Detection of Ochyromera ligustri (Coleoptera: Curculionidae: Curculioninae) in Ligustrum spp. (Oleaceae) Using Newly Developed PCR Primers

Simple Summary Privets (Ligustrum spp.) are popular hedge shrubs that escape from gardens and cause negative impacts by invading forests in the United States. The narrow host specificity of the seed-feeding weevil, Ochyromera ligustri, suggests that this weevil could be considered a fortuitous biological control agent of privets. To find out the host range of this weevil, time-consuming experiments involving rearing and dissections are often conducted. Herein, we developed PCR primers to rapidly and accurately detect the presence of the weevil in privet fruits. We confirmed that the novel PCR primers specifically work for O. ligustri even with a small quantity of DNA, which was 0.01 ng. Using the newly developed PCR-based detection saves time and labor in research related to the detection and impact of this weevil. Abstract Ligustrum spp. (Oleaceae) have become invasive species in the US and negatively affect native plant diversity and richness in forests. Ochyromera ligustri (Coleoptera: Curculionidae) is considered a potential biological control agent in the US because adults feed on the foliage and larvae are seed-feeders of Ligustrum spp. To discover the relationships between O. ligustri and Ligustrum spp., fruit dissections or rearing and field observations are required. In the current research project, novel PCR primers were developed to rapidly detect the DNA of O. ligustri in molecular analyses without rearing and observation. The developed PCR primers worked even with 0.01 ng of DNA and did not amplify the DNA of the other five curculionid species tested. When the novel primers were tested with three Ligustrum spp. species common in the southeastern US, the DNA of O. ligustri was detected from all three species. We expect that the novel primers will be utilized to find out the presence and impact of O. ligustri on Ligustrum spp rapidly and accurately.


