry at room temperature for 5–10 minutes. Lastly, the pellet was re-suspended in 30 µl ddH2O by pipette up and down for few moments. This extract was stored at -20 C and used 2 µl of the extracts for PCR. The extracted DNA concentrations were measured through Nano Drop before PCR.

Amplification of mitochondrial Cytochrome Oxidase I gene (mtCOI)

The mtCOI amplicon having the size of ~ 800bp was amplified through PCR by using primers: Bt-shF = TGRTTTTTTGGTCATCCRGAAGT and Bt-shR = TTTACTGCACTTTCTGCC (Mugerwa’s primers). The reactions were conducted in 25 µl vol containing RedTaq 12.5 µl, Bt-shF 0.5 µl, Bt-shR 0.5 µl, ddH2O 9.5 µl and DNA of whitefly 2.0 µl. The PCR conditions for the mtCOI amplicon fragment were 35 cycles of 95°C for 2 min, 95°C for 15 s, and 47°C for 15 s, 72°C for 50 s with a final extension step of 2 min at 72°C. The expected size of the amplicons was ~ 800bp which was confirmed by agarose gel (1%) electrophoresis in 1x TAE buffer, pH 8.0, containing 1x Gel Red dye, at 100 V for 40 min.

Cloning and Colony PCR

The cloning was done in pGEM-T Easy plasmid vector (Promega, Madison,WI) after getting the desired PCR products.

Transformation and Plating protocol (Brown’s lab protocol)

The standard protocol of Brown’s lab was adopted for transformation and plating and picked 2 white colonies as far apart from each other as possible followed by the colony PCR. Following primers were used for colony PCR.

M13F (5'-TGTAAAACGACGGCCAGT)

M13R (5'-AGGAAACAGCTATGACCATG)

The amplified DNA was run on gel electrophoresis to evaluate the DNA quantity and quality and stored as -20C. After confirmation of desired DNA quality, the samples were sent for sequencing to Eton Biosciences (San Diego, United States).

Identification of Bemisi tabaci Mitotypes

By comparing each of the COI segments to a group of global reference sequences from B. tabaci that are similar to the corresponding COI fragment, the B. tabaci mitotypes were identified. Reference sequences for both COI fragments were obtained from the laboratory COI DNA sequence database (JK Brown, unpublished data), which contained sequences for adult B. tabaci field collections identified using the recognized taxonomic (morphological) characters, or they were downloaded from the NIH NCBI-GenBank database (Benson et al., 2012), representing the seven major phylogeographic clades of B. tabaci (Takahashi 1936; Russell 1948).

Assembly and Alignment

The COI sequences were assembled and manually edited using SeqMan Pro software. The assembled fragments were exported as FASTA files. The sequences were aligned and the terminal gaps were treated as missing data. After removal of pseudogenes and chimeric sequences, a single mtCOI sequence per whitefly sample was selected for the identification of mitotypes and their spatial distribution pattern. The identical haplotypes (99–100%) were identified by reconstruct the phylogenies and redundant haplotype sequences were excluded from the alignment.

Phylogenetic Analysis

Phylogenetically, the mitochondrial COI sequences were examined (Huelsenbeck and Ronquist 2001, Ronquist et al. 2012). The regional distribution of B. tabaci mitotypes was displayed together with each individual's phylogenetic association with a well-supported clade in the 3′-mtCOI tree.

Insecticides

Five commonly used insecticides against whitefly including Buprofezin (Buprofezin 25% WP, Tara Imperial Pakistan), Diafenthiuron (Polo^®50 SC, Syngenta Pakistan), Flonicamid (Ulala^® 50% DF, ICI Pakistan), Spirotetramat (Movento^® 240 SC, Bayer Crop Sciences Pakistan) and Pyriproxyfen (Priority^® 10.8 EC, Kanzo Pakistan were purchased from local market for bioassays.

Nymphal bioassay

The experiment was performed on whole cotton plant at four true leaves stage. Fifty adult whiteflies were caged on individual cotton leaf in small clip cages for a 48-h oviposition period to allow synchronization of each developing stage (Pablo, 2018). Then the adult whiteflies were removed and plant was kept into a separate cage. On the 10th day after oviposition, when the majority of nymphs had reached second instar (N2), twenty 2nd instar nymphs were allowed and counted per leaf and marked with permanent marker (Peng 2017). Each infested leaf considered as a replicate for the treatment. Whole the leaf was dipped in serial dilutions of formulated insecticides for 10s & air dried. Each bioassay used four replicates at a minimum of five concentrations along-with a control. Control replicates were dipped in diluent only. Whole bioassay was placed under standard laboratory conditions (Basit, 2018). After 6 days nymphal mortality data was recorded by counting the healthy nymphs as living nymphs and whitefly exuviae with T-shaped exit holes, while unhealthy dead nymphs were scored by observing black, de-shaped or fall off dead nymphs under microscope.

