Genomic analyses of fall armyworm (Spodoptera frugiperda) populations from Uganda using mitochondrial cytochrome oxidase subunit 1 and nuclear triose-phosphate isomerase (Tpi) genes

doi:10.21203/rs.3.rs-468370/v1

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Genomic analyses of fall armyworm (Spodoptera frugiperda) populations from Uganda using mitochondrial cytochrome oxidase subunit 1 and nuclear triose-phosphate isomerase (Tpi) genes

https://doi.org/10.21203/rs.3.rs-468370/v1

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The fall armyworm (FAW), Spodoptera frugiperda; J.E. Smith (Lepidoptera: Noctuidae), has become a newly established economically important pest that causes huge losses to maize productivity in sub-Saharan Africa. In 2017, a survey was conducted to collect S. frugiperda specimens across 11 agro-ecological zones in Uganda. Cytochrome oxidase subunits 1 (CO1A and CO1B) and triose-phosphate isomerase (Tpi) partial gene segments of FAW were sequenced and subjected to comparative genomic analyses, to elucidate genetic identity, diversity and determine the existence of corn sub-haplotypes, distribution, and likely origin of invading populations. Results revealed the presence of both rice and corn strains (haplotypes h4 and h1), with rice strain being more predominant. The analyses of both mitochondrial and nuclear Tpi marker sequences showed that the FAW host strain haplotypes from Uganda were identical to those found in Florida, three Asian nations (China, India, and Pakistan) and Australia. A wide distribution of both strains was observed across all agro-ecological zones in Uganda. Comparative genomic analyses showed strain identification concordant results between CO1A and CO1B partial gene markers. Upon comparison with Tpi marker result, discordances in discrimination of FAW strains were observed. The study findings imply that Florida, China, India and Pakistan are possible centers of origin of invasion and support a possibility of multiple introductions into Uganda and the region. There is therefore a need for in-depth characterization of FAW populations in the region and other African nations to better understand its genetics and mechanisms of invasion.

Plant Molecular Biology and Genetics

Biotechnology and Bioengineering

General Cell Biology & Physiology

Computational Biology

Bioinformatics

Spodoptera frugiperda

Cytochrome oxidase subunits 1

triose-phosphate isomerase (Tpi)

genomic analyses

diversity

corn sub-haplotypes

distribution

origin of invading populations

Uganda

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a devastating polyphagous and highly migratory herbivorous insect pest, which is native to the Western Hemisphere ¹. Since its first detection in São Tomé and Príncipe, Nigeria, Togo, and Benin ², FAW has been reported in 46 (out of 54) countries in sub-Saharan Africa and 17 Asian nations ^3,4 and recently (2020) detected in Australia (https://bfvg.com.au/2020/04/06/when-was-fall-armyworm-detected-in-australia/). The pest feeds on 353 host plant species in 76 plant families ⁵, with a high preference for maize - a staple food crop for over 300 million African smallholder farmers ^6–8. FAW damages maize plants by voraciously feeding on leaves, stems, and reproductive parts, thus destroying their growth potential ^9,10. The young (small) caterpillars feed on young leaves, while older larvae penetrate the whorl (throat), buds, and stems ^10–12. The damage usually starts from the margin and proceeds to the midrib of the leaf creating clear perforations. Most often, older larvae extensively skeletonize the maize plant by consuming large amounts of leaf tissue, leaving behind a rugged and thorny appearance. In older maize plants, they also burrow into the ear and feed on kernels ^10,11.

The establishment of S. frugiperda species in Africa is of special concern. In 2017, 2.5 to 6 million US$ maize loss was estimated in Africa ¹². The annual loss estimates of maize due to FAW in 2018 was U S$177M and US$159M in Ghana and Zambia, respectively ¹³. Brazil spends approximately US$600 million annually to control this pest ¹⁴. In Uganda, FAW was reported to cause 15 and 75% yield loss ¹⁵. In only 2017 cropping season, Uganda lost about US$192M which directly affected 3.6 (about 10% of the population) million people ¹⁵. Without proper control measures, the FAW was estimated to have the potential to cause annual maize yield losses between US$2.48 billion and US$6.19 billion in 12 estimated African countries ¹². The invasion and rapid spread of FAW in Africa, therefore, threatens the food, nutrition, and income security of millions of smallholder farmers.

