Parent Washington Navel Orange Scale-Infested Rind cDNA Library UCRCS11

Vector: Lambda Uni-ZAP XR, excised phagemid; Site_1: EcoRI; Site_2: XhoI; In cooperation with Dr. Robert Luck and Dr. Joseph Morse, Department of Entomology, University of California Riverside, navel orange fruit was infested with red scale (Aonidiella aurantii [Maskell]). Dr. Lucks technician, Lisa Forster, maintained colonies of red scale. Dr. Morses technician, Paul Flores, cleaned and infested the fruit, bagged the fruit after the crawlers had time to settle, then harvested the fruit at the early second instar and late third instar phases of the insect. Claire Federici (Dr. Mikeal Rooses lab) cleaned off most of the insects (some remained firmly attached), cut the peel, then froze and stored at -80oC. The trees used for this set of samples were growing in field 16K of the University of California Citrus Experiment Station. This was the same field from which the tissue was collected for the navel shoot meristems and the peel for citrus thrips, Scirtothrips citri [Moulton]. The trees were planted 12 October 1992. The scion was Parent navel. The trees used for infesting with red scale all had Troyer rootstock. Three different trees were used for each of the two infestations, a total of 6 trees. This experiment did not take place according to the original schedule because the red scale colony became infested with mites, so too few insects were available to infest the fruit on the date originally planned. The plan had been to infest on or about June 30 and August 13. Instead the infestation dates were August 30 and September 27, 2004. Because this took us into the cool weather of fall, the insects applied on the second infestation date took about three times as long to develop to the same stage as the insects applied on the first infestation date. The insects were reared on lemons. Before infesting the fruit on the trees, Paul Flores cleaned the peel and checked to make sure it was not already infested. He placed about 200 crawler stage insects on the fruit using a soft paintbrush. After the crawlers had a day to move around and find a spot to attach, Paul placed a very fine mesh drawstring bag over the fruit to exclude predators and parasitoids of the scale. Bags were left in place for the duration of the field development period and also covered control fruit. Paul Flores infested fruit on 30 August and 1 September 2004 after cleaning the fruit on August 27. On 21 September the first set of red scale infested fruit were sampled. He brought Claire Federici 15 infested fruit and 12 uninfested control fruit. The insects were at the early second instar. The controls had been cleaned and bagged at the same time as inoculated ones, but had no scale introduced. The infestation was heavy, so the insects were not well separated on the fruit. A razor blade was used to slice off the flavedo with about half the thickness of the albedo included. The peel from only the stem half of the fruit was used because that was where the insects were most concentrated. The insects were impossible to wash off without severely damaging the peel; each fruit was washed with water and a sponge or bottlebrush and wiped dry with a paper towel before cutting it, but many of the insects were still intact and were included in the sample. The pulp had color but the fruit rind was still green. The peel from each half fruit was bagged separately in foil packets, and pressed between sheets of dry ice to freeze. All packets were placed in a paper bag with the date and information about control or infested written on the bag, and then placed at -80oC. The second sampling date from the first infestation was 11 October. Paul brought 19 infested and 9 uninfested fruit to Claire. These were all set up on 30 August. The insects were at the late third instar. Mikeal Rooses advice was to prepare three bulks of each, 5 infested and 3 uninfested fruit per bulk. The remaining four infested fruit were infested to a lesser degree and were discarded. Washing was done as before and the peel from each fruit frozen between sheets of aluminum foil pressed between sheets of dry ice. There were quite a lot of adhering insects in the frozen rind. After it was frozen, the peel from 3 uninfested or 5 infested fruit were pooled in a foil packet, then stored at -80oC. The pulp had color but the rind was mostly green. However, there was a little color developing on some fruit around the insects. The third sampling date was 2 December. Paul brought the first fruit from the second infestation, which were set up on September 27 or 28. He brought 15 infested and 10 uninfested fruit. Claire cut and froze them as before, making three pools of 5 infested fruit and three pools of 3 uninfested fruit. The control fruit all had green at the stem end, mostly about 1/4 of the circumference from stem to blossom end, but one fruit was green all over. The infested fruit were almost fully orange, with only a small amount of the surface still green, approximately the size of a quarter coin. Paul said it would be at least a month, probably 6 weeks or even more before the insects reach the late third instar, so this was the final sample for the cDNA library due to time constraints for EST sequencing. Mandal and Fenton (Close lab) purified RNA by the phenol method described in J. Japanese Soc. Hort. Sci. 1996. 64 (4): 809-814, purified poly(A) mRNA using PolyAT Tract mRNA Isolation Kit (Promega), produced a primary cDNA library using a lambda ZAP XR cDNA Synthesis Kit (Stratagene), then mass-excised 1 million pfu from the primary library to produce a phagemid population. The library was made from a mixture of RNA from each of the three treatments such that approximately equal amounts of early second instar and late third instar RNA were used. Phagemids were plated, plasmid DNA purified, cDNA clones archived, and DNA sequences determined bi-directionally using an ABI3730 at the Arizona Genomics Institute (Kim, Kudrna, Collura, Wissotski, Byrne, Stum, Smart, Muller, Wing). Chromatogram files were downloaded by FTP by Close, then processed by Wanamaker (Close lab) using the HarvEST pipeline (http://harvest.ucr.edu) to remove vector and cloning oligo sequences and various contaminants, and to trim to a high quality region. Sequences that retained a phred 17 region of at least 100 bases were assembled, then chimeras were removed following manual inspection of assemblies (Wanamaker, Close, Roose). Sequences that survived all removal steps were submitted to GenBank. Clones from this library are archived at the Arizona Genomics Institute (http://www.genome.arizona.edu/orders/).