#579 Molecular Markers for Detecting Rapid ʽŌhiʽa Death-associated Ambrosia Beetle Species from Frass
by Kylle Roy1, Kelly Jaenecke2, Carter Atkinson1, Dan Mikros2, and Bob Peck2 1U.S. Geological Survey and 2Hawaiʻi Cooperative Studies UnitPacific Island Ecosystem Research Center
Kylle Alohilani Minei Roy
Ambrosia beetles can be extremely invasive and have been associated with diseases worldwide, which may be attributed to their haploid-diploid biology and symbiosis with their ambrosia fungi. Most ambrosia beetles bore tunnels into stressed or dead trees, planting their symbiotic fungi in the xylem and remaining to feed and reproduce in the gallery for as long as conditions remain suitable. These beetles have recently been associated with the spread of Rapid ʽŌhiʽa Death (ROD) in Hawaiʽi, and knowledge of their life-history, community composition, and capacity to spread the disease have become essential. Unfortunately, species-level identification of these beetles can be difficult due to subtle differences in their morphological characteristics in addition to requiring excavation from trees. To facilitate species identification from frass and the beetles themselves, we designed species-specific molecular markers for ambrosia beetles associated with ROD in Hawaiʽi. Importantly, molecular-based identification of beetles from their frass can provide host-tree information whereas traditional lure-based trapping does not. This tool has potential be used with high throughput sequencing or mass Sanger sequencing to identify beetle species from frass collected directly from galleries on trees across the Hawaiian Islands. Identification of ROD-associated ambrosia beetle species that produce viable inoculum is critical to the development of proper ROD management strategies.
Most ambrosia beetle species (Coleoptera: Curculionidae: Scolytidae) are fungus farmers, planting their "ambrosia" fungi food source in their "homes" called galleries in the xylem of woody plants.
While creating their galleries, ambrosia beetles can expel ROD-causing fungi into the environment in their frass. Frass is a mixture of chewed wood pieces, beetle body parts, and beetle poop.
There are 5 main ambrosia beetle species on Hawaiʻi Island that brood in Metrosideros polymorpha (ʻōhiʻa lehua) associated with Rapid ʻŌhiʻa Death (ROD).
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Ambrosia beetles can be morphologically similar and often difficult to identify. For example, Xyleborus affinis and Xyleborus perforans differ by the obscure characteristic of shininess of the declivity. Additionally, a tool to facilitate the identification of ambrosia beetles from their frass would be beneficial to identify beetle species producing viable ROD-causing inoculum, negating invasive extraction and/or waiting for beetle emergence.
Introduction
Most ambrosia beetle species (Coleoptera: Curculionidae: Scolytidae) are fungus farmers, planting their "ambrosia" fungi food source in their "homes" called galleries in the xylem of woody plants.
While creating their galleries, ambrosia beetles can expel ROD-causing fungi into the environment in their frass. Frass is a mixture of chewed wood pieces, beetle body parts, and beetle poop.
There are 5 main ambrosia beetle species on Hawaiʻi Island that brood in Metrosideros polymorpha (ʻōhiʻa lehua) associated with Rapid ʻŌhiʻa Death (ROD).
Ambrosia beetles can be morphologically similar and often difficult to identify. For example, Xyleborus affinis and Xyleborus perforans differ by the obscure characteristic of shininess of the declivity. Additionally, a tool to facilitate the identification of ambrosia beetles from their frass would be beneficial to identify beetle species producing viable ROD-causing inoculum, negating invasive extraction and/or waiting for beetle emergence.
Ambrosia beetle DNA for all 5 target species was extracted using the Qiagen DNeasy Blood & Tissue extraction kit according to the manufacturers' protocol. The universal COI insect primers, LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify and sequence (bi-directionally) beetle DNA from a minimum of two beetles per species. All PCR’s were comprised of 12.5 µl of 2x NEB OneTaq master mix with standard buffer, .5 µl of each 10 µM primer, .5-20 ng of DNA, and nuclease-free water to 25 µl. Reactions were conducted in an BioRad DNAEngine Peltier Thermal Cycler according to parameters in Cognato et. al. 2011. All products were visualized on 1.5% Seakem agarose gels containing ethidium bromide. All PCR clean-up was conducted using the Exo-CIP Rapid PCR Cleanup Kit from New England Biolabs and Sanger sequencing was conducted by Sequetech (Mountain View, CA).
