Several genes were captured in this interval, including NADH:Ubiquinone oxidoreductase subunit A8 ; mucin-5AC-like, a gene putatively related to human mucin-5AC, and two hemomucin genes involved in hemocyte adhesion and innate immunity in insects. The PBS hotspot on contig 15 contained cryptochrome-2 , encoding a key component of the circadian clock . Finally, the large hotspot on contig 22 contained 69 genes, including multiple copies of juvenile hormone binding protein, ribosomal proteins, gustatory response genes, rhodopsins, andacetylcholinesterase , a gene implicated in organophosphate insecticide resistance . Interestingly, based on alignment data it appears the Central and Andes populations may have two copies of ache, while North populations only have one. We looked for known mutations conferring organophosphate resistance and found moderate frequencies in all three clusters as well .Using whole genome sequencing data, we found that Tuta absoluta samples collected from 11 locations in Latin America clustered into three basic regions, comprised of a North, Andes, and Central group. In addition, we see that Spanish populations likely originated from a Central Chilean source based on their low level of Fst with the Andes and location on the ML tree. Previous analyses with mitochondrial sequences were unable to differentiate populations ; however, large pots plastic analyses using microsatellite data was able to identify these same three clusters and suggest a Central Chilean source for the European migration as well .
In agreement with this conclusion, looking at fresh tomato export data we see Chile is a worldwide exporter, shipping 12 tons of tomatoes an average distance of 11,700 km in 2018, while most other countries in South America tend to export within the continent . The Andes Mountains represent an obvious geographic barrier that would separate the Central population from the North and Andes populations. Population structure between Andes and North populations may be due to factors related to the changing latitude, including temperature and daylength. While PCA groups our Ecuador samples with Andes populations, admixture analysis and Treemix both provided some evidence that Ecuador may represent an admixture zone between the two regions. The weighted Fst between the North and Andes is also lower than between North and Central, suggesting that North and Andes are indeed more closely related. This general Fst pattern was also observed based on microsatellite analyses . Sequencing of more samples fromPeru and Ecuador might be needed to further elucidate the extent of an admixture zone between these clusters. While Tuta absoluta was first discovered in Peru in 1917, its native range is not well established. One hypothesis is that Tuta absoluta migrated out of the Andes region and across South America through the 1960s-80s because of human transport by agricultural shipping. This aligns with the surge in domestic tomato agriculture in South America at the same time . However, based on the similar nucleotide diversity levels between clusters, as well as high levels of Fst, we hypothesized it might be more likely that this migration across South America may have happened prior to tomato commercialization, with populations of Tuta absoluta later adapting to the appearance of commercial tomato agriculture.
Based on our simple 3 population model, it appears the ancestral population diverged twice tens of thousands of years ago. Relative to the ancestral population size, the combined effective population size is roughly three times larger, although this is heavily weighted toward a very large Andes population, relative to the North and Central regions. The fact that the estimated Andes population size is nearly 10 times larger than that of Central or North populations, ,as well as its slightly higher level of genetic diversity, could suggest that the Andes cluster represents the ancestral population range. Given that the wild ancestors of tomatoes and potatoes are also native to the Andes region , the Andes region would be an ideal place to search for native parasitoids for biocontrol. To date, biocontrol methods in South America have relied on non-natives or generalist parasites, with only a handful of native specialists identified in the literature and none that are commercially available . Thus, knowledge of Tuta absoluta’s native range may help focus efforts to identify more natural parasitoids. Using the PBS, we found multiple genomic windows under apparent selective forces. Not surprisingly, one of the highest PBS windows contained the para gene, which encodes a sodium ion channel that is targeted by pyrethroids. The extremely low allele diversity in the Andes and Central populations relativeto the North suggest a hard selective sweep occurred here. Heavy pyrethroid use in Brazil led to the appearance of resistant strains starting in the 1990s . We found that Central and Andes populations were completely fixed for the L1014F mutation, one of the most common causes of knock-down-resistance to pyrethroids , while the North had an intermediate frequency. A study looking at Brazilian populations found a similar pattern of fixed L1014 , while another study looking at multiple populations in South America also found the same pattern of L1014F fixation in Central and Andes populations but not in the North .