Spodoptera frugiperda exists in two morphologically identical but genetically distinct strains: the corn strain designated as “C” strain and the rice strain designated as “R” strain that are considered to differ in host preference, physiology, and behavior ^16–19. The corn strain is thought to be associated with maize, cotton, and sorghum, whereas the rice strain is thought to feed on rice and other pasture grasses though this is not always consistent as observed in Argentina ²⁰. The only accurate method of identifying these two morphologically indistinguishable strains is by the use of molecular markers ^4,21,22.

Molecular markers such as polymorphisms in mitochondrial cytochrome oxidase subunit 1 (mtCO1A and mtCO1B) and triosephosphate isomerase (Tpi) gene in the nuclear genome are the most accurate identifiers of S. frugiperda strains so far ²³. These are the most common and currently used markers though some disagreements have been reported among them ^19,24. Recently, attempts have been made to identify the strains through whole-genome sequencing ⁴. This preferably good approach is expensive and thus unaffordable for many scholars. In this study, we used a combination of mtCO1A and mtCO1B, and nuclear Tpi sex-linked DNA markers to elucidate the genetics of FAW populations in Uganda.

Although S. frugiperda has been reported in sub-Saharan Africa including Uganda, there is limited knowledge on its genetic diversity (genetic composition and linkage) and distribution. The existing information in Uganda only indicates the occurrence of two strains, identified using CO1 markers ²². Besides, lack of knowledge on the presence/absence of FAW strain hybrids and limited comparative genomic information to trace areas of similar invasions have led to limited molecular evidence to fully determine the pest origin, possible route (s) of incursion (s), and dynamics of prevailing FAW populations in Uganda and Africa’s agricultural scenarios. This study reports the molecular diversity, genomic comparisons, and genetic distribution of S. frugiperda investigated by sequencing a combination of mtCO1 and nuclear Tpi genes. The study answered the following questions: i) what is the distribution of FAW haplotypes in Uganda? ii) What is the likely origin of fall armyworm invading Uganda and African continent? And iii) How do results of the Tpi gene marker compare with those of the CO1 marker?

Morphological identification of S. frugiperda

Most of the larvae collected had an inverted “Y” mark (Fig. 1A); reddish-brown head, brownish body bearing white sub-dorsal, and lateral lines, which match with described morphological identification criteria ^12,25. Four black dots arranged in a square pattern on the 8^th abdominal segment (Fig. 1B) and rough granular epidermal structure were seen upon close examination. Male adult moths were identified by the presence of shaded gray and brown forewing with triangular white spots at the tip and near the center of the wing (Fig. 1C) whilst females had a plain gray forewing with brown, obscure lines and spots (Fig. 1D).

Corn symptoms expression due to FAW damage

A range of FAW damage symptoms expressed on leaves and stems of infested maize plants were observed during the survey. The most frequently encountered symptoms included elongated lesions on whorl, furl leaves, and irregular shaped holes injury (Fig. 2) with many cases of extensive foliar damage. Skeletonized corn plants with ragged and thorny appearance were seen. Larval penetration into the whorl (throat), buds, stems, and fed on kernels were also observed.

PCR amplification

All primer sets used in this study amplified successfully and gave PCR products of CO1A, CO1B, and Tpi genes of the expected band sizes (Fig. 3A-3C). Of the 95 suspected S. frugiperda samples analyzed, 93 samples gave positive results and these were sequenced to enable genetic diversity studies.

