Wisconsin diversity panel phenotypes: spoken descriptions of plants and supporting data.

OBJECTIVES: Phenotyping plants in a field environment can involve a variety of methods including the use of automated instruments and labor-intensive manual measurement and scoring. Researchers also collect language-based phenotypic descriptions and use controlled vocabularies and structures such as ontologies to enable computation on descriptive phenotype data, including methods to determine phenotypic similarities. In this study, spoken descriptions of plants were collected and observers were instructed to use their own vocabulary to describe plant features that were present and visible. Further, these plants were measured and scored manually as part of a larger study to investigate whether spoken plant descriptions can be used to recover known biological phenomena. DATA DESCRIPTION: Data comprise phenotypic observations of 686 accessions of the maize Wisconsin Diversity panel, and 25 positive control accessions that carry visible, dramatic phenotypes. The data include the list of accessions planted, field layout, data collection procedures, student participants' (whose personal data are protected for ethical reasons) and volunteers' observation transcripts, volunteers' audio data files, terrestrial and aerial images of the plants, Amazon Web Services method selection experimental data, and manually collected phenotypes (e.g., plant height, ear and tassel features, etc.; measurements and scores). Data were collected during the summer of 2021 at Iowa State University's Agricultural Engineering and Agronomy Research Farms.

Geographic Distribution of Bacillus thuringiensis Cry1F Toxin Resistance in Western Bean Cutworm (Lepidoptera: Noctuidae) Populations in the United States.

The western bean cutworm (WBC), Striacosta albicosta (Lepidoptera: Noctuidae), can be a severe pest of transgenic corn in the western Plains and Great Lakes regions of North America, including on hybrids expressing the Bacillus thuringiensis (Bt) Cry1F toxin. The level and geographic distribution of Cry1F resistance are not completely known. Neonate S. albicosta from 10 locations between Nebraska and New York state were subjected to dose-response trypsin-activated native Cry1F toxin overlay bioassays. In 2017, the mean estimated lethal concentration causing 50% larval mortality (LC50) ranged from 15.1 to 18.4 µg Cry1F cm-2, and were not significantly different among locations. In 2018, LC50 estimates at Scottsbluff, NE (22.0 µg Cry1F cm-2) and Watertown, NY (21.7 µg Cry1F cm-2) were significantly higher when compared to locations in Michigan (15.8 µg Cry1F cm-2). Significantly lower 14-day larval weight among survivors was correlated with higher Cry1F dose. Results from this study indicate that S. albicosta survivorship on purified Bt Cry1F toxin shows a relatively even distribution across the native and range expansion areas where seasonal field infestations typically occur.

Temporal Variation in Genetic Composition of Migratory Helicoverpa Zea in Peripheral Populations.

Migrant populations of Helicoverpa zea (Boddie) captured during 2002, 2005, 2016, and 2018 from Landisville and Rock Springs in Pennsylvania, USA were genotyped using 85 single nucleotide polymorphism (SNP) markers. Samples (n = 702) genotyped were divided into 16 putative populations based on collection time and site. Fixation indices (F-statistics), analysis of molecular variance, and discriminant analysis of principal components were used to examine within and among population genetic variation. The observed and expected heterozygosity in putative populations ranged from 0.317-0.418 and 0.320-0.359, respectively. Broad range of FST (0.0-0.2742) and FIS (0.0-0.2330) values indicated different genotype frequencies between and within the populations, respectively. High genetic diversity within and low genetic differentiation between populations was found in 2002 and 2005. Interestingly, high genetic differentiation between populations from two collection sites observed in 2018 populations was not evident in within-site comparisons of putative populations collected on different dates during the season. The shift of H. zea population genetic makeup in 2018 may be influenced by multiple biotic and abiotic factors including tropical storms. Continued assessment of these peripheral populations of H. zea will be needed to assess the impacts of genetic changes on pest control and resistance management tactics.

Differentiation of European Corn Borer (Lepidoptera: Crambidae) and American Lotus Borer (Lepidoptera: Crambidae), Ostrinia penitalis, from North American Field Collections.