While both studies also found M918T and T929I at elevated frequencies in all populations, we additionally detected the resistance allele V1016G in the North. We also found L925M in Central and I254T in Andes. While these have not been characterized as resistance alleles, mutations at these same positions have been shown to confer resistance in Drosophila melanogaster and Bemisia tabaci . Other regions under selection were less obvious. We found one region in contig 2 containing Ndufa8 and several hemomucin/mucin genes with a similar low genetic diversity in the Central and Andes populations and high PBS in North, indicating a hard selective sweep. Ndufa8 produces a nuclearencoded subunit of the NADH dehydrogenase complex I, part of the electron transport chain in the mitochondria used to generate ATP. Mutations here are known to cause mitochondrial complex I deficiency in humans , although a few studies have found evidence of positive selection occurring in other species, potentially related to metabolism . The hemomucin genes are a component of the insect immune system that are involved in endocytosis . Selection here could be in response to the increased use of parasitoids and predators such as Trichogramma evanescens and Nesidiocoris tenuis as an biological control alternative to insecticides .The elevated PBS region in contig 22 was relatively large at approximately 1Mb in size, containing over 60 genes. Interestingly, we noticed two copies of ache contained within this window, which codes for acetylcholinesterase, a gene which encodes a protein which degrades the neurotransmitter acetylcholine . As this enzyme is the main target of organophosphates and carbamate insecticides, alleles in ache have been documented to confer resistance. Using the amino acid numbering scheme based on T. california , the resistance allele A201S has previously been reported to be present in European populations , and we found this allele to be at moderate to high frequency in all three regions. We also found moderate frequencies of the mutation F290V and F290N. The F290V resistance allele has been documented in mosquitoes and moths , while other mutations such as F290Y have been documented in Drosophila and M. domestica . Interestingly, based on mapping read depth the North population only contained a single copy of ache. As duplication of ache has been implicated in improved organophosphate resistance , square planter pots this large structural duplication may be the reason for elevated PBS levels across such a large interval. Follow-up work with long-read methods or higher sequencing coverage will be needed to confirm the presence of structural duplication at this locus. We expect that addition of a new contiguous genome assembly with annotations will be of benefit to the Tuta absoluta and Lepidopteran research community. Previous studies have worked to develop potential RNA interference strategies to use as an alternative to traditional pesticides . Work is also being conducted to develop Cas9 gene-editing techniques for Tuta absoluta to facilitate future genetics studies . These developments in combination with an accurate assembly and gene annotations will allow for accelerated research towards understanding Tuta absoluta biology and methods to contain its economic impacts and spread.The tomato plant represents a massive economic industry worldwide, with an estimated 252 million metric tons of tomatoes harvested in 2020 .
This production is concentrated in a few major producing countries, with 70% of the world’s production currently accounted for by China, the European Union, India, the USA, and Turkey . However, this industry is quickly becoming threatened by the invasive gelechiid moth Tuta absoluta Meyrick, commonly known as the tomato leafminer. T. absoluta is an agricultural pest of the nightshade family, including peppers, eggplants, and potatoes, but primarily represents a serious threat to tomatoes. Left untreated, the larvae will consume leaf tissue, bore into flower buds, or burrow into fruit with infestations causing crop losses as high as 80 to 100% . While T. absoluta was first identified in Peru in 1917 , it was not considered an agricultural pest until the 1960s when T. absoluta was detected in farms in Argentina, causing significant crop loss . From there it rapidly spread throughout South America, causing severe agricultural losses everywhere it was found. In 2006, T. absoluta was identified in a greenhouse in Spain, marking the first intercontinental migration of the pest, likely due to anthropological transportation . From Spain, it rapidly spread throughout Europe, SubSaharan Africa, the Middle East, and most recently into Asia . Without rapid identification and quarantine strategies, T. absoluta will likely continue to spread to North America. Preventing initial establishment of an invasive species in a risk ecosystem can be an effective strategy but requires vigilant monitoring of imported products as well as an ability to rapidly detect the presence of the target . In the United States, policies such as federal orders issued by the Animal and Plant Health Inspection Service have been used to prevent accidental introduction of T.absoluta by regulating import of tomato fruit or propagation material from infested countries . Correctly detecting and identifying the insect, however, remains challenging due to the presence of other gelechiid moths such as P. operculella and Keiferia lycopersicella in the United States, which look nearly identical to T. absoluta and are also commonly found on tomato and potato crops . Distinguishing these species morphologically requires dissection of adult male genitalia, which requires entomological expertise and increases waiting times for identification. Molecular diagnostics, on the other hand, would allow for accurate and rapid identification without entomological expertise. To date, several molecular diagnostics have been developed, all using Polymerase Chain Reaction to amplify target DNA presence before detecting signal either by fluorescence, gel electrophoresis, or mass spectrometry . A potential limitation of these PCR-based diagnostics is the requirement for a thermocycler, as well as specialized equipment to visualize results. For field detection, a system that requires minimal specialized equipment would be ideal. The CRISPR-Cas system has recently become an attractive option for molecular diagnostics . CRISPR-Cas originated as a bacterial immunity system, in which the Cas protein complexes with a CRISPR RNA that is complementary to a target DNA sequence . When the Cas-crRNA complex binds to its target it cleaves it, causing a double-strand break in the DNA. While Cas9 is the most commonly used Cas protein for its genomic editing capabilities, the Cas12a enzyme was discovered to exhibit indiscriminate single-stranded DNA nuclease activity upon binding to its target DNA . If a single-strand oligonucleotide probe modified with a fluorophore and quencher is present when Cas12a-crRNA binds its DNA target, Cas12a will proceed to also cleave the single-strand probe, separating the fluorophore from the quencher and allowing a fluorescent signal to be measured. As ssDNAse activity is proportional to the number of target DNAmolecules and can occur isothermally between 15℃ to 50℃, using an isothermal DNA amplification method such as Recombinase Polymerase Amplification to amplify target DNA allows for sensitive detection with Cas12a at constant temperature, eliminating the need for an expensive thermocycler. Additionally, detection of probe cleavage is flexible as several methods have been published on alternative visualization methods, including using flow strip assays, gold-conjugated DNA probes, and naked-eye fluorescence detection . In this study, we leveraged prior sequencing data to develop two molecular diagnostic assays.