Sequences analysis results

Out of the 93 sequences (for both CO1 and Tpi markers), 5 sequences (which were poor in quality) were not considered in downstream analyses. Upon cleaning, only 88 samples were therefore used for downstream analyses.

The initial BLAST search for CO1 genes of the analyzed Uganda’s FAW sequences showed that the sequences were highly similar by an overall range of 99-100% nucleotide sequence identity to S. frugiperda species. However, one sequence for sample UG_A38 was omitted because preliminary analysis showed it was a different species from the rest of the samples. The analysis of CO1A sequences for Uganda’s FAW populations revealed a majority (65) of samples to be 100% similar to the nucleotide sequences of the four (04) FAW rice haplotypes RS9 (HM136601.1), RS10 (HM136602.1), (MN075831.1), and (MW454865.1) in the GenBank. Additionally, a portion (22) of samples analyzed showed 100% nucleotide sequence identity to five FAW corn haplotypes (HM136586.1, HM136591.1, and HM136592.1), (MN075830.1), and (MW454866.1) (Fig. 5) found in the Florida, China and Australia, respectively.

Notably, analysis of a 560-bp partial gene segment of CO1B revealed 100% nucleotide sequence identity with other sequences in India (MH779586.1, MH779590.1, and MW357423.1), and Pakistan (MW349493.1, and MW455103.1).

The percentage nucleotide identity of the TPi sequences ranged from 98.7 to 100% with Tpi haplotypes in the Florida (KT336239) and China, (MN066368.1 and MT767446.1).

Phylogenetic linkage analysis

Phylogenetic linkage analysis (by neighbor-joining method) of Ugandan samples using the CO1A gene revealed two main clades. The first clade was composed of 22 samples (28.4%) while the second clade was composed of 65 samples (71.6%) (Fig. 4). Sequences within two respective clades were identical to each other (Fig. 4). The outgroup (H. armigera) clustered away from Uganda FAW samples (in-groups) and other S. frugiperda species. This enabled relevant phylogenetic comparisons and proper tree rooting to be done (Fig. 4).

Host strain genetic identity of Spodoptera frugiperda populations across agro-ecological zones of Uganda

Based on a 770bp phylogenetic comparative analysis of CO1A barcode region sequences, Uganda’s populations of S. frugiperda species formed two clusters corresponding to the corn- (CS) and rice-(RS) strains (Fig. 5). Out of the 87 FAW sequences analyzed, 22 (28.4%, - Fig. 6a) were identified as corn-strains and were identical to five (05) FAW corn haplotypes from Florida (HM136586.1, HM136591.1, and HM136592.1), China (MN075830.1), and Australia (MW454866.1) (Fig. 5).

Similarly, 65 (71.6%) samples (Fig. 6a) were identified as rice strains and clustered with four (04) other rice strains CO1A haplotypes from the gene bank (Fig. 5). Uganda’s FAW rice haplotypes were also identical to those in Florida RS9 (HM136601.1), and RS10 (HM136602.1), China (MN075831.1), and Australia (MW454865.1) (Fig. 5).

In addition to host strains phylogenetic clustering patterns, an irregular distribution pattern of host strains was observed across agro-ecological zones in Uganda (Fig. 6B). The Eastern Savannah (ES) zone revealed the highest proportion of rice strains (16.05%) while the lowest (1.23%) rice strain proportion was observed in Lake Victoria Crescent (LVC) and Southwestern highlands (SWH). The highest proportion (6.17%) of corn strain samples was recorded in the Northern farming system (NFS) and ES zone while the lowest proportion (1.23%) was observed in Busoga Farming System (BFS), West Nile farming system (WFS), Karamoja dry zone (KDZ), Eastern highlands (EH) and Karamoja wet zone (KWZ). All S. frugiperda samples collected from the SWH zone were rice strains while the entire KWZ was composed of corn strain samples only.

