MOLECULAR DIAGNOSIS AND COMPARISON STUDY TO THE RED PALM WEEVIL RHYNCOPHOROUS FERRUGINEUS ( OLIVIER , 1790 ) IN BASRAH PROVINCE-IRAQ

The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) is well-known as serious insect pest of date palm and other palm species. The pest was firstly recorded in Iraq in 2015. This study was confirmed the identification of this pest using molecular diagnostic (PCR and sequence analysis) and investigate the genetic relationship by comparison of the sequences with other recorded copies worldwide. The results confirmed the identity of the R. ferrugineus (Olivier) molecularly. Ten copies of mitochondrial COI gene of Iraqi samples (MF092880.1MF092889.1) were firstly registered in National Center of Biotechnology Information (NCBI). The sequences comparison of Iraqi sample genes with worldwide genes showed high similarity with Chinese and Mediterranean COI copies especially the Tunisian one, which may assumes that they were descended from the same origin. The study concluded that the invention of this insect to Iraq could be occurred directly from farms in border area between Iraq and Kuwait or by infested offshoot that imported from other countries.

The eggs, which is mostly, sized 2.62 × 1.12 mm, creamy white, shiny and oblong, will hatch within 2-5 days into larva.Larvae body are pyriform comprised of 13 segments, about 50 mm long and 20 mm wide, legless, creamy white with brown-russet-red to brown-black head capsule with strongly chitinized mouthparts.The larvae starts boring and feeding on the soft succulent tissues around apical meristems at the early stages then move to the crown or trunk edges to form cocoon.The larval stage may range from one to three months depending on temperature and host species.The pupation occurs in cylindrical fibrous cocoons for two to three weeks ended with adult weevil emergence, so the life cycle will completed in about 4 months (5, 7).Adults are large, about 35 mm long and 10 mm wide, although they can be up to 42 mm and 16 mm wide with a long rostrum contains short brownish seta (hairs) on the anterior dorsal half in meals and disappearance of this structure in females.They are reddish-brown in colour with variable dark markings on the pronotum.The RPW adults have the ability to fly for distances ranging from 100 to 800 m because of well-developed wings that they have.The adults usually stay in the same palm until complete consumption of the meristem of the palm or its offshoots then the weevil left the dead palm to a new one (5, 7).The detection of the infestation is very difficult unless the Palm been severely damaged as larvae and adults mostly destroy the interior parts of the palm without showing any detectable signs.The early symptoms are eggs lying notches, cocoons in the base of the palm, irregular crown growing, wilting, desiccation and necrosis of the fronds as a result of lacking water, and tunnelling in the trunk and stem which leads to hollowing out it that resulting in reduce the mechanical resistance of the trunk which exposing palm to collapse.The injured tissues could be infected with opportunistic bacteria and/or fungi to speed up the deterioration of the palm (1, 5, 7).The objective of this study was to confirm the diagnostic of RPW (R. ferrugineus) molecularly and compare the COI gene sequences with other wroldwide copies to investigate the source of infestation in Basrah province.

MATERIALS AND METHODS Samples :
The samples were goten from previous study (2), which were collected from a farm in Safwan border region about less than 1 km from Iraq-Kuwait border line (Figure 1) in 1 st of October 2015.The farm included several cultivars of date palm involved Berhi, Hylawy, Sayer and Boraim but the severe injury was in Berhi and Hylawy cultivars.The collected samples placed in sealed plastic bags then saved in 70% ethanol for next experiments.

Sequence Analysis
The sequences were processed using Chromas version 2.6.2 (14) and the assembly of sequences (forward and reverse) was performed using CAP3-PRABI-Doua software (13).The assembled sequences were submitted to the National center of biotechnology information (NCBI) for registration.The molecular identity of the samples performed by multiple alignment of each sample sequence with NCBI database using Basic local alignment search tool (BLAST) software (3).A comparison among all our identified sequences with all NCBI copies was carried out using BLAST and the best compatible sequences of BLAST throughput were processed by performing new multiple alignment to build neighbor joining tree using MEGA 5 software (15) to determine the similarity relationship among related COI copies.