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Ambrosia beetle COI sequences were edited and concatenated in Geneious 8.1.6. All Folmer-amplified beetle sequences were aligned using Genious Pairwise Alignment, and primers were designed for regions that were heterogeneous between species and homogeneous within species for a desired amplicon product of 209 bp using Primer3. Based on the alignment, two primer pairs were designed: COI 342 F, COI 582 and COI 342 AMB F, COI582 AMB R. One pair contained degenerate or ambiguous nucleotides and the other without.
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Standard desalted custom DNA oligos were synthesized by Integrated DNA Technologies. Optimal annealing temperatures were obtained by testing all primer combinations (4 total, PP1-4) on beetle DNA extractions of two of the species using a temperature gradient ranging from 48-58°C. Optimal cycling and temperatures were found at 30 sec at 94°C initial denaturing, followed by 35 cycles of at 94°C for 30 sec (denature), 55°C for 1 min (anneal), 68°C for 40 sec (extension) and a final extension of 68°C for 5 minutes for all primer pair combinations.
Primer Pair Combinations:
PP1 = COI342F + COI582R
PP2 = COI342AMBF+COI582AMBR
PP3 = COI342F + COI582AMBR
PP4 = COI342AMBF + COI582R
Primer pair combinations with optimal annealing temperature were then tested on Xyleborinus saxesenii DNA, the most distantly related of the 5 beetles. PP4 not amplify X. saxesenii, therefore this primer pair set was eliminated and not used for further testing. PP1-3 amplified all 5 ambrosia beetle species, but PP3 produced the brightest bands on a check-gel.
Frass collected in emergence traps with known beetle species sources for all 5 ambrosia beetle species was obtained from various studies, extracted, amplified, and visualized using both PP1 and PP3.
Methods
Ambrosia beetle DNA for all 5 target species was extracted using the Qiagen DNeasy Blood & Tissue extraction kit according to the manufacturers' protocol. The universal COI insect primers, LCO1490 and HCO2198 (Folmer et al. 1994) were used to amplify and sequence (bi-directionally) beetle DNA from a minimum of two beetles per species. All PCR’s were comprised of 12.5 µl of 2x NEB OneTaq master mix with standard buffer, .5 µl of each 10 µM primer, .5-20 ng of DNA, and nuclease-free water to 25 µl. Reactions were conducted in an BioRad DNAEngine Peltier Thermal Cycler according to parameters in Cognato et. al. 2011. All products were visualized on 1.5% Seakem agarose gels containing ethidium bromide. All PCR clean-up was conducted using the Exo-CIP Rapid PCR Cleanup Kit from New England Biolabs and Sanger sequencing was conducted by Sequetech (Mountain View, CA).
Ambrosia beetle COI sequences were edited and concatenated in Geneious 8.1.6. All Folmer-amplified beetle sequences were aligned using Genious Pairwise Alignment, and primers were designed for regions that were heterogeneous between species and homogeneous within species for a desired amplicon product of 209 bp using Primer3. Based on the alignment, two primer pairs were designed: COI 342 F, COI 582 and COI 342 AMB F, COI582 AMB R. One pair contained degenerate or ambiguous nucleotides and the other without.
Standard desalted custom DNA oligos were synthesized by Integrated DNA Technologies. Optimal annealing temperatures were obtained by testing all primer combinations (4 total, PP1-4) on beetle DNA extractions of two of the species using a temperature gradient ranging from 48-58°C. Optimal cycling and temperatures were found at 30 sec at 94°C initial denaturing, followed by 35 cycles of at 94°C for 30 sec (denature), 55°C for 1 min (anneal), 68°C for 40 sec (extension) and a final extension of 68°C for 5 minutes for all primer pair combinations.