Phylogenetic comparative analyses of Uganda’s CO1B FAW host strain haplotype sequences

Three Unique FAW haplotypes (representatives) in Uganda exhibited different clustering patterns (Fig. 7). The Uganda rice strain haplotype (Fig. 7) was identical to other sequences in India (MH779586.1, MH779590.1, and MW357423.1), Pakistan (MW349493.1, and MW455103.1) and the Florida CO1-RS consensus. Uganda CS-h4 clustered with CO1-CS consensus from the Florida and Uganda CS-h1 appeared Unique (Fig. 7).

Comparative molecular genomics of CO1B haplotype gene segment revealed the Florida and Caribbean-Corn strain haplotype profile in Ugandan fall armyworm collection

Upon comparison of the CO1B gene segment, 21/22 (27.16%) samples in Uganda (Fig. 8A) which were identified as CO1-CS (by both CO1A barcode region and CO1₁₁₆₄; CO1₁₂₈₇ polymorphic sites) expressed G₁₁₆₄ and G₁₂₈₇ haplotype (h4) combination.

Only one sample collected from Iganga district in the BFS agro-ecological zone (Fig. 8B) showed haplotype one (1)-h1= A₁₁₆₄ and A₁₂₈₇(Fig. 8A). Haplotypes h2 and h3 were not observed in any sample in this study. An irregular distribution of S. frugiperda haplotypes in Uganda was observed (Fig. 8B), giving concordant results between CO1B polymorphic sites (CO1₁₁₆₄ and CO1₁₂₈₇₎ and CO1A barcode region marker. The agreement between these two markers further indicates that 71.6% (65/87) of the samples (Fig. 8A) analyzed were CO1-RS expressing T₁₁₆₄ and A₁₂₈₇combination.

Relative abundance and distribution of Spodoptera frugiperda strains in Uganda

Both corn and rice strains of S. frugiperda occurred in all districts of Uganda surveyed during the 2017 cropping season. The most dominant strain revealed by CO1 markers was rice strain (16.05%) observed in ES agro-ecological zone covering all the corn growing districts of Amuria, Budaka, Bukedea, Busia, Butaleja, Kaberamaido, Katakwi, Kumi, Ngora, Pallisa, Serere, and Soroti (Fig. 9). Lake Victoria crescent region and southwestern highlands had the lowest incidence (1.2%) of rice FAW strain. The highest incidence (6.02%) of corn strain was observed in NFS agro-ecological zone in districts of Amolatar, Apac, Dokolo, Kitgum, Lira, Oyam, and Pader (Fig. 9) in addition to ES districts.

Discordances revealed by Tpi and CO1 gene markers

Comparative analysis of sequences revealed a 99% disagreement between CO1 and Tpi markers with respect to Uganda’s FAW strain discrimination. All sequences (except one identified as rice strain by Tpi gene marker) of Ugandan populations identified as either corn or rice strains using CO1 gene markers were classified as corn strain by the Tpi marker. Further analysis of the Tpi gene segment in this study did not reveal the FAW hybrids population and FAW Tpi variants.

The fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuid), has become a new economically important pest of maize in sub-Saharan Africa ²⁵. In this study, we report on the genetic diversity and distribution of S. frugiperda, the existence of corn sub-haplotypes, and possible origin of invasion into Uganda and the region.

Based on the use of mitochondrial CO1 partial gene markers, our study revealed shared genetic similarities among Uganda’s FAW populations with those found in Florida, Asia (China, India and Pakistan) and Australia. The results further demonstrated the existence of both rice and corn-strains of S. frugiperda species, with rice strain being more predominant in the country. Similar results (on the existence of both strains) using the same mitochondrial CO1 partial gene have been previously reported by Otim et al.,²². Furthermore, the results showed a wide distribution of both corn and rice strains across all agro-ecological zones in Uganda. It further revealed the presence of two corn strain sub-haplotypes (h4 and h1), and a disagreement between CO1 and Tpi gene markers with respect to the Uganda’s FAW strain discrimination. Despite the wide distribution of FAW strains, a higher percentage (71.6%) of rice strain (CO1-RS) was noted across the country. This suggests that most of the rice strains of FAW populations as compared to corn strain populations could have invaded Uganda and the African continent at large. This similar predominance pattern of CO1-RS was also previously reported in India, East, and South Africa²⁷.