Introduction
Ligustrinae Koehne 1893 is a monophyletic subtribe comprising Ligustrum (Oleaceae; common name: Privet) and Syringa (Oleaceae; common name: Lilac) [1].Many members of the two genera are ornamental shrubs, and Ligustrum includes species frequently used as hedge plants [2].Since the late 18th century, at least nine species of Ligustrum have been introduced into the United States (US) for ornamental purposes [3], and various hybrids and varieties have also been developed [4].Planted Ligustrum spp.escape from gardens to the wild, and they are considered invasive species or potential problem species in the US [3].
Insects 2024, 15 The invasion of Ligustrum sinense in the US has resulted in negative impacts on forest ecosystems [5].The imported plants were especially well-established in the southeastern US [3], and many native plants and shrub species have been replaced by L. sinense in floodplains [6].Moreover, the aggressiveness to L. sinense thickets has been associated with a reduction in forest regeneration, suggesting a change in ecosystem communities [7].In the forests near the Wolf River in Tennessee, where the river was channelized, resulting in dry areas, L. sinense became more abundant in the post-channelized conditions.Additionally, a noticeable deceleration in the growth of canopy oaks was observed [8].The growth of other native species commonly distributed in floodplain forests, Acer negundo L. subspe.negundo (Sapindaceae; common name: box elder), Boehmeria cylindrica (L.) Swartz (Urticaceae; common name: false nettle), Carex tribuloides Wahlenburg var.tribuloides (Cyperaceae; common name: blunt broom sedge), and Chasmanthium latifolium (Michaux) Yates (Poaceae: common name: northern wood oats), were also inhibited by L. sinense in floodplain forests along the Piedmont ecoregion of South Carolina [6].
To manage the populations of L. sinense and confirm the usefulness of conventional control methods for the plants, numerous studies have been conducted.The effectiveness of physical and chemical controls was compared, and chemical treatment was more successful than physical treatment [9].Among chemicals, glyphosate was more effective than triclopyr, but the former did not provide 100% control of L. sinense populations from the test plots [10].In a study investigating the effectiveness of burning and chemical treatments, neither treatment method was effective in reducing L. sinense populations [11].Due to the negative environmental effects and other limitations of conventional control methods, biological control has been considered as an alternative method for the management of L. sinense and some other Ligustrum species [12][13][14].
In June 1959, the seed-feeding weevil Ochyromera ligustri Warner 1961 (Figure 1) was first detected in Wake County, North Carolina, US, and was confirmed to be feeding on L. japonicum Thunberg 1780 [15,16].The adults of this weevil are 3.0 to 4.7 mm in body length and feed on the fruits of L. amurense Carrière, 1861 [15,16], L. japonicum [15,16], L. lucidum WT Aiton, 1810 (Warner 1961) [15,16], L. sinense [15][16][17], Syringa spp., and Vitis spp.(Vitaceae; common name: grapes) [16].Even though the exact origin of O. ligustri is unknown, the oriental or far east Paleartic regions are considered the weevil's origin [15,17].After its first discovery in North Carolina, O. ligustri has been reported from most states in the southeastern US, New Jersey, and North and South Dakota (Figure S1) [15,16,18,19].In North Carolina, adult females were noted to oviposit a single egg in the fruit seeds or flesh, and the emerging larvae feed on the fruit and seeds during the fall and winter [16].The larvae mature by April, and adult weevils emerge from the fruit found on the ground below the privets in May [16].The complete development of O. ligustri was confirmed from only L. japonicum [16,17], but O. ligustri in the US was presumed to be adapted to L. sinense and be able to complete its development [20].These previous investigations were conducted based on observation, rearing, and dissection, which are time-consuming steps to determine host associations [21].Due to the uncertainty of the host associations from these reports, it is important that future studies define the host plants suitable for adult feeding and immature development [22].
With the advancement of molecular techniques, diverse molecular analyses are being utilized in biological control research.In particular, polymerase chain reaction (PCR)based detection methods are widely used to identify target organisms [23][24][25], confirm host ranges and trophic relationships [26][27][28][29], and detect gut content [30,31].Since the traditional rearing and dissection of insects are laborious and lengthy experimental processes, the utilization of PCR-based methods allow investigators to rapidly detect and correctly identify target organisms [32][33][34].In a tachinid parasitoid study, a PCR-based method identified parasitism rates that were three times higher than rearing methods [34].
We propose to utilize a PCR-based method since molecular-based work can improve on the weakness of traditional rearing and dissection experimental methods, which are time-consuming, laborious, and error-prone.The potential of O. ligustri as a fortuitous biological control agent (BCA) of invasive Ligustrum spp. or a pest of the Oleaceae species is unknown.Therefore, the objectives of this study were (a) to develop species-specific PCR primers for the quick detection and identification of O. ligustri and (b) to confirm the host range of O. ligustri based on the molecular data.We propose to utilize a PCR-based method since molecular-based work can improve on the weakness of traditional rearing and dissection experimental methods, which are time-consuming, laborious, and error-prone.The potential of O. ligustri as a fortuitous biological control agent (BCA) of invasive Ligustrum spp. or a pest of the Oleaceae species is unknown.Therefore, the objectives of this study were (a) to develop species-specific PCR primers for the quick detection and identification of O. ligustri and (b) to confirm the host range of O. ligustri based on the molecular data.