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is a perennial insect pest of cultivated maize that was inadvertently introduced into North America in the early 1900s, but population densities have decreased since the widespread adoption of transgenic hybrids that express Bacillus thuringiensis (Bt) toxins. The native American lotus borer, Ostrinia penitalis (Lepidoptera: Crambidae), is among the most ancestral species described in the genus Ostrinia, and has a geographic range that coincides with that of O. nubilalis across major maize growing regions of North America. Due to the recent decrease in O. nubilalis populations, O. penitalis has become more pronounced in light trap samples intended to monitor O. nubilalis. A molecular tool based on variation in restriction endonuclease digestion pattern of a polymerase chain reaction amplified fragment of the mitochondrial cytochrome c oxidase subunit I (coxI) gene was developed and validated to differentiate these two species. This method was applied to light trap samples over a 2-yr period and achieved accurate quantification of species, and shows that O. penitalis can be prevalent in O. nubilalis first flight sampling. These methods are useful for contemporary O. nubilalis field research in North America.

Development and application of a quantitative bioassay to evaluate maize silk resistance to corn earworm herbivory among progenies derived from Peruvian landrace Piura.

Corn earworm (CEW), Helicoverpa zea (Boddie), (Lepidoptera: Noctuidae), is a major insect pest of corn (Zea mays spp. mays L.). CEW larvae feed on silks, kernels and cobs, causing substantial yield and quality losses both through herbivory and by vectoring pathogens. The long-term goal of this work is to elucidate the genetic and biochemical basis of a potentially novel CEW resistance source discovered in silk tissue of Piura 208, a Peruvian landrace of maize (PI 503849). We developed a quantitative CEW bioassay and tested it on four populations that contrast alleles from Piura 208 with those from GT119, a CEW-susceptible maize inbred line. In replicated analyses of two populations of F1:2 families, corn genotype accounts for 84% and 68% of the variance in CEW larval weights, and up to 60% of the variance in CEW pupation percentage, demonstrating both the success of the quantitative bioassay and the strength of the Piura 208 resistance mechanism. Analyses of two corresponding populations of BC1:2 families revealed substantially diminished effects of corn genotype on CEW weight gain and pupation. This loss of Piura 208-derived CEW resistance during backcrossing suggests complex (multi-genic) inheritance of a threshold-dependent mechanism. Technical factors in bioassay performance were also assessed, often by analyzing the 1,641 CEW larvae that were raised on control diet (meridic with no corn silks added). Minor, but statistically significant impacts on CEW weight gain, pupation, and mortality were attributable to multiple technical factors in the preparation, incubation and evaluation phases of the bioassay, demonstrating the importance of randomization, stratification, replication, and variable-tracking across the many steps of this quantitative CEW bioassay. Overall, these findings indicate that this scaled-up, quantitative CEW bioassay is fundamentally sound and that Piura 208-derived resistance alleles are experimentally tractable for genetic and mechanistic research using this approach.

Estimation of long terminal repeat element content in the Helicoverpa zea genome from high-throughput sequencing of bacterial artificial chromosome pools.

The lepidopteran pest insect Helicoverpa zea feeds on cultivated corn and cotton across the Americas where control remains challenging owing to the evolution of resistance to chemical and transgenic insecticidal toxins, yet genomic resources remain scarce for this species. A bacterial artificial chromosome (BAC) library having a mean genomic insert size of 145 ± 20 kbp was created from a laboratory strain of H. zea, which provides ∼12.9-fold coverage of a 362.8 ± 8.8 Mbp (0.37 ± 0.09 pg) flow cytometry estimated haploid genome size. Assembly of Illumina HiSeq 2000 reads generated from 14 pools that encompassed all BAC clones resulted in 165 485 genomic contigs (N50 = 3262 bp; 324.6 Mbp total). Long terminal repeat (LTR) protein coding regions annotated from 181 contigs included 30 Ty1/copia, 78 Ty3/gypsy, and 73 BEL/Pao elements, of which 60 (33.1%) encoded all five functional polyprotein (pol) domains. Approximately 14% of LTR elements are distributed non-randomly across pools of BAC clones.

Peripheral genetic structure of Helicoverpa zea indicates asymmetrical panmixia.

Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F-statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low F ST (0.01 to 0.02) and high F IS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species.

The mitochondrial genome of the American lotus borer, Ostrinia penitalis (Lepidoptera: Crambidae).

The American lotus borer, Ostrinia penitalis, is the most ancestral among more than 20 species described in the genus Ostrinia, and the near complete mitochondrial genome sequence described here is important for phylogenetic comparisons. The 12,612 bp contiguous fragment contains 13 protein coding genes, 20 tRNAs and a partial rRNA gene complement. Compared to two previously sequenced Ostrinia mitochondrial genomes, gene order and orientation remains identical. In contrast, complete stop codons of cox2 (TAG) and atp6 (TAA) in O. penitalis show that completion of truncated stop codons in other Ostrinia are derived.

