Marco A. Toapanta, David J. Schuster, Philip A. Pepper weevil, Anthonomus eugenii Cano, is the major arthropod pest of peppers, Capsicum spp. Adult weevils feed and oviposit in buds, flowers, and, especially, fruit. Larvae develop and feed inside those plant structures, thus reducing crop yields.
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The pepper weevil, Anthonomus eugenii , is a major pest on Capsicum species. Apart from natural spread, there is a risk of spread via international pepper trade. In the Netherlands, a pepper weevil outbreak occurred in and affected six greenhouses producing different sweet pepper varieties.
The following year, a pepper weevil outbreak occurred in Italy. To trace the origin of the Dutch outbreak and to establish if the Dutch and Italian outbreaks were linked, we determined the mitogenomes of A. The circular 17, bp A. Intra-species variation of these mitochondrial genes revealed four main mitochondrial lineages encompassing 41 haplotypes. The highest diversity was observed for specimens from its presumed native area i. The Dutch outbreak specimens represented three highly similar haplotypes, suggesting a single introduction of the pest.
The major Dutch haplotype was also found in two specimens from New Jersey. As the Netherlands does not have pepper trade with New Jersey, it is likely that the specimens sampled in New Jersey and those sampled in the Netherlands originate from a shared source that was not included in this study.
In addition, our analysis shows that the Italian and Dutch outbreaks were not linked. The mitochondrial genome is a useful tool to trace outbreak populations and the methodology presented in this paper could prove valuable for other invasive pest species, such as the African fruit moth Thaumatotibia leucotreta and emerald ash borer Agrilus planipennis.
This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Gene sequences of atp6, atp8, cytb, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6 of the Anthonomus spp. Cees Waalwijk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. The pepper weevil, Anthonomus eugenii Cano Coleoptera: Curculionidae , is considered as one of the most important pests on cultivated peppers Capsicum spp.
Larvae feed on soft tissues inside the developing fruit, whereas adults feed on flowers and young fruits. Cultivated peppers are the main host for A. Because of its impact, A. Based on the earliest reports of the pepper weevil and its hosts, it is generally believed that the species originates from Mexico or surrounding regions in Central America [ 7 ].
Today, A. Apart from its natural range, the pest has been found outdoors in Hawaii in [ 1 ], French Polynesia pre [ 9 ], Puerto Rico in [ 10 ], and the Dominican Republic in [ 11 ]. The pest was also encountered in greenhouses in southern Canada in [ 12 ] and again in and [ 13 ]. Similarly, the pest was found in greenhouses in the Netherlands [ 14 ] and Italy [ 15 ], where in the latter country the pest was also found outdoors.
For the European Union the risk of infestation from the import of Capsicum and Solanum fruits was initially regarded as low but with a high uncertainty , as import of host fruits from countries in which the pest is endemic occurs since at least and no outbreaks had taken place since [ 16 ].
In the Netherlands, A. The first and only outbreak of A. In October and November of the same year, two additional greenhouses were found to be infested. All infested greenhouses were situated close to each other 2 km radius and a surveillance program was initiated. Eradication measures were taken against A.
The National Plant Protection Organization declared that the pest was successfully eradicated in the Netherlands in December [ 18 ]. In Italy, the pest was declared eradicated in [ 19 ]. The mitochondrial genome is frequently used in phylogenetic [ 20 , 21 ] and population-level studies [ 22 , 23 ] as it typically represents a single maternally inherited molecule with low levels of recombination while being present in high copy numbers [ 24 , 25 ].
With the introduction of next generation sequencing technology, the number of annotated complete mitogenomes has increased tremendously [ 26 ]. For instance, higher level phylogenetic relationships in the weevils Curculionoidea , were determined by generating and analyzing close to one hundred near complete mitochondrial genomes from a single Illumina MiSeq run [ 27 ].
For the weevil tribe Anthonomini, one complete mitogenome, namely that of Bradybatus kellneri KX , and two partial mitogenomes, those of Anthonomus pomorum JN and Anthonomus grandis MG are present in the public domain.
To place the A. Following the Dutch outbreak, 32 specimens of A. Over fifty production sites of sweet pepper C. These traps were also placed in greenhouses producing tomatoes, egg-plants or ornamental Solanaceae within an area of 2 x 3 km around the infested sites.
In addition, specimens from regular import inspections performed by the Dutch National Plant Protection Organization NPPO-NL , and from collections as well as and specimens obtained from external contacts were included to create a reference panel of A. Three A.
Genomic DNA was extracted from whole or partial Anthonomus spp. Insect tissue was ground in lysis buffer with a micro-pestle prior to DNA extraction. Each sample was tagged to create an unique index-flowcell combination, and libraries were split over four sequencing lanes in one of six flowcells resulting in a median yield of 2, Mb per sample S1 Table. NextSeq reads of the Anthonomus spp.
The circular reference mitogenomes were annotated using the online MITOS tool using the invertebrate mitochondrial code [ 33 ]. The circular conformation of the A. For each of the specimens included, NextSeq reads were mapped to the annotated reference mitogenome sequence of the respective species in CLC genomic Workbench length fraction: 0.
