Molecular identification of Helicoverpa armigera (Hubner) associated with tomatoes (Solanum lycopersicum L.) in Ogun State

The tomato fruit worm Helicoverpa armigera is a key polyphagous agricultural pest with a worldwide distribution. Apart from tomatoes, H. armigera also infests cotton, maize, chickpea and pigeon-pea. Its larvae affect almost all the aerial parts of tomato plants from the vegetative stage to the fruit maturation stage. Due to its high mobility, it causes a huge damage on crops and, as such, early detection of H. armigera from different agroecosystems is crucial for implementing control measures. Herein, we used a molecular marker approach to identify Helicoverpa spp. collected from twelve different locations in Ogun State. The H. armigera larvae used for the molecular identification were collected on tomato fruits from the 12 different locations from three agro-ecological zones in Ogun State. Genomic DNA of Helicoverpa was extracted and its purity was checked using Nanodrop spectrophotometer. Cytochrome Oxidase-I (CO-I) region of the DNA was subjected to Polymerase Chain Reaction (PCR) amplification. The PCR amplification of the CO-I gene after gel electrophoresis of PCR products of H. armigera showed a similar banding pattern at 700 base pairs which confirmed the insect as H. armigera.


Introduction
Tomato fruit worm Helicoverpa armigera is one of the most damaging and hard to control bollworm insect pests. It feeds on important agricultural crops such as tomato, maize, chickpea, pigeon pea, cotton, sorghum, sunflower, soybean and groundnut (Fitt, 1989, Regupathy et al., 1997. The average crop damages due to this polyphagous pest are valued to be US $350 million annually (Lammer and Macleod, 2007). The insect was reported to cause estimated yield loss of over US$ 5 billion annually in Africa even with application of pesticides (Sharma, 2005). The larvae of the insect affect almost all parts of the tomato plant from the vegetative stage to the reproductive stage (Tripathy et al., 1999). H. armigera damage varies among different varieties of tomato, suggesting that some tomato varieties could be highly damaged while others could resist the effect of the pest (Osipitan et al., 2017). It has been reported that H. armigera has high movement and productiveness and it also revealed a great capability to develop resistance to insecticides used for its control (Ramasubramaniam and Regupathy, 2004). The cosmopolitan and polyphagous nature of the insect makes it have a wide host range and geographical distribution with the ability to adapt to new environments (Djihinto, 2012). H. armigera was reported to possess the ability to migrate from one fruit to the other without fully consuming it; and this explains the widespread damage of the insect to crops even when the larvae population is low (Zalucki et al., 1986). Molecular markers are a convenient tool to reveal population genetic structure, and to fingerprint the individuals and the populations by eliminating the anomaly related to the standard morphology based markers (Heckel, 2003, Fakruddin et al., 2006. Molecular tools find variation at DNA sequence level and are evidenced to be an effective tool for identifying closely related genotypes. The performance of this insect could be due to the presence of a strong genetic makeup that makes it a serious pest on several crops. (Zhou et al., 2000, Scott et al., 2003 There is the need to understand the genetic variations of polyphagous pests like H. armigera. The knowledge of the genetic variation can be very helpful in determining the population dynamics, mode of action and control measures for the insect. The early detection of H. armigera infestation in different agro-ecosystem is crucial to implement control measures. The observation of morphological characteristics alone (e.g., wing pattern design) may not be adequate to identify Helicoverpa species, especially when the integrity of pheromone-trapped specimens cannot be assured. This is particularly true for H. armigera and H. zea, which, besides their similar morphology and overlapping host range, are attracted by the same volatile compounds (Pogue, 2004). Increase in the development of DNA based markers has reduced the use of morphological, physical and cytological markers for evaluating genetic diversity and population dynamics (Cruickshank, 2002).
Information on molecular identification of H. armigera in Ogun State is scarce. This study is therefore tailored towards identifying H. armigera molecularly in Ogun state.