The mitochondrial genome of the western bean cutworm, Striacosta albicosta (Lepidoptera: Noctuidae).

The complete 15,553 bp mitochondrial genome of the western bean cutworm, Stricosta albicosta, (Lepidoptera: Noctuidae) was assembled from paired end Illumina HiSeq2500 read data. Annotation showed 13 predicted protein coding genes (PCGs), 22 tRNAs and 2 rRNAs have an order and orientation typical of insect mitochondrial genomes, and the derived rearrangement of tRNA-Met, -Ile and -Gln upstream of nad2 as in other Lepidoptera. A 79.3% A + T content resulted in a bias for codons with A or T in the 3rd position, and prevalence of synonymous substitutions suggest the effects of purifying selection on the mitochondrial genome sequence. Two microsatellite repeat motifs, (CA)10(AT)19 and (AT)12, are respectively located in intergenic spaces between tRNA-Glu and -Phe and tRNA-Leu and 16S rRNA. Mitochondrial phylogenomics was able to resolve sub-families within the Noctuidae, and suggest analogous analyses may be applicable across other lepidopteran Families.

Aflatoxin and Fumonisin in Corn (Zea mays) Infected by Common Smut Ustilago maydis.

Corn infected with Ustilago maydis (common smut) produces galls that are valued as a delicacy in some cultures. During a 4-year period, aflatoxin levels in asymptomatic kernels of smutted ears were, on average, 45-fold higher than in kernels harvested from smut-free control ears and 99-fold higher than in smut galls. Aflatoxin levels in smut galls were lower than in kernels of smut-free control corn in all years combined. Fumonisin levels in asymptomatic kernels harvested from smutted ears were 5.2-fold higher than in kernels from smut-free control ears and 4.0-fold higher than in smut galls. Fumonisin levels in smut galls were not significantly different than in kernels of smut-free control corn. These studies indicate that, although corn smut was relatively free of the mycotoxins studied, the asymptomatic kernels of those ears contained mycotoxins at levels much higher than usually considered safe for direct human consumption.

Effect of maize lines on larval fitness costs of Cry1F resistance in the European corn borer (Lepidoptera: Crambidae).

Crops producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) are widely planted and enable management of key insect pests while reducing the use of conventional insecticides. However, the evolution of Bt resistance could diminish these benefits. Fitness costs of Bt resistance occur in the absence of Bt toxin when individuals with resistance alleles show a reduction in fitness relative to susceptible individuals, and they can delay the evolution of resistance. Ecological factors including host-plant variety can affect the magnitude of fitness costs, and consequently, the degree to which fitness costs delay resistance. In this study, we measured fitness costs of resistance to Bt toxin Cry1F in the European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Crambidae) using Cry1F-resistant and Cry1F-susceptible strains sharing a similar genetic background. Fitness costs were tested on three lines of maize, Zea mays L., by measuring larval survival and development in two greenhouse experiments with plants in either the vegetative or reproductive stage. Both experiments showed that maize line significantly affected larval survival and developmental rate. However, larval survival, mass, and developmental rate did not differ between the Cry1F-resistant and susceptible strains, indicating a lack of fitness costs of resistance to Cry1F for the larval fitness components measured in this experiment. Future experiments should test for fitness costs of Cry1F resistance affecting survival to adulthood and adult life-history parameters.

Unlinked genetic loci control the reduced transcription of aminopeptidase N 1 and 3 in the European corn borer and determine tolerance to Bacillus thuringiensis Cry1Ab toxin.

Transgenic expression of Bacillus thuringiensis (Bt) crystalline (Cry) toxins by crop plants result in reduced insect feeding damage, but sustainability is threatened by the development of resistance traits in target insect populations. We investigated Bt toxin resistance trait in a laboratory colony of the European corn borer, Ostrinia nubilalis, selected for increased survival when exposed to Cry1Ab and correlated survival on Cry1Ab toxin with a constitutive ∼146.2 ± 17.3-fold reduction in midgut aminopeptidase N1 (apn1) transcript levels. A 7.1 ± 1.9-fold reduction apn3 transcript level was also correlated with Cry1Ab resistance. Quantitative trait locus (QTL) mapping identified a single major genome region controlling Cry1Ab resistance on linkage group 24 (LG24), and a minor QTL on LG27. Both QTL were independent of apn1 and apn3 loci on LG02. Positional mapping identified genetic markers that may assist in the identification of causal gene(s) within QTL intervals. This study indicates that genetic factor(s) may act in trans to reduce both apn1 and apn3 expression in Cry1Ab resistant O. nubilalis larvae, and suggest that gene regulatory pathways can influence Bt resistance traits. These findings show that gene interactions (epistasis) may influence Bt resistance in target insect populations.