Adult bioassay

Each single leaf was considered a single replicate for the treatment which was dipped in insecticides serial dilutions for 10 s and then air dried at room temperature. For adult bioassay twenty sedated whitefly with CO₂ were confined with small clip cage. Each bioassay was conducted with five serial dilutions with three repeats. The bioassays were maintained at 28°C and mortality data was recorded after 72 h.

Statistical analysis

Mortality data of adult B. tabaci and nymphs was analyzed by Probit analysis by using Polo plus (LeOra Software, 2003) to obtain LC50 which was later used to calculate resistance ratio. LC values for the field resistant strain were divided by the corresponding LC values for the laboratory susceptible strain for each insecticide to determine the Resistance factors (RF). Values were considered as significant if 95% CI will not be overlapping (Chen et al., 2018). As previously described by Ahmad and Khan (2017), generally resistance was classified as RF = 1 none, RF = 2–10 very low, RF = 11–20 low, RF = 21–50 moderate, RF = 51–100 high, and RF > 100 very high.

Identification of B. tabaci Mitotypes from South Punjab-Pakistan

The PCR amplification of the 5′ and 3′COI fragments yielded a 700 and 850 bp amplicon, respectively. These amplicons were obtained after removing the primer sequences at both ends and conducting quality trimming. Trimmed sequences were run and examined in the sequencing data from Brown's lab.

The global Bayesian analysis of the 5′ and 3′-fragment sequences revealed that all haplotypes belonged to the Asia II clade. Based on pairwise identity, all the samples of South Punjab cotton agro ecosystem belong to one subgroup which was identified as Asia II-1 mitotype. Four haplotypes of Asia II-1 was identified through SNP pattern analysis (Fig. 2). The haplotype 4 is predominant (56%) among all the haplotypes followed by haplotype 2 (23%). However, haplotype 1 is least commonly found in cotton agro ecosystem of Punjab (Fig. 3).

By classifying the genetic variants Asia II (1, 2, 3, 4) as a sister group to harboring mitotypes II-5 and II-6, which themselves were sister groups to the II-7. The phylogenetic framework based on the 3′-COI fragment resolved phylogenetic relationships that were consistent with earlier findings (Frohlich et al., 1999, Brown 2010; Dinsdale et al., 2010).

A mixture of three haplotypes of Asia II-I including haplotypes-2, haplotype-3 and haplotype-4 was identified from Jampur and Bahawalpur samples. However, Multan population was identified with two haplotypes (haplotype-4 and haplotype-3) and Vehari samples had mixture of haplotype-4 and haplotype-2.

Resistance Monitoring Results

The LC50 and RR values for all of the insecticides were very high in all of the field collected strains, indicating that these commonly used conventional compounds lost their efficacy against B. tabaci. Buprofezin gave highest values of LC50 (229.96-327.79 mg L^− 1) and RR (25.75–36.71 folds) while maximum adult mortality was observed in Flonicamid showed minimum values of LC50 (9.46–20.31 mg L^− 1) among all the treatments during the years 2020 and 2021 respectively.

Diafenthiuron

Field collected population of B. tabaci exhibited moderate level of resistance against diafenthiuron in all the four locations. No significant difference was observed among all tested populations (95% FL overlapped) in 2020 and 2021. The RR values ranged from 20.59–28.12 folds in district Vehari and Jampur respectively.

Buprofezin

Field population of B. tabaci also showed moderate level of resistance which ranging from 25.75–36.71 fold in Bahawalpur and Jampur district respectively. The LC50 values were differ significantly with each other but the LC₅₀ values were significantly high from spirotetramat, flunicamid and Pyriproxyfen respectively.

Spirotetramat

Spirotetramat exhibited very low to low level of resistance in populations collected from four locations. The RR values ranged from 7.45–12.36 in districts Multan and Jampur respectively. The LC50 values were not significantly different from each other but significantly different from Jampur (44.84–76.79) population collected in 2021.

Flonicamid

Field populations collected form five districts exhibited very low to low level of resistance. The level of resistance ranged from 7.57–14.14 fold in 2021 while 10.16–16.25 in 2021. The LC50 value of Multan population was significantly high as compared to Vehari and Bahawalpur but the LC50 values of other populations were similar with each other in 2020 and 2021.

Pyriproxyfen

Field collected population of B. tabaci also exhibited very low to low level of resistance. The LC50 values were non-significant from each other. The RR values ranged from 7.98–12.78 fold in populations of four districts in both years.