Moreover, the co-occurrence of both strains of S. frugiperda in many agro-ecological zones of Uganda, and the existence of rice and corn strains as single infestation in SWH and KWZ, respectively is worth noting. Our data suggests that the corn damage in nine agro-ecological zones (ES, NFS, BFS, WFS, LAC, WRL, KDZ, EH, and LV) of Uganda, was attributed to the co-occurrence of voracious strains of S. frugiperda while SWH and KWZ had single strain infestation. Whether corn or rice strain is associated with high severity and damage in corn and other plants attacked is unclear, and this requires further investigation. Such studies coupled with understanding various host plant and pest molecular resistance mechanisms could clarify this issue for better pest management strategies.

Favorable agro-ecological conditions of sub-Saharan Africa ²⁵ are likely to favor the establishment of S. frugiperda as an endemic pest for several generations. The presence and wide distribution of these two strains in Uganda could potentially result into future hybridization between S. frugiperda strains as evolutionary pressures continue to shape and favor their reproduction, adaptation, subsequent survival, and further spread.

Although it is commonly believed that rice and corn strains associate with their host plants ²⁸²⁴²⁶, all samples used in this study were collected from corn plants and many of them displayed the highest proportion (71.6%) of rice strains based on mitochondrial markers. These results are similar to the recent observations in Myanmar where a majority of corn strain host plant collections expressed CO1-RS haplotypes ²⁷. This could be attributed to either the plasticity in plant choice or the inability of the markers to perfectly and accurately discriminate between these strains as previously suggested by Nagoshi et al.,²⁹. Nonetheless, results from this study further support previous reports that the association between rice and corn strains to their hosts is not absolute ²⁰.

Two single nucleotide polymorphic sites (CO1₁₁₆₄ and CO1₁₂₈₇) variants of CO1B segment present unique mitochondrial allele frequencies that showed geographical differences, and further divided CO1-CS into four haplotypes (h1-h4) which vary by either A or G (Fig. 1A). Upon comparison, the present study revealed the presence of corn strain haplotype four h4 = CO1₁₁₆₄G and CO1₁₂₈₇G combination which is known to be more frequent in the Florida and Caribbean ^19,27, and was previously reported in Togo ¹⁹. Recently, similar results have been reported in most countries in Africa, Myanmar, India, and southeastern Asia ²⁷. Thus, suggesting that Florida could be a common and primary origin of S. frugiperda infestations into the African continent in addition to other regions. Given this similarity, it is important to determine whether the Africa invading populations came from either Florida or Asia or both countries.

Our data suggests that Florida is the primary origin of infestations, and further demonstrates that both corn and rice strains from Uganda also shared genetic identities with haplotypes in China ³⁰, India, Pakistan, and Australia .

The question of whether invasion into the African continent involved either single or multiple introductions can only be answered by a wide range of genomic data. Previous studies ^21,27, supported the possibility of a single incursion into the African continent. This means, if the first detection of FAW in West Africa ² represents the exact time of pest arrival into the continent, then FAW populations in West Africa are serving as “donor” species which are rapidly spreading and becoming established in the rest of sub-Saharan Africa, Asia and Australia.

