Preliminary Molecular Identification of a Predatory Bug, Orius albidipennis, Collected from Ornamental Plants

Orius albidipennis (Reuter) (Hemiptera: Anthocoridae) is a generalist predator used for biological control of insects attacking ornamental plants. Molecular identification of this species using internal transcribed spacer 1 (ITS1) of ribosomal DNA was conducted for the first time. The complete sequence of ITS1 and fragments of its flanking 18S and 5.8S rDNA genes are reported herein. The estimated length of ITS1 of O. albidipennis was 305 bp. This spacer was nearly identical to its counterpart of Orius sp-Taif strain in spite of the difference in their length. The phylogentic relationships were determined using the maximum-likelihood method supported with strong bootstrap probabilities clustering of both taxa together. Further molecular markers could be useful to identify the Taif strain and support its sister relationship to the Egyptian O. albidipennis.

Some Orius species are sold commercially for augmentive biological control releases. Orius have been used successfully as a biological control in a number of greenhouse crops and open fields including sweet pepper, cucumber, and some ornamentals (Van de Veire and Degheele 1992; Chambers et al. 1993;Lorenzana et al. 2010). Accordingly, it is very important to evaluate this generalist predator systematically for managing its pest control. Narita and Ohno (2000) identified adult females of the genus Orius independent of cuticular hydrocarbons by gas chromatography-mass spectrometry The molecular diversity and systematics of Orius are poorly understood. Honda et al. (1998) used the internal transcribed spacer 1 (ITS1) and its flanking rDNA genes to infer the relationship among 10 Orius species. Hinomoto et al. (2004) developed multiplex polymerase chain reaction (PCR) methods to identify five Orius species that occur commonly in Japan. They amplified ITS1 of the nuclear ribosomal DNA. Jung et al. (2010) studied the family Anthocoridae, including 9 species of Orius, by using molecular markers. Other investigations also used molecular markers to examine the the anthocorid family (Muraji et al. 2000a, b;. Sayed and Montaser (2012) used the ITS1 to identify an Orius species inhabiting west Saudi Arabia, but they could not identify which species it was. The authors however, named this taxon Orius sp-Taif Strain. To the best of our knowledge, there are no molecular systematic studies of O. albidipennis so far. This work, therefore, aimed to sequence ITS1 and its flanking rDNA genes for the Egyptian O. albidipennis and compare it to its counterparts of Orius sp-Taif strain, as well as other sequenced species, in order to construct a preliminary identification of O. albedipennis and relate it to the Taif strain.

Materials and Methods
A well-identified and recorded specimen was taken from the laboratory of predators mass production, Faculty of Agriculture, Cairo University. The insect was reared as a commercial product and was morphologically identified as O. albidipennis. It was originally collected from a field in Cairo, Egypt. An individual O. albidipennis was dissected, the gut was removed and discarded, and the remainder was ground in 100 µl lysis buffer (5% Chelex-100 and 4µl of proteinase K (20µg/µl) in a 1.5-mL tube. The tube was incubated for 6 hours at 56° C followed by 10 minutes at 95° C, then centrifuged at 12000 rpm for 10 minutes ). DNA in the supernatant was spectrophotometrically measured (at 260/280nm), then used for PCR.
The resultant solution was electrophoresed on a 1.5% agarose gel in TAE (40mM Tris, 40mM acetic acid and 1mM ethylenediaminetetra acetic acid) and the gel was stained with ethidium bromide. 100 bp DNA Ladder (Biolabs) was used as a marker for the molecular weight size. The PCR product was then purified from gel with the use of a spin column according to the kit manual.
Sequencing reactions were performed in a MJ Research PTC-225 Peltier Thermal Cycler using a ABI PRISM. BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq-DNA polymerase (FS enzyme) (Applied Biosystems, http://www.appliedbiosystems.com/) following the protocols supplied by the manufacturer were used. A single-pass sequencing was performed on each template using the last mentioned PCR-primers. The fluorescent-labeled fragment was purified from the unincorporated terminators with an ethanol precipitation protocol. The sample was resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems). The obtained sequence was analyzed with the DNASIS-Mac program version 3.5 (Hitachi, http://www.hitachi.com/). After the sequence was obtained, its feature was identified by both Sequencher version 4 and MacClade version 4.08 programs (Sinauer Associates, Inc., http://www.sinauer.com/). Wollastoniella rotunda (AF061375) was used as an outgroup to root the constructed relationship. The obtained sequences were aligned separately and manually using MacClade version 4. The major portion of ITS1 was unalignable among the different taxa, and only 43 bp were alignable and used in the analysis besides 131 bp from 18S gene. The tree analysis was done by the maximum-likelihood method in PAUP* 4.0b10 (Swofford 2002). In this analysis, heuristic searches with the TBR branch swapping and 10 random taxon additions were adjusted. The bootstrapping replicates were set to 1000 with simple additions.