Insecticide Resistance to Adults of B. tabaci

Results of toxicity for field population of B. tabaci collected from four districts in 2021, revealed very low to moderate level of resistance of to Spirotetramat (7.77–12.36 fold), Pyriproxyfen (9.59–12.78 fold), Flonicamid (10.16–16.25 fold), Diafenthiuron (23.03–28.12 fold) and Burpofezin (27.88–36.71 fold) as to susceptible strain. The slope of the regression line ranged 1.93–2.14 fold, 1.71–1.95 fold, and 2.79–2.86 fold for Spirotetramat, Pyriproxyfen, and Flonicamid respectively. Among the tested insecticide Diafenthiuron and Buprofezin exhibited high level of resistance with a slope of regression 1.42–1.71 fold and 1.97–2.22 fold respectively. Highest toxicity was observed for Flonicamid which was significantly different from all other tested insecticides (95% FL not overlapped) followed by spirotetramte, pyriproxyfen diafenthiuron and Buprofezin respectively.

The results obtained during the year 2020, evident slight but non-significant increase in resistance ratio in 2021. Low level of resistance to Spirotetramat (7.45–11.02 fold), Flonicamid (7.57–14.44 fold), and Pyriproxyfen (7.98–10.97 fold) was recorded compared to susceptible lab strain. The slope of the regression line was ranging in 2.08–2.25, 2.87–3.26, and 1.8–2.12 for Spirotetramat, Flonicamid, and Pyriproxyfen respectively. While moderate level of resistance to Diafenthiuron and Buprofezin of (20.59–25.83 fold) and (25.75–34.01 fold) was recorded with the slope of regression line 1.46–1.96 fold and 1.94–2.22 fold respectively. (Table 2).

Insecticide Resistance to Nymphs of B. tabaci

The toxicity of different insecticides against nymphs of B. tabaci was evaluated during the year 2020-21. The collected resistant populations showed a very low to low level of resistance in nymph of B. tabaci to Spirotetramat (2.98–4.01 fold), Diafenthiuron (6.15–7.68 fold), and Buprofezin (6.67–13.22 fold) as compared to susceptible lab strains. The slope of the regression line ranged 2.58–3.9, 3.07–4.35, and 1.68–2.19 for Spirotetramat, Diafenthiuron, and Buprofezin respectively. Opposite to these insecticides moderate level of resistance to Pyriproxyfen (17.6-22.92 fold) and Flonicamid (16.43–24.04 fold) was recorded with the slope of regression line ranging 1.53–1.85 and 1.6–2.11 respectively.

The resistant populations of B. tabaci collected from different districts of Punjab during 2020, revealed a very low to low level of resistance to Spirotetramat (2.2–3.67 fold), Diafenthiuron (4.63–5.65 fold), and Buprofezin (6.40-8 fold) as compared to susceptible lab strains. The slope of the regression line ranged 3.53–7.21, 3.17–4.33, and 1.93–2.10 to Spirotetramat, Diafenthiuron, and Buprofezin respectively. Similarly moderate level of resistance to Pyriproxyfen (14.02–21.33 fold) and Flonicamid (11.65–18.32 fold) was observed. The slope of the regression line ranged 1.83-2 and 1.67–2.33 to Pyriproxyfen and Flonicamid respectively (Table 3).

Table 2 Toxicity of different insecticides against adults of B. tabaci collected from different Districts of Punjab-Pakistan during 2020-2021

Table 3 Toxicity of different insecticides against nymphs of B. tabaci collected from different Districts of Punjab-Pakistan during 2020-2021

The present study is significant because it gives a summary of the current levels of insecticide resistance expressed by B. tabaci populations belonging to different haplotypes of Asia-II-1, drawn across cotton zone areas of Southern Punjab-Pakistan. B. tabaci is reported to be resistant against more than 40 active ingredients including many new chemistry insecticides including Pakistan (Naveen et al., 2017). A number of studies reported that B. tabaci has become resistant to Imidacloprid, Acetamaprid, Thiamethoxam (Bass et al., 2015), Spirotetramat (Bielza et al., 2019), Diafenthiuron (Zhang et al., 2017), Buprofezin (Khalid et al., 2021a), Pyriproxyfen (Roy et al., 2019) and Flonicamid (Roditakis et al., 2014). It has been reported by Naveen et al., (2017) that Asia II-1 populations exhibited more resistance against pyrethroids, organophosphate and neonicotinoids as compared to Asia-II-7. Our study endorsed these findings as that all the whitefly populations (Asia II-1) exhibited considerable resistance against tested insecticides.

The prevalence of resistance is depends up on suitable host and the subsequent model of insecticide-based management program and their consequences (Gao and Reits, 2017). Results of our study exhibited that the field population of B. tabaci showed a very low to moderate level of resistance to different insecticides against adult and nymphal stages of B. tabaci. The nymphal stages are slightly less resistant as compared to adult stages. Similar study was also reported previously in which nymphal stages were found more sensitive as compared to other stages (Peng et al., 2017). However, low to moderate level of resistance to conventional and new chemistry insecticides was also reported (Zheng et al., 2021; Saleem et al., 2021; Bielza et al., 2019; Khalid et al., 2021).