Contrary to this observation, this current study detected haplotypes from three Asian nations (China, India, and Pakistan) in addition to the Florida and Australia. These results suggest a possibility of multiple introductions from these regions, confirming the previous report by Otim et al. (2018) that the invasion into the continent involved at least three matrilineal lineages. Due to this, Asian populations particularly China, India and Pakistan could be the possible source/route for the FAW invasion in Uganda and possibly the African continent in addition to Florida. This observation is supported by a recent genome-wide study which indicated that FAW has been in Asian nations before 2016 when it was first reported in West Africa ⁴. It is, therefore, possible that sub-populations of FAW from China, India and Pakistan are also invading Africa, thus confirming the previous genome-wide evidence reported by Tay et al., (2020). The recent (2020) detection of FAW in Australia (https://bfvg.com.au/2020/04/06/when-was-fall-armyworm-detected-in-australia/) seems to rule out the possibility of invasion of Australian FAW populations into Uganda and the region. Despite the similarity with the Ugandan FAW populations, the data would then suggest that the FAW populations invading Australia could possibly be coming from Asia and the Florida, with a chance of future invasion by African populations as well depending on their migratory behaviors and scenarios presented. However, a more holistic genome-wide study would help to clarify this issue. In addition, similar studies involving more analysis of both CO1A and CO1B of FAW populations in different African countries, and genetic comparisons with FAW populations from Asia, Florida and Australia would be beneficial in understanding the source and the actual number of incursion pathways of invading populations in the entire continent of Africa. Whether or not the invasion into the African continent was precisely due to either the migratory power of the insect or human activities is yet unclear. Studies aimed at understanding and estimating the true fate of these events would be beneficial in designing pest risk control strategies in Africa and the rest of the newly infested regions.

Although both CO1A and CO1B reveal shared similarities with Asian FAW populations in addition to Florida and Australia, the CO1A marker result suggests that both rice and corn strains that invaded Uganda came from China, Florida and Australia. On the other hand, it is worth noting that CO1B marker results further suggest that the rice strains from India and Pakistan could have added the Chinese FAW population diversity at the time of invasion into Uganda and the region, with the Florida being the primary source. It is therefore not known whether this observed rice strain movement pattern is either accidental or strain-specific, although both corn and rice strains of FAW are present in India ³¹. Therefore, it becomes necessary to find out whether the migratory behavior and new ecological colonization of FAW is strain-dependent, to enable both future accurate mapping and monitoring of FAW movements across continents and nations of the world.

We also sequenced a portion of sex-linked Tpi nuclear gene that encodes for the glycolytic enzyme triose-phosphate isomerase (EC 5.3.1.1) ³² and segregates independently from a maternally inherited CO1 gene ^19,21 Tpi gene located on the Z-sex chromosome in all lepidopteran species ¹⁹ is conserved in noctuid moths and is known to distinguish between strains of FAW and detect potential cases of hybridization ^19,33,34. In this study, we observed a 99% identification disagreement between CO1 and Tpi markers. Much as this result is not completely new due to earlier observations and reports of disagreement between nuclear Tpi and CO1 gene markers ^19,35, the disagreement observed in this study is uniquely pronounced in the sense that all samples (except one) that were identified by the CO1 marker as either rice strains or corn strains were categorized as Tpi-corn strain (Tpi-CS). Given this result, it would be deduced that the FAW populations invading Uganda and other nations could have come from a hybrid, which is a result of crossing between female rice strain and male corn-strain, possibly due to nuclear genome of corn-strain becoming more dominant to form a unique corn-strain as postulated in China ³⁰. However, the absence of both Tpi hybrids and variants in this study, coupled with previous reports that the disagreement could be attributed to either the plasticity in plant choice or inability of the markers to perfectly and accurately discriminate between these strains (Rodney et al., 2019), does not support this observation. Although previous studies have shown the Tpi marker to accurately identify FAW in the Americas ( Rodney, 2012; Nagoshi & Meagher, 2016), its use in the identification of African FAW population is faced with inconsistencies when compared to western Hemisphere Tpi FAW identification pattern. This may further confirm the previous suggestion that the current rice strains in Africa could be unique ²⁴, and could have significant implications of pest management in Africa’s crops attacked. However, these results present the need to either reconsider the Tpi marker to perfectly determine the exact cause of this (“ese”) change (s) at gene (s) level or to develop a consistently reproducible method for future accurate routine identification of S. frugiperda populations in Africa and beyond.