Bioinformatics:
The comparison of the studied sequence samples with all deposited cytochrome c oxidase subunit I (COI) gene copies of R.ferrugineus in the NCBI database presented high similarity (99-100% identity) with five non-Iraqi R.ferrugineus COI gene copies (KX228866.1 -Tunisia, KT428893.1 -China, KM503130.1 -Greece, GU581319.1 -Egypt and KF413073.1 -China) respectively.Furthermore, among the mentioned five copies, the Tunisian one was the most similar to all Iraqi samples (Table 1).A phylogenetic neighbor joining tree results (Figure 2) showed grouping of examined samples to two main related groups within differences ranged from (0 -0.66%) among examined COI copies.Previously, RPW considered as a major palm pest in all Iraq surrounding countries that share the same boarders especially Saudi Arabia (since 1987) and Kuwait (since 1988), which were the oldest invented countries in this area followed by Iran (since 1990) (9, 10).The molecular identification confirmed the pest identification; furthermore, it connected the collected samples with the most related worldwide samples, which gave us preliminary perception about the source of this infestation.There are two possible scenarios may investigates the appearance of this weevil in Iraq, that were either through direct invention by flying adults from the nearby farms of Kuwait or by infested offshoots or tissue culture date palm clones that imported to Iraq.The first scenario, which is the most accepted, is the direct transfer of the pest from nearby farms of Kuwait, which are distributed beneath the Iraq -Kuwait borders especially that the infested Iraqi farm located in less than 1km away from borders and less than 2 km from some Kuwaiti farms in Al-Abdily border area.The second scenario proposed that RPW entered Iraq through uncontrolled importing of infested palm plants especially in the last few years when a huge number of UAE, Iranian and even Saudi tissue culture plants (clones) were imported and grown in Iraqi orchards especially in Basrah province.The comparison results showed high genetic similarity with RPW samples that collected from Mediterranean area especially the Tunisian one.According to (9), there is no available details about RPW in Kuwait, UAE and Iran adding to that, there is no registered (COI) copies in the NCBI related to these regions, so we think that could explain why the Mediterranean and chines samples were the most related to Iraqi ones.Furthermore, when we tried to inspect the source of the first infestation of RPW in all mentioned countries in the literature, we'll found that it related nearly to the same source, "the ornamental palm offshoots imported from Middle East" (9, 10).Similar study reported that the haplotypes of COI gene showed significant relatedness to their geographic regions and suggested that the invasion history of RPW for last thirty years followed three main routes; the east haplotype and two of west haplotypes, which were the Middle East and Mediterranean ones (8).The mentioned study also reported that the Middle East and Mediterranean haplotypes were descended from different geographical populations.The results of this study could be support the possibility that the related samples in our neighbor-joining tree returned to the same origin, which is may clearly explain the high genetic similarity among them.

Fig. 2 .
Fig. 2. Neigbour joining tree to determine the relationship among Iraqi samples of Rhynchophorus ferrugineus mitochondrial cytochrome c oxidase subunit I (COI) gene copiesand the most similar worldwide copies Red palm weevil was not recorded among the usual and/or local pests on palms in Iraq until 2015 when it was first recorded in Safwan, Basrah by the Agricultural Directorate of Basra (4).Previously, RPW considered as a major palm pest in all Iraq surrounding countries that share the same boarders especially Saudi Arabia (since 1987) and Kuwait (since 1988), which were the oldest invented countries in this area followed by Iran (since 1990) (9, 10).The molecular identification confirmed the pest identification; furthermore, it connected the collected samples with the most related worldwide samples, which gave us preliminary perception about the source of this infestation.There are two possible scenarios may investigates the appearance of this weevil in Iraq, that were either through direct invention by flying adults from the nearby farms of Kuwait or by infested offshoots or tissue culture date palm clones that imported to Iraq.The first scenario, which is the most accepted, is the direct transfer of the pest from nearby farms of Kuwait, which are distributed beneath the Iraq

9). The red palm weevil was firstly mentioned by (11) in Basrah province
P.dactylifera about one meter from the ground (12).The wounds and injured tissues following date palm service operations like offshoot removal and frond shaving considered as assistant factors to RPW infestation (7).The females of RPW usually lays about 300 eggs individually in the separate holes in the wounded or injured areas.