Sample Collection and Processing
Multiple adults of Ochyromera ligustri were collected from Ligustrum sinense found along hiking trails at the Forest Community Park located in the southeastern part of East Baton Rouge Parish (Baton Rouge, LO, USA; site A) on 6 August 2022, using a beating sheet by the corresponding author.Fruit samples of Ligustrum sinense were collected at the same place on 4 October 2022.Weevil specimens were directly preserved in 100% ethanol.At the collecting site, L. sinense was the most abundant among Ligustrum spp., and L. lucidum was the second most abundant.
We targeted seemingly unhealthy fruit samples or samples with feeding or oviposition marks by O. ligustri.The fruit samples were separated into three groups using a stereomicroscope: i. unhealthy brown; ii.green with feeding or oviposition marks; iii.green without any feeding or oviposition marks (Figure 2).Fruit samples of L. lucidum and L. japonicum were collected near the University Lake at Louisiana State University (Baton Rouge, Louisiana; site B) and forest on a private property (Rosedale, Louisiana; site C).Fruit samples of non-Ligustrum spp.having a similar fruit shape living near L. sinense on site A were collected by the authors.Fruits of the following plant species were included: Ardisia crenata Sims, 1818 (Primulaceae; common name: coral ardisia), Ilex vomitoria Aiton (Aquifoliaceae; common name: yaupon holly), Nandina domestica Thunberg (Berberidaceae; common name: heavenly bamboo), and Sambucus canadensis L. (Adoxaceae; common name: American black elderberry).We preserved the fruit samples in a refrigerator at 4 °C.The fruit samples were used in molecular analyses within two weeks of collection.

Sample Collection and Processing
Multiple adults of Ochyromera ligustri were collected from Ligustrum sinense found along hiking trails at the Forest Community Park located in the southeastern part of East Baton Rouge Parish (Baton Rouge, LO, USA; site A) on 6 August 2022, using a beating sheet by the corresponding author.Fruit samples of Ligustrum sinense were collected at the same place on 4 October 2022.Weevil specimens were directly preserved in 100% ethanol.At the collecting site, L. sinense was the most abundant among Ligustrum spp., and L. lucidum was the second most abundant.
We targeted seemingly unhealthy fruit samples or samples with feeding or oviposition marks by O. ligustri.The fruit samples were separated into three groups using a stereomicroscope: i. unhealthy brown; ii.green with feeding or oviposition marks; iii.green without any feeding or oviposition marks (Figure 2).Fruit samples of L. lucidum and L. japonicum were collected near the University Lake at Louisiana State University (Baton Rouge, Louisiana; site B) and forest on a private property (Rosedale, Louisiana; site C).Fruit samples of non-Ligustrum spp.having a similar fruit shape living near L. sinense on site A were collected by the authors.Fruits of the following plant species were included: Ardisia crenata Sims, 1818 (Primulaceae; common name: coral ardisia), Ilex vomitoria Aiton (Aquifoliaceae; common name: yaupon holly), Nandina domestica Thunberg (Berberidaceae; common name: heavenly bamboo), and Sambucus canadensis L. (Adoxaceae; common name: American black elderberry).We preserved the fruit samples in a refrigerator at 4 • C. The fruit samples were used in molecular analyses within two weeks of collection.Mississippi, and Auburn, Alabama; and L. sinense berries were collected from Kathleen, Perry, and Thomaston, Georgia; and Houston, Beaumont, and High Island, Texas.Weevil specimens were preserved in 100% ethanol, while the berry samples were stored in a freezer at −20 °C until used for processing.

Primer Design
To develop the COI species-specific primers, a LepF1/LepR1 primer set was used to obtain a 658 bp-long COI sequence of Ochyromera ligustri (Table 1).After obtaining the COI sequence of O. ligustri, we searched for similar sequences using the MegaBLAST al- For a primer specificity test, specimens of five other curculionid species were included.Specimens of Anthonomus quadrigibbus Say, 1831, Tychius picirostris (Fabricius, 1787), and Lignyodes sp. were provided by the Louisiana State Arthropod Museum (LSAM; Baton Rouge, LO, USA), and specimens of Cyrtobagous salviniae Calder and Sands, 1985 and Tanysphyrus lemnae (Paykull, 1792) were reared in an experimental pond in St. Gabriel, Louisiana by the LSU Entomology Laboratory and included in the test.Five to ten live adults of C. salviniae and T. lemnae were transferred into vials containing 70% ethanol and preserved until processing.To confirm the effectivity of the primers on O. ligustri collected outside of Louisiana, weevils were collected in 2023 from Wesson, Mississippi, and Auburn, Alabama; and L. sinense berries were collected from Kathleen, Perry, and Thomaston, Georgia; and Houston, Beaumont, and High Island, Texas.Weevil specimens were preserved in 100% ethanol, while the berry samples were stored in a freezer at −20 • C until used for processing.