The widespread distribution of FAW strains has huge implications in crop improvement efforts in Africa. This is because, their genetic arsenal favor polyphagy and xenobiotics detoxification ¹⁴. Given the possibility of slow adaptation of FAW to newly identified natural enemies (Otim et al., 2021) in Africa and the likely existence of new FAW resistance traits in Africa, massive host plant resistance screening of corn and other crop varieties is key to guide emerging and existing breeding programs geared toward crop protection and improvement.

Specimen collection

The specimens were collected during a survey which was conducted between August and October 2017 in ninety-four (94) maize growing districts across eleven (11) agro-ecological zones of Uganda (http://www.fao.org/agriculture/seed/cropcalendar/aezones.do?isocode=UGA). These agro-ecological zones included Busoga Farming System (BFS), Eastern Highlands (EH), Eastern Savannah (ES), Karamoja Dry Zone (KDZ), Karamoja Wet Zone (KWZ), Lake Albert Crescent (LAC), Lake Victoria Crescent (LVC), Northern Farming System (NFS), South Western Highlands (SWH), West Nile Farming System (WFS), and Western Range Lands (WRL). Each agro-ecological zone had its unique soil type, topography, rainfall pattern, and agricultural practices. Upon field inspection, specimens (both larvae and adults) were handpicked and stored in a 5 mL screw-cap tubes containing absolute ethanol. The samples were packed in Ziploc bags and then transported to the National Crops Resources Research Institute (NaCRRI), Namulonge molecular laboratory and kept in -20⁰C prior to DNA extraction.

DNA extraction

Total genomic deoxyribonucleic acid (DNA) was extracted from a total of 95 specimens (one sample per district) using a modified Chelex method ³⁸ previously reported by Otim et al.,²². Prior to DNA extraction, each sample was rinsed twice with sterile molecular grade water (Thermo Fisher Scientific, UK) to remove the ethanol which was used for preservation. Using a clean sterile surgical blade, a portion of each specimen was excised and placed in a sterile 1.5 mL micro-centrifuge tube. Fifty microliters of 10% Chelex 100 solution were added followed by 10μL (20mg/mL) of Proteinase K solution. Each sample was incubated at 56⁰C overnight, followed by brief vortex and heat inactivation at 100⁰C for 15 min. The samples were then centrifuged at 15,900 relative centrifugal forces (rcf) for 3 minutes. Forty microliters of supernatant (genomic DNA) of each sample were collected into new sterile 1.5 mL microfuge tube and stored at -20⁰C until PCR amplification. The unused portions of the specimens were stored in absolute ethanol and kept at -20⁰C for future.

PCR amplification and sequencing of FAW CO1 and Tpi partial gene segments

All partial gene sequences of CO1 and Tpi were amplified using three different primers sets, previously designed and used by Rodney et al., ¹⁹. Primers CO1_101F, 5’-TTCGAGCTGAATTAGGGACTC-3’ and CO1_911R, 5’-GATGTAAAATATGCTCGTGT-3’ that give amplicons length of 811bp, were used to amplify the FAW CO1A barcode region (Fig. 10A). This fragment was used in FAW identification at both species and host strains level (CO1 corn-strain, CO1-CS, and CO1 rice-strain, CO1-RS). Amplification of a 603bp fragment of CO1B gene segment with CO1_891F, 5’-TACACGAGCATATTTTACATC-3’, and CO1_1472R, 5’-GCTGGTGGTAAATTTTGATATC-3’ primer pair (Fig. 10A), was performed to determine haplotype profiles of S. frugiperda and confirm FAW species host strain identity in Uganda. The CO1B single nucleotide polymorphisms at sites CO1₁₁₆₄and CO1₁₂₈₇ were used to identify rice-strain (CO1₁₁₆₄ = T, CO1₁₂₈₇ = A) and four corn-strain haplotypes (h1-h4) defined by polymorphisms at two loci, together forming the corn-strain group (Fig. 10A).