Primer Pair Combinations:
PP1 = COI342F + COI582R
PP2 = COI342AMBF+COI582AMBR
PP3 = COI342F + COI582AMBR
PP4 = COI342AMBF + COI582R
Primer pair combinations with optimal annealing temperature were then tested on Xyleborinus saxesenii DNA, the most distantly related of the 5 beetles. PP4 not amplify X. saxesenii, therefore this primer pair set was eliminated and not used for further testing. PP1-3 amplified all 5 ambrosia beetle species, but PP3 produced the brightest bands on a check-gel.
Frass collected in emergence traps with known beetle species sources for all 5 ambrosia beetle species was obtained from various studies, extracted, amplified, and visualized using both PP1 and PP3.
1. We designed primers that can be used to identify all 5 ambrosia beetle species that are associated with ROD.
2. We have identified ambrosia beetle species from their frass using PCR in 3 of the 5 beetle species so far and have obtained successful amplification for all 5 species.
All 5 whole-beetle species DNA extractions were successfully amplified and sequenced using PP1 and PP3. However, PP1 produced some non-specific binding for Xyleborinus saxesenii and PP3 yielded low quality, yet accurate sequencing for Xyleborus affinis for reverse sequencing using COI 582 AMB R.
Three sets of DNA extracted from Xyleborus ferrugineus and perforans frass obtained from a previous field study (Roy et al. 2018) were successfully amplified and sequenced using both PP1 and PP3, successfully matching their respective ambrosia beetle source. Frass from X. similimus, X. affinis, and X. saxesenii has been successfully amplified and visualized on a check-gel but has not yet been sequenced. Frass from 2 unknown frass samples from emergence traps in Olaʻa Forest Reserve have been amplified and sequenced using PP1, matching 100% to Xyleborus similimus.
Results
1. We designed primers that can be used to identify all 5 ambrosia beetle species that are associated with ROD.
2. We have identified ambrosia beetle species from their frass using PCR in 3 of the 5 beetle species so far and have obtained successful amplification for all 5 species.
All 5 whole-beetle species DNA extractions were successfully amplified and sequenced using PP1 and PP3. However, PP1 produced some non-specific binding for Xyleborinus saxesenii and PP3 yielded low quality, yet accurate sequencing for Xyleborus affinis for reverse sequencing using COI 582 AMB R.
Three sets of DNA extracted from Xyleborus ferrugineus and perforans frass obtained from a previous field study (Roy et al. 2018) were successfully amplified and sequenced using both PP1 and PP3, successfully matching their respective ambrosia beetle source. Frass from X. similimus, X. affinis, and X. saxesenii has been successfully amplified and visualized on a check-gel but has not yet been sequenced. Frass from 2 unknown frass samples from emergence traps in Olaʻa Forest Reserve have been amplified and sequenced using PP1, matching 100% to Xyleborus similimus.
Increased annealing temperature for X. saxesenii using PP1 may decrease the non-specific binding. PP3 may be best suited for all 5 ambrosia beetle species, using the non-ambiguous reverse primer for reverse sequencing of X. affinis specifically.
Once all beetle species identifications from frass has been confirmed by sequencing, field-collected “fresh” frass from a single gallery will be tested. However, field collected frass from emergence traps may prove to be more fruitful than random sampling due to guarantee of less-degraded DNA and avoiding the mixing of multiple species' frass particles, preventing mixed sequencing signals. Trap-free collections will also be limited by quantity of frass.
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Although more work is necessary for this study, results are promising and further down-stream applications of these molecular markers appears obtainable.
Discussion and Future Directions
Increased annealing temperature for X. saxesenii using PP1 may decrease the non-specific binding. PP3 may be best suited for all 5 ambrosia beetle species, using the non-ambiguous reverse primer for reverse sequencing of X. affinis specifically.
Once all beetle species identifications from frass has been confirmed by sequencing, field-collected “fresh” frass from a single gallery will be tested. However, field collected frass from emergence traps may prove to be more fruitful than random sampling due to guarantee of less-degraded DNA and avoiding the mixing of multiple species' frass particles, preventing mixed sequencing signals. Trap-free collections will also be limited by quantity of frass.
Although more work is necessary for this study, results are promising and further down-stream applications of these molecular markers appears obtainable.