DNA Extraction and Polymerase Chain Reaction Amplification
The DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) was used to extract the genomic DNA from the weevils and the weevil DNA in Chinese privet fruits.The entire weevil body (one to three individuals) was ground using Fisherbrand™ RNase-Free Disposable Pellet Pestles (Fisher Scientific, Carlsbad, CA, USA) in 180 µL of ATL buffer and 20 µL of proteinase K solution (Qiagen, Hilden, Germany).For the fruit samples, we put one to ten fruit samples in each microcentrifuge tube (1.5 mL).When we used one to two fruit samples, we ground the fruit samples using pestles, and 180 µL of ATL buffer and 20 µL of proteinase K solution or 1.5 times of each buffer were used (Table S1).When we put ten fruit samples in a microcentrifuge tube, we did not grind the fruit samples but cut each fruit sample in half instead.A total of 900 µL of ATL buffer and 100 µL of proteinase K solution was used for a tube including ten fruit samples (Table S1).We incubated the prepared samples overnight at 56 • C. The tubes with weevils or one to two fruit samples were directly used in the remaining Insects 2024, 15, 320 5 of 12 steps to extract the DNA following the manufacturer's protocol.For the tube with ten fruit samples, ~400 µL of the incubated supernatant was relocated to new microcentrifuge tubes and processed (Table S1; tubes 6, 8, and 10).In the following two steps, 100 µL of AL buffer (Qiagen, Hilden, Germany) and 100 µL of 100% ethanol were used (Table S1).In the remaining steps, we followed the manufacturer's protocol.
The PCR reaction was performed using a T100™ thermal cycler (Bio-Rad, Hercules, CA, USA).The total volume of each PCR sample was 25 µL, containing 12.5 µL of DreamTaq Green PCR Master Mix (2X) (Thermo Scientific, Waltham, MA, USA), 1-2 µL of template genomic DNA, 7.5-8.5 µL of nuclease-free water, 0.5 µL MgCl2 (Thermo Scientific), and 1.0 µL of each primer at 5 µM resuspended in a low TE buffer (10 mM Tris, 0.05 mM EDTA, pH 8).Using a gradient PCR method ranging from 45 to 65 • C, an optimal annealing temperature for a newly developed primer set was found.A PTC-200 thermal cycler was used (MJ Research, Watertown, MA, USA).The PCR conditions were 95 • C for 3 min; 35 cycles of 95 • C for 30 s, 58 • C for 30 s, and 72 • C for 1 min; and a final extension at 72 • C for 7 min for a newly developed species-specific primer set.For LepF1 and LepR1, the PCR conditions were 95 • C for 3 min; 5 cycles of 95 • C for 30 s, 45 • C for 30 s, and 72 • C for 1 min; and 35 cycles of 95 • C for 30 s, 51 • C for 30 s, and 72 • C for 1 min; and a final extension at 72 • C for 7 min.Using a 2.0% agarose gel stained with 1X SYBR™ Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), we confirmed the amplified products.The PCR products were cleaned using a primer depletion clean-up method developed at the LSU genomics facility (Baton Rouge, LA, USA) and sequenced on the 3130xl genetic analyzers (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v 3.1 chemistry (Applied Biosystems) at the same facility.

DNA Sequence Assembly
Using Geneious Prime (version 2022.0.1) (https://www.geneious.com;accessed on 2 September 2022) [39], we obtained the DNA sequences with high quality, which were edited.The primer regions were confirmed and trimmed.The edited sequences were assembled via de novo assembly.The consensus sequences were compared with the sequences deposited in the GenBank database using BLASTn.

