# Rapid Molecular Identification of Scolytinae (_Coleopterara: Curculionidae_)
## 2 Results
### Primer Pairs for Molecular Identification of Diverse Genera  
The usefulness of primers D2F1/D3R2, eflafor1/eflarev1, ets149/efa754, ITS2F/ITS2R, and LCO1490/HCO2198 (Table 1) for identification of beetles in the _Coptoborus_, _Xyleborus_, _Theoborus_, _Hypothenemus_, and _Araptus_ genera (Figure 3), is quantified as the proportion of each that amplified with a given primer pair, and the proportion that yielded a correct species or genus match following BLASTn analysis with the resulting sequence (Table 2). Primer pair D2F1/D3R2 amplified all samples except B20 (_Hypothenemus_ sp.) and provided high-quality sequences (91.9% quality, 420 bp length) even with PCR product concentrations as low as 2.3 ng/\(\upmu\)L (B16). However, sequences from _Hypothenemus_ and _Araptus_ isolates returned incorrect matches in GenBank (Table 3). The _Hypothenemus_ isolates were 99.7% identical to sequences of _Theoborus_ sp., with no _Hypothenemus_ sp. accessions within the 100 best matches. There was only one 28S sequence from an _Araptus_ specimen in GenBank but it had a lower % identity (87%) with the generated sequences. Figure 2: Amplification of decreasing amounts of DNA from three _Theoborus_ sp. beetles using primers D2F1 and D3R2 (1% agarose gel _w/v_) was performed to determine the smallest quantity of DNA that could still be successfully amplified. DNA quantity started at (**A**) 1.5 ng and was decreased by a factor of five each time. Subsequent levels had (**B**) 0.3 ng, (**C**) 0.06 ng, (**D**) 0.012 ng, (**E**) 0.0024 ng, (**F**) 0.00048 ng, and (**G**) 0.000096 ng of DNA. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Primer Pair** & **Variable Assessed** & _Xyleborus_ & _Coptoborus_ & _Theoborus_ & _Hypothenemus_ & _Araptus_ \\ \hline D2F1 & Amplification & 1/1 & 5/5 & 4/4 & 3/4 & 3/3 \\ D3R2 & Quality & 1/1 & 5/5 & 4/4 & 3/4 & 3/3 \\  & Identification & 1/1 & 5/5 & 4/4 & 0/4 & 0/3 \\ \hline eflafor1 & Amplification & 1/1 & 4/5 & 4/4 & 3/4 & 2/3 \\ eflafor1 & Quality & 1/1 & 4/5 & 4/4 & 2/4 & 2/3 \\ eflafor1 & Identification & 1/1 & 4/5 & 4/4 & 3/4 & 0/3 \\ \hline eflafor1 & Amplification & 1/1 & 4/5 & 4/4 & 3/4 & 2/3 \\ efarev1 & Identification & 1/1 & 4/5 & 4/4 & 3/4 & 0/3 \\ \hline est149 & Amplification & 1/1 & 4/5 & 4/4 & 3/4 & 3/3 \\ efa754 & Quality & 1/1 & 3/5 & 3/4 & 2/4 & 0/3 \\  & Identification & 1/1 & 4/5 & 3/4 & 2/4 & 0/3 \\ \hline ITS2F & Amplification & 1/1 & 3/5 & 0/4 & 0/4 & 0/3 \\ ITS2R & Quality & 1/1 & 3/5 & 0/4 & 0/4 & 0/3 \\  & Identification & 0/1 & 0/5 & 0/4 & 0/4 & 0/3 \\ \hline LCO1490 & Amplification & 1/1 & 5/5 & 2/4 & 4/4 & 3/3 \\ HCO2198 & Quality & 1/1 & 2/5 & 1/4 & 0/4 & 2/3 \\  & Identification & 1/1 & 2/5 & 2/4 & 1/4 & 2/3 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Proportion of amplification, generation of quality sequences, and positive identification in members of five Scolytinae genera using different primer pairs. Amplification is based on visible bands at 150 ms exposure and at least 2 ng/uL. Generation of quality sequences is defined as sequences of \(\geq\)150 bp and a quality score \(\geq\) 80% as determined by the Geneious Prime software (Ver 2019.2.1.). Identification is whether a BLASTn search gave correct matches to the genus level. Figure 3: Representatives from the five genera tested in this study: (**A**) _Coptoborus_, (**B**) _Xyleborus_, (**C**) _Theoborus_, (**D**) _Hypothenemus_, and (**E**) _Araptus_. Size bar is representative of 250 μm. Elongation factor sequences were capable of identifying all groups except _Araptus_ sp. Primer pair eflafor1/efarev1 amplified in all genera tested and yielded high-quality sequences (80-97%) (Table 4). However, they did not unambiguously identify members of the _Araptus_ genus, with 91.6% identity with _Phelloterus_ sp. (KY805860) vs. only 89.6% and 89.7% for _Araptus costaricensis_ (KY852964) and _A. attenutatus_ (FJ347565), the best matches within this genus.