Results and Discussion
Using the primers of Hinomoto et al. (2004), O. albidipennis gave a PCR product that showed similar size to its counterpart of some Orius species (data not shown). Sequencing of the PCR product showed that the amplified fragment acquired the typical arrangement of all Orius species sequenced so far. The first 131 bp were located in 3' end of 18S rRNA gene, followed by 306 bp for the complete ITS1 and 74 bp for the 5' end of 5.8S rRNA gene (Figure 1). The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB624550.
After the sequence of the present study was obtained, individual features were identified in light of their counterparts from other Orius species. As for identification of ITS1, the conserved sequences of both 18S and 5.8S rDNA genes that are typically located on either boundaries of the spacer were searched. The start of ITS1 was identified by searching for 5'-ACCTGCGGAAGGATCATT-3', which was found at the 3'-end of 18S rDNA gene as shown in Figure 1. For the 3'-end of the spacer, the sequence 5'-TACCCTGAGCGGCGG-3' was located at the 5'-end of 5.8S rDNA gene.  ITS1 showed variable lengths among the different Orius species and it exhibited great ambiguity in its features; therefore, it was unalignable. Several investigations revealed variable lengths for ITS1 in the different species of the genus (Honda et al. 1998;Hinomoto et al. 2004;Muraji et al. 2004 The sequence of ITS1 for the different Orius species was not able to be aligned because of the very high mutations this spacer exhibited. Meanwhile, the flanking gene of 18S rDNA was conserved within the genus. Because of this ambiguity, the relationship among the Orius species could not be constructed with clear resolution. In spite of the high ambiguity in the sequence of ITS1, nearly complete identity of this spacer between O. albidipennis and Orius sp-Taif strain was found. This identity supports that Taif strain clustered to the Egyptian O. albidipennis in spite of length difference and mutations (deletion and substitution) this spacer exhibited between the two taxa. Therefore, we recommend sequencing ITS1 and other mitochondrial DNA markers (cytochrome b gene) for more samples of both taxa in a further study in order to identify the unknown species of Taif and to fix its relation to the Egyptian species.
Analyses of ITS1 for different Orius species were conducted to assess their relative relationships after resolving the alignment problems for ITS1 of anthocorids (Honda et al. 1998) with the sequence identified here. 131 bp from 18S rRNA gene were concatenated with the alignable 43 bp from ITS1 ( Figure 3) and were used for analysis. Therefore, unambiguous 174 nucleotides were used in analysis by maximum-likelihood method. Fifty-six substitution models were tried by using the Modeltest (Posada and Crandall 1998), and the best fit model that explained the data was GTR+I. A single maximum-likelihood tree was found with a negative log likelihood score -lnL = 430.6143 ( Figure 4). The base frequencies were A = 26.5%, C = 21.8%, G = 31.0% and T = 20.7%. In the constructed tree, the Orius species found in Taif was segregated with the Egyptian O. albidipennis (99% bootstrap). The Japanese Orius species (O. minutus, O. sauteri, and O. strigicollis) were grouped together as previously shown (Honda et al. 1998). In general, the relationship among different Orius species agreed with the previous constructed relationship (Honda et al. 1998;Jung et al. 2010;Shapiro et al. 2010) with differences in the positions of some taxa in our tree. These differences could be because only the manually alignable fragment of ITS1 was used in the present study, whereas the previous studies used more data from ITS1 and aligned it automatically by Clustal W program.
In conclusion, Orius sp.-Taif strain is closely related to the Egyptian Orius albidipennis, and both taxa could be from the same species. This result should be confirmed with sequencing more molecular markers for more samples of both taxa.