Effects of foliar boron application on seed composition, cell wall boron, and seed δ(15)N and δ(13)C isotopes in water-stressed soybean plants.

Limited information is available on the effects of foliar boron (B) application on soybean seed composition. The objective of this research was to investigate the effects of foliar B on seed composition (protein, oil, fatty acids, and sugars). Our hypothesis was that since B is involved in nitrogen and carbon metabolism, it may impact seed composition. A repeated greenhouse experiment was conducted where half of the soybean plants was exposed to water stress (WS) and the other half was well-watered. Foliar boron (FB) in the form of boric acid was applied twice at a rate of 1.1 kg ha(-1). The first application was during flowering stage, and the second application was during seed-fill stage. Treatments were water stressed plants with no FB (WS-B); water stressed plants with FB (WS+B); watered plants without FB (W-B), and watered plants with FB (W+B). The treatment W-B was used as a control. Comparing with WS-B plants, B concentration was the highest in leaves and seed of W+B plants (84% increase in leaves and 73% in seed). Seeds of W+B plants had higher protein (11% increase), oleic acid (27% increase), sucrose (up to 40% increase), glucose, and fructose comparing with W-B. However, seed stachyose concentrations increased by 43% in WS-B plants seed compared with W-B plants. Cell wall (structural) B concentration in leaves was higher in all plants under water stress, especially in WS-B plants where the percentage of cell wall B reached up to 90%. Water stress changed seed δ(15)N and δ(13)C values in both B applied and non-B applied plants, indicating possible effects on nitrogen and carbon metabolism. This research demonstrated that FB increased B accumulation in leaves and seed, and altered seed composition of well-watered and water stressed plants, indicating a possible involvement of B in seed protein, and oleic and linolenic fatty acids. Further research is needed to explain mechanisms of B involvement in seed protein and fatty acids.

Implications of Bt traits on mycotoxin contamination in maize: Overview and recent experimental results in southern United States.

Mycotoxin contamination levels in maize kernels are controlled by a complex set of factors including insect pressure, fungal inoculum potential, and environmental conditions that are difficult to predict. Methods are becoming available to control mycotoxin-producing fungi in preharvest crops, including Bt expression, biocontrol, and host plant resistance. Initial reports in the United States and other countries have associated Bt expression with reduced fumonisin, deoxynivalenol, and zearalenone contamination and, to a lesser extent, reduced aflatoxin contamination in harvested maize kernels. However, subsequent field results have been inconsistent, confirming that fumonisin contamination can be reduced by Bt expression, but the effect on aflatoxin is, at present, inconclusive. New maize hybrids have been introduced with increased spectra of insect control and higher levels of Bt expression that may provide important tools for mycotoxin reduction and increased yield due to reduced insect feeding, particularly if used together with biocontrol and host plant resistance.

Frequency of hybridization between Ostrinia nubilalis E-and Z-pheromone races in regions of sympatry within the United States.

Female European corn borer, Ostrinia nubilalis, produce and males respond to sex pheromone blends with either E- or Z-Δ11-tetradecenyl acetate as the major component. E- and Z-race populations are sympatric in the Eastern United States, Southeastern Canada, and the Mediterranean region of Europe. The E- and Z-pheromone races of O. nubilalis are models for incipient species formation, but hybridization frequencies within natural populations remain obscure due to lack of a high-throughput phenotyping method. Lassance et al. previously identified a pheromone gland-expressed fatty-acyl reductase gene (pgfar) that controls the ratio of Δ11-tetradecenyl acetate stereoisomers. We identified three single nucleotide polymorphism (SNP) markers within pgfar that are differentially fixed between E- and Z-race females, and that are ≥98.2% correlated with female pheromone ratios measured by gas chromatography. Genotypic data from locations in the United States demonstrated that pgfar-z alleles were fixed within historically allopatric Z-pheromone race populations in the Midwest, and that hybrid frequency ranged from 0.00 to 0.42 within 11 sympatric sites where the two races co-occur in the Eastern United States (mean hybridization frequency or heterozygosity (H O) = 0.226 ± 0.279). Estimates of hybridization between the E- and Z-races are important for understanding the dynamics involved in maintaining race integrity, and are consistent with previous estimates of low levels of genetic divergence between E- and Z-races and the presence of weak prezygotic mating barriers. This work describes the development of new single nucleotide polymorphism (SNP) markers within the pheromone gland expressed fatty acyl reductase (pgfar) gene of Ostrinia nubilalis. These SNPs were shown to segregate based upon female pheromone production, and thus provide the first description of an assay for genetic determination of O. nubilalis pheromone strain from field-collected samples. These assays were applied to estimate hybridization within field populations, and represent valuable tools for future population genetic studies of this species.

Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing.

Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5 kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58 Gb (2.80 pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17 Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding.

Mobilizing the genome of Lepidoptera through novel sequence gains and end creation by non-autonomous Lep1 Helitrons.

Transposable elements (TEs) can affect the structure of genomes through their acquisition and transposition of novel DNA sequences. The 134-bp repetitive elements, Lep1, are conserved non-autonomous Helitrons in lepidopteran genomes that have characteristic 5'-CT and 3'-CTAY nucleotide termini, a 3'-terminal hairpin structure, a 5'- and 3'-subterminal inverted repeat (SIR), and integrations that occur between AT or TT nucleotides. Lep1 Helitrons have acquired and propagated sequences downstream of their 3'-CTAY termini that are 57-344-bp in length and have termini composed of a 3'-CTRR preceded by a 3'-hairpin structure and a region complementary to the 5'-SIR (3'-SIRb). Features of both the Lep1 Helitron and multiple acquired sequences indicate that secondary structures at the 3'-terminus may have a role in rolling circle replication or genome integration mechanisms, and are a prerequisite for novel end creation by Helitron-like TEs. The preferential integration of Lep1 Helitrons in proximity to gene-coding regions results in the creation of genetic novelty that is shown to impact gene structure and function through the introduction of novel exon sequence (exon shuffling). These findings are important in understanding the structural requirements of genomic DNA sequences that are acquired and transposed by Helitron-like TEs.

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes.

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.

Offspring from sequential matings between Bacillus thuringiensis-resistant and Bacillus thuringiensis-susceptible Heliothis virescens moths (Lepidoptera: Noctuidae).

The tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), is an economically important pest of the Americas. Females of this species copulate multiple times during their lifetimes, and the presence of sperm from multiple males inside them could allow for a diversity of paternal genotypes in the offspring, unless there was complete precedence of sperm from the first mating. If a female copulates with a male that is insecticide-susceptible and another male that is insecticide-resistant, her progeny could vary in their resistance phenotypes. In some cases, this could impact the evolution of insecticide resistance in a population. We designed a series of experiments to determine whether Bacillus thuringiensis susceptibility is maintained when an H. virescens female that is homozygous for a genetically recessive form of B. thuringiensis resistance copulates with a Cry1Ac-susceptible and a Cry1Ac-resistant males. During the lifetime of double-copulated females, a proportion of F1 progeny were Cry1Ac-resistant. This indicates that when a B. thuringiensis-resistant H. virescens female copulates with two males, with one male being resistant to Cry1Ac, some of the progeny will carry resistance to this insecticide. Due to the polyandrous nature of this species, the above-mentioned scenario is not unrealistic; therefore, results from this study may help understand and manage the evolution of B. thuringiensis-resistance in field populations.

Identification of two haplotypes of cytochrome oxidase subunit II (COII) gene of the Formosan subterranean termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), in southern Mississippi.

BACKGROUND: Coptotermes formosanus Shiraki was accidentally introduced into the United States from Asia. The introduction of the pest has brought significant economic consequences. During the past decade, Mississippi has become a significantly infested state, partly due to the proximity to coastal port cities such as New Orleans. This study was initiated to investigate the origin and infestation route of C. formosanus in southern Mississippi. RESULTS: Twenty-eight colonies (21 colonies from Mississippi, six from Louisiana, one from China) were collected. Sequencing and analysis of 112 sequences revealed 15 haplotypes of cytochrome oxidase subunit II (COII) gene in the world. Two haplotypes of COII were identified in Mississippi. In addition, specific primers were designed and tested differentially to amplify characteristic fragments for verifying and surveying different genotypes of C. formosanus in the future. CONCLUSION: Of the two haplotypes identified in Mississippi, the GA group was identical to those reported previously in Georgia, Louisiana, Alabama and other infested states. The second haplotype, the AT group, was identified for the first time in southeastern United States. Sequence identity of the AT-group C. formosanus with those reported mainly in southeastern Asian countries provided evidence of at least two introductions of C. formosanus into the United States.