Primer Specificity
The DNA from the other five curculionid species was extracted using the method described in 2.3.To confirm the success of DNA extraction, ~650 bp of the COI region was amplified using LepF1/LepR1 and visualized on a 2.0% stained agarose gel.We then tested the newly developed primers in PCR and gel electrophoresis using the DNA extracted from O. ligustri and the other five curculionid species to see whether the primer set only amplified the DNA of O. ligustri.In addition, the PCR products amplified using the newly developed primers were sequenced and assembled using the same method mentioned above to confirm whether the DNA was amplified from O. ligustri.

Primer Sensitivity
To confirm the sensitivity of the newly developed primer set, we ran a PCR amplification with different quantities of DNA extracted from O. ligustri.After confirming a raw DNA sample concentration (5.05 ng/µL) using the Qubit™ 4 fluorometer using the Qubit™ dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), we then prepared eight DNA samples for the sensitivity test (10 ng, 5 ng, 1 ng, 0.5 ng, 0.1 ng, 0.05 ng, 0.01 ng, and negative).For the samples with 10 ng and 5 ng, we loaded 2 µL and 1 µL into 25 µL PCR samples, respectively.For the samples with ≤ 1 ng DNA concentrations, we diluted the raw DNA sample (5.05 ng/µL) with nuclease-free water and prepared the appropriate DNA concentrations.In the negative control, only nuclease-free water was loaded.After 35 cycles of the PCR reaction, the PCR samples were visualized on a 2.0% stained gel.

Application to Fruit Samples of Ligustrum spp.
To confirm the ability of the newly developed primers to detect O. ligustri DNA in the infected fruits of L. sinense in the PCR and gel electrophoresis, we prepared the fruit samples following the method in 2.1 and extracted the DNA using the method described in 2.3 (Table S1).We amplified the DNA of O. ligustri with the species-specific primers in the PCR and confirmed the presence of the DNA of O. ligustri as well as the ability of the species-specific primers in gel electrophoresis.After we confirmed that the novel primers amplified the DNA of O. ligustri in the fruit samples of L. sinense, we utilized the primers to determine if the DNA of O. ligusti was present in the fruits of L. lucidum and L. japonicum (Table S2).To test for O. ligustri's DNA presence in the fruit samples of the other plant species having similar fruit shapes and sizes living near L. sinense in site A, the same molecular methods were used (Table S3).

Distribution Map
Distribution records of Ligustrum spp.(31,392), Syringa spp.(14,615), and O. ligustri (123) in the US were available from the Global Biodiversity Information Facility (GBIF) (26 June 2023).We downloaded all GPS coordinate data from the GBIF and generated a map presenting the distribution of Ligustrum spp., Syringa spp., and O. ligustri in the US using ArcGIS Pro 3.0 (version 3.0.0)[40] (Figure S1).

Species Primer Design and Optimal PCR Conditions
A species-specific primer set, COI_O_ligustri_F1/COI_O_ligustri_R1 (Table 1), was successful in detecting O. ligustri DNA in the tested fruits.The primer set produced 182 bp of a target sequence after trimming the primer regions (Figure S4).The obtained 182 bp sequence (sequences with the following GenBank accession numbers: OQ199864; OQ199865) was a 100% match with an internal region of the 658 bp sequence of O. ligustri (sequences with the following GenBank accession numbers: OQ199862; OQ199863).Using a gradient PCR, we found that the optimal annealing temperature for the species-specific primers was 58 • C (Figure S4) when the T-100 thermal cycler (Bio-Rad) was used.

Primer Specificity
In order to confirm whether the raw DNA of O. ligustri, A. quadrigibbus, Ta. lemnae, Lignyodes sp., Ty.Picirostris, and C. salviniae were successfully extracted from the specimens, we amplified the standard COI region (658-bp) of the six curculionid species and ran a gel (Figure 3A).The COI DNA sequences of all curculionid species were successfully amplified and obtained (Figure 3).Using the raw DNA of the six curculionid species, we tested the specificity of the newly developed PCR primers (COI_O_ligustri_F1 and COI_O_ligustri_R1).The novel PCR primers only amplified 182 bp of O. ligustri DNA (Figure 3B).