Materials and methods
Collection of larva Different larvae of Helicoverpa ranging from green to dark brown used for molecular identification were collected from tomato fruits from 12 different farms at three agricultural zones namely; Ijebu-Imushin, Imodi-Imosan, Ipebi and Ijebu-Ode (Ijebu zone), Anigbajo, Ayetoro, and Abule Oje (Ilaro zone), Sogbade, Odeda, Olugbo, Latemo and FUNAAB (Abeokuta zone) Ogun State. Each collected larva was placed in a 1.5 ml Eppendorf tube , labelled and kept in a Haier Thermocool freezer at -20 o c.

DNA extraction
Total genomic DNA was extracted using a Qiagen DNeasy insect Blood and Tissue Kit (Qiagen, Valencia, Calif.). The larva was macerated using mortar and pestle; the macerated larva was placed in a 1.5 ml microcentrifuge tube, 180 μl of tissue lysis buffer (sodium dodecyl sulfate) and 20 μl Proteinase K enzyme were added and mixed by vortexing and incubated at 56°C (in a dry bath) for 1 hour to denature the proteins. About 200μl lysis buffer was added and mixed thoroughly by vortexing and incubated at 56°C for 10 minutes to isolate the DNA ; 200 μl ethanol was added and mixed thoroughly by vortexing.
The mixture was then pipetted into DNeasy mini spin column placed in a 2 ml collection tube and centrifuged at ≥ 6000g for 1 minute. T he collection tube and the flow through were discarded, and the spin column was placed in another 2 ml collection tube. 500μl wash buffer (AW1) was added and centrifuged at ≥ 6000g for 1 minute and the collection tube and the flow through were also discarded. T he spin column was placed in another 2 ml collection tube, 500μl wash buffer (AW 2) was added again and centrifuged at ≥ 20,000g for 3 minutes to remove protein residues. T he collection tube and the flow through were discarded and the spin column was placed in a new 1.5 ml micro centrifuge tube. The DNA was eluted in 200 μl elution buffer (AE buffer), incubated for 1 minute at room temperature (15°C -25°C) and centrifuged for 1 minute at ≥ 6000g. The spin column was discarded and the DNA was stored

Agarose gel electrophoresis
Agarose gel electrophoresis was carried out using an electrophoresis set-up (Bio-Rad®) and the steps were as follows. Agarose (2% -2 g in 100ml) solution was prepared in a conical flask with the addition of 49 ml 1X TAE buffer. It was kept in a micro wave oven for 60 seconds until the agarose was completely dissolved and the solution was clear. The solution was allowed to cool and 5 µl ethidium bromide solution was added and mixed well. The solution was poured into a gel casting tray (which was placed on a horizontal surface and comb was placed properly in the gel caster) and allowed to solidify for 45 minutes at room temperature. The comb was removed gently and the gel casting tray was placed in an electrophoresis tank and submerged (just until wells were submerged) with electrophoresis buffer (1X TAE).
The DNA sample was prepared by mixing 3 μl of tracking dye (loading buffer) with 10μl of the DNA solution. The samples were mixed well and loaded at 13μl per well. The set-up was connected to the power source and the gel was run at constant voltage of 80 volts for 30 minutes. The gel containing the electrophoresed DNA was viewed photographed in a UV transilluminator.
Purity and concentration of DNA The purity and concentration of the DNA was checked using a Nanodrop spectrophotometer model NanoDrop-1000 (Thermo Scientific TM). Absorbance was recorded at both wavelengths and the purity was indicated by the ratio OD260/OD280. A value between 1.8 and 2.0 indicates that the DNA was pure and free from proteins and RNA. When the ratio is <1.8, the sample is contaminated with RNA, but when the ratio is >2.0 the sample is protein contaminated.

