2017

The 2017 full report (in Romanian) can be found here.

After an analysis of the published data, we concluded that worldwide there are at least 54 parasitoid species from the order Hymenoptera that can parasitize Nezara viridula eggs. We compiled a table with all these parasitoids and a list of references (see full report).

We selected adults of egg parasitoids of Pentatomidae, including Nezara viridula, performed DNA extractions and amplified selected markers with PCR. This was done so far for a total of 63 specimens of Anastatus, Ooencyrtus (Chalcidoidea) and Trissolcus (Platygastroidea).The specimens originated from: Bulgaria, South Korea, France, Republic of Moldova, Romania, Turkey, and Ukraine.

Genomic DNA was extracted from whole specimens using a non-destructive protocol using the DNeasy® blood and tissue kit (Qiagen, Hilden, Germany). This is a modification of the one described by the producer, as described in Polaszek et al. (2014), being further optimised to obtain high DNA yelds from small specimens such as those in the genera Anastatus, Ooencyrtus and Trissolcus.

Briefly, 180 µl ATL buffer is pipetted in previously labelled, sterile 1.5 ml micro tubes followed by the addition of 20 µl proteinase K. If the specimens are very small, the volume can be reduced to half of the recommended one. Specimens are removed from ethanol and dried on filter paper for about 15–30 sec. as an excess of ethanol could inhibit proteinase K. Specimens are placed in the microtubes and the content is mixed by turning the tube upside down several times. Even if vorthexing is recommended at this step, it is avoided to prevent eventual damage to the specimen.

The micotubes are centrifuged briefly for about 10-15 sec (short spin) to ensure that the specimen is submerged. The microtubes are incubated for a minimum of 8 h (better 12 h) in a water bath at 55C. The ATL buffer is then removed to a new tube, and the specimen is left in the original tube to avoid its damage in the following steps. Frome here one manufacturer’s protocol id followed with the modifications as described below:

  1. Roth or Merk expensive alcohol to add to the wash buffers. Cheap alcohol contains traces of isopropyl alcohol, benzene etc.

  2. After the last wash with AW2 buffer open centrifuge, rotate tubes by 180 degrees, centrifuge again. This ensures that there is no remaining alcohol on the plastic ring that holds the membrane.

  3. Add polyA RNA. 2 uL of 2ug/uL solution per extraction. This is done after incubation and just before adding AL and alcohol.

  4. During elution, two succesive elutions of 50 uL each are done. Pipette the elution buffer directly on the silica membrane. Incubation 15 minutes or so before centrifugating. Use warm (56˚C) elution buffer. Rewarm buffer between the two consecutive centrifugations. You obtain 100 uL in the end, not 200 uL. If it is a rare sample, you can freeze the columns from the extraction in a zip bag and in the future re-elute the remaining residual DNA.

  5. The DNA is eluted in LoBind tubes (Eppendorf).

The amplification of the standard barcode region in Chalcidoidea (COI) is known to be problematic (Kaartinen et al. 2010, Fusu 2017). Beside the standard primer pair LCO1490 and HCO2198 we alternatively used the primers COI PF2 and COI 2437d (Kaartinen et al. 2010) where the first pair fails. The yield of the PCR reaction is increased, and the nonspecific amplification is decreased. By using these primers, the poly-T region at the 5’ end of the COI gene is avoided as sequencing this can lead to polymerase slippage and ambiguous electrophoregrams.