Primer Sensitivity
In the primer sensitivity test, we confirmed that strong visible bands were produced when ≥0.05 ng of raw DNA was used in a 25 µL PCR reaction (35 cycles), and a slightly visible band was produced with 0.01 ng of raw DNA in the PCR reaction with the same conditions (Figure 4).

Primer Sensitivity
In the primer sensitivity test, we confirmed that strong visible bands were produced when ≥0.05 ng of raw DNA was used in a 25 µL PCR reaction (35 cycles), and a slightly visible band was produced with 0.01 ng of raw DNA in the PCR reaction with the same conditions (Figure 4).

Primer Sensitivity
In the primer sensitivity test, we confirmed that strong visible bands were produced when ≥0.05 ng of raw DNA was used in a 25 µL PCR reaction (35 cycles), and a slightly visible band was produced with 0.01 ng of raw DNA in the PCR reaction with the same conditions (Figure 4).

Application to the Fruit Samples of Ligustrum spp.
The DNA of O. ligustri was detected in the fruit samples of L. sinense, especially from the fruit group i (brown and unhealthy) and group ii (green with feeding or oviposition marks), but not from the fruit group iii (healthy and green without any marks) (Figure 5B).The DNA of O. ligustri was found in all three Ligustrum species included in the test (L.sinense, L. lucidum, and L. japonicum) (Figure 5A).The DNA of O. ligustri was only detected from L. sinense, but not from the other non-ligustrum species collected in site A, including Ardisia crenata, Sambucus sp., unidentified sp., and Nandina sp., which have fruits with a similar shape and size (Figure 6).The primers were also successful in detecting the DNA in weevils from both MS and AL and in berries from MS and GA (Figure S5).The DNA of O. ligustri was detected in the fruit samples of L. sinense, especially from the fruit group i (brown and unhealthy) and group ii (green with feeding or oviposition marks), but not from the fruit group iii (healthy and green without any marks) (Figure 5B).The DNA of O. ligustri was found in all three Ligustrum species included in the test (L.sinense, L. lucidum, and L. japonicum) (Figure 5A).The DNA of O. ligustri was only detected from L. sinense, but not from the other non-ligustrum species collected in site A, including Ardisia crenata, Sambucus sp., unidentified sp., and Nandina sp., which have fruits with a similar shape and size (Figure 6).The primers were also successful in detecting the DNA in weevils from both MS and AL and in berries from MS and GA (Figure S5).S2.

Discussion
The newly developed primers successfully amplified the DNA of O. ligustri, not only from the weevils but also from the O. ligustri larvae-infested fruit samples of all three Ligustrum spp.species (L.sinense, L. lucidum, and L. japonicum) (Figures 5 and 6).All the steps (sample preparation, DNA extraction, PCR, gel electrophoresis) were completed in two days.The novel primer set for O. ligustri will be useful for the rapid and accurate identification and detection of O. ligustri, enabling future studies to rapidly confirm the presence of O. ligustri in the newly infested regions and measure the initial dispersal and establishment rates of O. ligustri.
Researchers can save time in confirming the presence of O. ligustri with direct PCR or time-efficient DNA extraction methods.However, sensitivity tests should be performed when raw DNA are extracted using different isolation methods.To detect the specific DNA from a targeted organism, finding the optimal PCR conditions is important for specificity and efficiency [41].Our gradient PCR results showed that the novel primer set visualized fairly bright bands at an annealing temperature range of 46-64 °C (Figure S4).However, we recommend the use of annealing temperatures of 55-58 °C because (a) the  S3.