Polymerase Chain Reaction (PCR) amplification
Good quality genomic DNA isolated from Helicoverpa larva was used for PCR amplification of the cytochrome oxidase subunit -1 (CO1) locus using the LCO52 (GGTCAACAAATCATAAAGATATTGG) Forward primer and HCO52 (TAAACTTCAGGGTGACCAAAAAATCA) Reverse primer. The amplification was carried out in Verti Thermal Cycler (Applied Biosystems®). PCR amplification was performed in a 20 μl reaction mixture which consisted of a) Genomic DNA -2.0 μl b) Fire pol master mix -4.0 μl c) Forward Primer -0.5 μl f) Reverse Primer -0.5 μl g) Nuclease Free Water -13.0 μl Total volume -20.0μl Several PCR conditions were tested in the experiments to determine the effects of annealing temperature, annealing time, and cycle number on the marker. The PCR conditions were programmed as; Lid temperature 98 o C, initial denaturation 94 o C for 5 minutes, 40 cycles each of denaturation at 94 o C for 45 seconds, primer annealing at 55.8 o C for 45 seconds and primer extension 72 o C for 45 seconds, followed by 7 minutes' extension at 72 o C.
The second round of amplification (i.e. re-amplification) was undertaken as follows: 2µl of each of the above PCR products was used as a template. The reaction was carried out using the procedure of the first amplification.
Electrophoresis of PCR products PCR products were analyzed by electrophoresis in 1% agarose gel electrophoresis run at 80 W for 30 min in 1x TAE buffer. The molecular size of the amplified products was estimated using a 100 bp DNA marker (Norgen Biotek Corp). Agarose gel electrophoresis of the PCR products of samples collected from the three agro ecological zones

Concentrations of DNA extracted from
The a garose gel electrophoresis of the PCR products to identify the species of Helicoverpa collected on tomatoes from the three agro ecological zones in ogun state is shown in Plate 3. The electrophoresed PCR products of the DNA extracted revealed 700 bp for the amplified cytochrome oxidase subunit -I region. The mitochondrial cytochrome oxidase 1 region of the DNA obtained after amplification with universal DNA -based markers in a thermal cycler gave an intact band of 700 base pairs when resolved at 1 per cent agarose gel. Gel electrophoresis of the PCR products of 11 Helicoverpa out of the 12 Helicoverpa CO-I gene amplified showed a similar banding pattern. The DNA from one of the larvae could not be amplified ; this failure could have been due to poor gDNA and/or the fact that it belonged to other lepidopteran species. There was variation in the quantity of DNA extracted from the 12 locations; this could be due to variation in the larva color which ranges from green color to dark brown. The results showed that there was no significant variation in the CO-I sequences of H. armigera from the eleven geographical locations, suggesting that the larvae are the same molecularly, though they differ in color morphologically . The result is similar to an earlier report by Asokan et al. (2012) which indicated that H. armigera sourced from various hosts and from different geographical locations in India has no significant variation when mtCO1 gene of Helicoverpa was used to reveal their species identity. Behere et al. (2007) reported that DNA barcoding has been successfully used to assign lepidopteran specimens into taxonomic category and to distinguish between closely related Helicoverpa species, H. armigera and H. zea. Floyd et al. (2010) reported that DNA barcoding has the potential for easing the identification of aggressive insect pests affecting crops. Nagoshi et al. (2011) reported that DNA barcoding supplemented the morphological methods for identifying the invasive armyworm, Spodoptera species in Florida. In the present study, DNA barcoding based on mitochondrial cytochrome oxidase 1 gene confirmed the species level of identity of tomato fruit borer as H. armigera. H. armigera identification based on molecular traits could be helpful in revealing the divergence of species, evolution of biotypes and species complex. Arnemann et al. (2016) reported that knowledge of mtDNA genes such as the widely used cytochrome oxidase subunit I will greatly assist in the confirmation of species apart from providing an opportunity to ascertain the population diversity and to infer possible patterns associated with widespread infestation of the insect. Omaththage et al. (2015) reported that diversity of morphologically similar species using standard taxonomic features is challenging, time wasting, and needs technical know-how.
H. armigera larvae identification based on molecular traits could be used as the primary diagnostic tool to reveal the divergence of species, evolution of biotypes and species complex where the requisite morphological keys are unavailable.