Annotations were transferred from the reference sequences to the resulting specimen specific sequences. Datasets of fourteen specimens did not meet these requirements and were excluded from the analysis. Gene sequences of atp6 , atp8 , cytb , cox1 , cox2 , cox3 , nad1 , nad2 , nad3 , nad4 , nad4L , nad5 , and nad6 from the specimens included in the analysis were submitted in NCBI under accession numbers MK to MK Extracted mitochondrial coding sequences representing in total A.
The ML tree was provided with scientific name and origin of the specimens, and for A. A concatenated alignment of the thirteen mitochondrial genes of A.
Individual haplotypes within a subgroup were numbered sequentially e. Following the Dutch outbreak, 70 specimens were collected from the six outbreak locations, referred to as greenhouse 1 to 6. NextGen sequence data was generated for all of the Anthonomus specimens included in this study, and de novo assembly of this data resulted in 26, scaffolds on average. For A. The GRAbB assembly for this specimen resulted in a 17, bp circular mitogenome with The A.
Top: The A. Bottom: comparison of the A. Orthologous elements are linked, using green links for protein coding genes, purple links for tRNAs, and red links for rRNAs. Similarly, chimeric mitochondrial consensus sequences were created for A.
The complete mitogenomes of the four reference specimens show identical gene, rRNA and tRNA content, with conserved organization and orientation Fig 1 , and an overall GC-content ranging from Based on the primer sites designed with the 17, bp A.
The amplicons lengths obtained for the other primer pairs corresponds with the in silico design S1 Fig. In addition, read mapping of the A. This suggests a repetitive sequence is present in the trnI—trnQ intergenic region which was not resolved in the GRAbB assembly. The thirteen protein coding mitochondrial genes were used to determine the phylogenetic relation between the A. Gaps in the alignment were the result of gene length differences which were manually verified.
Intra-species variation observed for the protein coding genes in A. Interspecies variation between A. Clustering of A. Maximum Likelihood cladogram based on thirteen mitochondrial protein coding gene sequences of four Anthonomus species using Bradybatus kellneri as outgroup.
Branches are colored according to species identity and bootstrap values are shown for the major nodes. The inner colored ring represents the country of origin, whereas the outer colored ring represents the main A. The specimen names are displayed as labels on the inner node layer and A. Read mapping of NextSeq data of A. A median joining haplotype network was created from the concatenated alignments of the thirteen mitochondrial protein coding genes.
The resulting alignment comprised 10, nt and contained parsimony informative sites. Haplotype analysis shows that the A. A total of 41 unique haplotypes was obtained, of which fifteen consisted of more than one representative specimen S3 Table. Median Joining haplotype network representing A. Nodes in the network are colored based on the origin of the samples, and black nodes represent hypothetical ancestors.
Haplogroups are subdivided when they represent multiple localities of the same country. The specimens from Mexico display the highest diversity and are represented by 15 haplotypes covering all four major haplogroups. Specimens collected in the province of Jalisco cluster in haplogroups 1 and 4, whereas specimens from Chihuahua are found in both haplogroups 3 and 4 S3 Fig. Only single specimens from Tamaulipas and Aguascalientes were included in the study, which represent haplotypes in haplogroups 1 and 3, respectively.
No locality information was available for other Mexican specimens which represented haplotypes in haplogroups 1, 2, and 4, and were obtained from regular import inspections.
The high diversity displayed by the Mexican specimens is almost matched by specimens from New Jersey USA which were present in two major haplogroups and 14 different haplotypes. No link was identified between the sampling locality and the haplotype identity for samples from New Jersey and Georgia S4 Fig Specimens from the Dominican Republic were confined to subgroups 4F and 4G which represented 10 different haplotypes.
The two specimens from the Italian outbreak were found in haplotype 4F10, and cluster with 18 specimens from the Dominican Republic. All 32 specimens collected at the Dutch outbreak locations cluster in subgroup 1D, which consists of three haplotypes i.
Specimens with all three haplotypes were identified in greenhouse 2, whereas specimens from greenhouses 1, 4, 5 and 6 represented haplotypes 1D2 and 1D3.
List of symptoms / signs
The pepper weevil, Anthonomus eugenii Cano, is the most important insect pest of pepper in the southern United States. Figure 1. Adult pepper weevil, Anthonomus eugenii Cano. Photograph by Paul M. Choate, University of Florida.
EPPO Global Database
The pepper weevil, Anthonomus eugenii , is a major pest on Capsicum species. Apart from natural spread, there is a risk of spread via international pepper trade. In the Netherlands, a pepper weevil outbreak occurred in and affected six greenhouses producing different sweet pepper varieties. The following year, a pepper weevil outbreak occurred in Italy. To trace the origin of the Dutch outbreak and to establish if the Dutch and Italian outbreaks were linked, we determined the mitogenomes of A. The circular 17, bp A.