. In our study spirotetramat proved to be the most effective insecticides with very low level of resistance followed by diafenthiuron and buprofezinin both adult and nymphal stages. The similar findings were reported by Prabhaker et al., (2014) where the field populations from California exhibited limited variation in susceptibility to spirotetramat in general both the study years(1.02–7.02 µg [AI] ml − 1). Spirotetramat is a new chemistry and systemic insecticide. It is a tetramic acid derivative. It inhibits the Acetyl-CoA carboxylase in the lipid biosynthesis pathway (Nauen etal., 2019). Frequent use of spirotetramat is evident for the population management of juvenile and adult stages of sucking insect pests including whitefly. Application of this insecticide results in growth retardation and low reproductive potential (Maus, 2008; Nauen et al., 2008).

Our study demonstrated that Pyriproxyfen exhibited very low level of resistance ratio (9.59–12.78 fold) against the adult field populations and similar results were observed by Shah et al., (2020) where they found that all B. tabaci populations showed no resistance to very low level of resistance to pyriproxyfen (2.4–3.5 fold). The use of juvenile hormone analogue like Pyriproxyfen is supposed to be persuasive in embryogenesis and further developmental phases of B. tabaci (Karatolos et al., 2012).

The response of novel insecticides like Flonicamid found effective in reducing B. tabaci under field conditions (Roditakis et al., 2014). Despite being effective, the resistance has also been recorded in the field populations of B. tabaci (Ahmad and Khan, 2017; Roy et al., 2019). Rakotondravelo (2013) found that there is no significant difference in the mortality of both resistant and susceptible stages. The level of resistance of B. tabaci was recorded 3.09–20.85 in field populations of eastern India (Roy et al., 2019). The finding of previous published data coincides with the results figure out by our study. Similar results were reported earlier when very low to medium level resistance was observed against to Pyriproxyfen, Flonicamid and Spirotetramat (Ma et al., 2010; Roditakis et al., 2014; Zheng et al., 2021).

The amount of information about the resistance and susceptible level of field population of B. tabaci against Spirotetramat, Pyriproxyfen, Flonicamid, Diafenthiuron and Buprofezin is still very limited. The possible reasons in Pakistan scenarios, lack of specified resistance management schemes and injudicious use of insecticides are supposed to be the reasons in development of resistance in B. tabaci (Banerjee et al., 2014). The continuous relying on limited group of insecticides has lead and resulted in resistance development to B. tabaci (Khalid et al., 2021a).

It is quite interesting that the areas (Jampur and Bahawalpur) where a mixture of three haplotypes of Asia-II-I were found, these populations showed comparatively high level of resistance against all the tested insecticides as compared to the Multan and vehari. This is indicating that different haplotypes have different level of capability to interact with spray chemicals. As per findings suggest, the immature stage is found to be more sensitive that must be targeted to kept resistance under desired control. The studies related to genetic bases, their inheritance and approaches like these might help to understand resistance development at molecular level. There is a need for regular monitoring of insecticide resistance status in diverse B. tabaci genetic groups and haplotypes in Pakistan.

The authors declare that compliance along ethical standards is followed.

Ethics approval and consent to participate

Consent for publication

All authors have agreed to submit the article in International Journal of Tropical Insect Science.

Availability of data and materials

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

Not applicable.

Authors' contributions

Shamraiz RM executed the experiment. Saeed S and Qayyum MA helped in research planning and analytical work. Khan Z helped in planning and writing the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors are thankful to Prof. Dr. Judith K Brown, School of Plant Sciences and Jorge R. Paredes-Montero Research Professional II, University of Arizona, USA for providing Bemisia tabaci molecular characterization facilities, also grateful to the Higher Education Commission of Pakistan and Institute of Plant Protection, MNS-University of Agriculture, Multan for supporting as financial grant.

Conflict of interest

The authors declare that they have no conflict of interest

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Insecticidal Resistance Monitoring in mitotypes of Bemisia tabaci in South Punjab region of Pakistan

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Abstract

Figures

Introduction

Materials and Methods

Insect Collection and Rearing

DNA extraction protocol from single whitefly

Amplification of mitochondrial Cytochrome Oxidase I gene (mtCOI)

Cloning and Colony PCR

Transformation and Plating protocol (Brown’s lab protocol)

Assembly and Alignment

Phylogenetic Analysis

Insecticides

Nymphal bioassay

Adult bioassay

Statistical analysis

Results

Resistance Monitoring Results

Diafenthiuron

Buprofezin

Spirotetramat

Flonicamid

Pyriproxyfen

Discussion

Declarations

References

Status:

Version 1