Discussion
The newly developed primers successfully amplified the DNA of O. ligustri, not only from the weevils but also from the O. ligustri larvae-infested fruit samples of all three Ligustrum spp.species (L.sinense, L. lucidum, and L. japonicum) (Figures 5 and 6).All the steps (sample preparation, DNA extraction, PCR, gel electrophoresis) were completed in two days.The novel primer set for O. ligustri will be useful for the rapid and accurate identification and detection of O. ligustri, enabling future studies to rapidly confirm the presence of O. ligustri in the newly infested regions and measure the initial dispersal and establishment rates of O. ligustri.
Researchers can save time in confirming the presence of O. ligustri with direct PCR or time-efficient DNA extraction methods.However, sensitivity tests should be performed when raw DNA are extracted using different isolation methods.To detect the specific DNA from a targeted organism, finding the optimal PCR conditions is important for specificity and efficiency [41].Our gradient PCR results showed that the novel primer set visualized fairly bright bands at an annealing temperature range of 46-64 • C (Figure S4).However, we recommend the use of annealing temperatures of 55-58 • C because (a) the brightest bands were produced at 55 • C and 58 • C, (b) low annealing temperatures frequently produce non-specific bands [42], (c) band production is decreased when high annealing temperatures are used [42].Regarding the PCR cycles, we confirmed that 40 PCR cycles produced a brighter band compared to 35 PCR cycles when 0.01 ng of DNA of O. ligustri was used.However, using more than 40 PCR cycles is undesirable due to the possibility of producing non-specific bands [43].
The newly developed PCR primers can be employed for the streamlined confirmation of the DNA presence of O. ligustri in Ligustum spp.and Syringa spp.established in the US.The widespread distribution of Ligustum spp.and Syringia sp. in the central southeastern and northeastern US, respectively (Figure S1), emphasizes the importance of understanding the impact of O. ligustri across this vast climatic gradient.We envision utilizing the novel primers to conduct a citizen science project to determine the current distribution of O. ligustri in the US.The specific primers will allow the prompt testing of fruit samples for the presence of weevil DNA within one week.
While these PCR primers facilitate swift and straightforward O. ligustri DNA detection from fruit samples, gauging the actual infection rates necessitates suitable sampling strategies.Drawing from the approach of diverse studies [44][45][46][47][48], the refinement of the optimal sampling and measurement strategies is crucial to assess the impact of O. ligustri on Ligustrum spp.For expeditious and cost-effective O. ligustri screening, the adoption of pooled testing, akin to SARS-CoV-2 (COVID-19) monitoring [47, 49,50], holds promise.Implementing pooled testing entails gathering seemingly unhealthy fruit, enhancing the detection of O. ligustri DNA, and ultimately economizing both the time and expenses associated with screening processes.
We also acknowledge certain limitations of the developed method.Specifically, identifying the sex of O. ligustri and discerning whether the extracted weevil DNA originated
were provided by the Louisiana State Arthropod Museum (LSAM; Baton Rouge, LO, USA), and specimens of Cyrtobagous salviniae Calder and Sands, 1985 and Tanysphyrus lemnae (Paykull, 1792) were reared in an experimental pond in St. Gabriel, Louisiana by the LSU Entomology Laboratory and included in the test.Five to ten live adults of C. salviniae and T. lemnae were transferred into vials containing 70% ethanol and preserved until processing.To confirm the effectivity of the primers on O. ligustri collected outside of Louisiana, weevils were collected in 2023 from Wesson,

Figure 2 .
Figure 2. Ligustrum sinensis fruits of different conditions: (A) group i (unhealthy brown); (B) group ii (green with feeding or oviposition marks); (C) group iii (green without any feeding or oviposition marks).

Figure 2 .
Figure 2. Ligustrum sinensis fruits of different conditions: (A) group i (unhealthy brown); (B) group ii (green with feeding or oviposition marks); (C) group iii (green without any feeding or oviposition marks).

3. 4 .
Application to the Fruit Samples of Ligustrum spp.

Table 1 .
Primer information used in this project.LepF1/LepR1 was used to obtain the standard COI DNA sequences of the curculionid species, and COI_O_ligustri_F1/COI_O_ligustri_R1 were the newly developed species-specific primers for O. ligustri in the current project.