Single nucleotide polymorphisms (SNPs) site (e4₁₈₃) in the Tpi gene (Fig. 10B) was also used to identify host strain identity and its results were compared with CO1 markers. Approximately 500bp fragment of the Tpi gene segment was amplified using Tpi_ 282F, 5’-GGTGAAATCTCCCCTGCTATG-3’ and Tpi_850R, 5’-AATTTTATTACCTGCTGTGG-3’ primer pairs. The FAW host strains were identified with the presence of either nucleotide base letter “C” or “T”, for corn-strain (Tpi-C) or rice- strain (Tpi-R), respectively. The PCR products for primers Tpi_282F and Tpi_850R were sequenced with primer Tpi_412F, 5′-CCGGACTGAAGGTTATCGCTTG-3’, to avoid overlap of two genes which may result in data ambiguity from heterozygous males due to insertion or deletion (indels) ¹⁹.

A PCR mixture of 16.0 µl of nuclease-free water (ThermoFisher Scientific, UK), 2.5 µl PCR buffer, 2.5 µl MgCl₂ (2.5mM), 0.5 µl dNTPs (10 mM), 1 µl of each primer (10 µM), 0.5 µl (2.5 units) of Dream Taq DNA polymerase ® (ThermoFisher-Scientific) and 1 µl of the template DNA was used. The PCR thermo-cycling conditions consisted of 94⁰C (2 minutes), followed by 35 cycles of 94⁰C (30s), 56⁰C (35s), and 72⁰C (45s) for denaturation, annealing, and extension, respectively. The PCR products were analyzed by electrophoresis in 1XTAE buffer on a 1.4% (w/v) agarose gel stained with 5 µl of ethidium bromide (0.5 μg/mL) and run at 80 volts for 1 hour. The gels were then visualized under a UV light source and photographed using gel documentation system (U: GENIUS3-SYNGENE). The PCR products that gave positive results of the expected band sizes were then sequenced at Macrogen Europe BV. (Meiberg dreef 31, Amsterdam, the Netherlands).

Sequence analysis and phylogenetic comparisons of sequencing data

Sequence cleaning and editing were done in Chromas software (Version 2.6.6). Mega blast Nucleotide Basic Local Alignment Search (BLASTn) was performed in the NCBI database to determine homologous sequences in the database ^39,40 . Cleaned sequences were then imported to Geneious program 8.05 for further analyses. Multiple sequence alignment and consensus sequence building were performed using Multiple Sequence Comparison by Log- Expectation (MUSCLE).

To infer the evolutionary relationship between Ugandan sequences, and retrieved sequences (out-group, other Spodoptera species, and S. frugiperda corn and rice strains) from NCBI ⁴⁰, which were previously reported by Nagoshi et al.,⁴¹, we generated neighbor-joining consensus trees using Jukes-Cantor genetic distance model with 70% support threshold and 1,000 number of replicates in a bootstrap resampling method. Both CO1 and Tpi DNA sequences of FAW selected were compared with DNA sequences of the fall armyworm host strains and other Spodoptera species that are available in the GenBank ⁴⁰

Statistical data analysis

Haplotype strain identification scoring data were subjected to descriptive analysis in R software version 3.5.3 (R Core Team, 2019). Bar plots were constructed to present the distribution of host strains and CO1 haplotypes across agro-ecological zones in Uganda.

Genetic analyses of this study provide the most fundamental information on the genetic identity and diversity of the S. frugiperda populations across all agro-ecological zones in Uganda. We observed two FAW groups which were later identified as corn and rice strains, with the latter being more predominant according to the CO1 marker. Both strains are widely distributed across all agro-ecological zones of Uganda and our study identified Florida, and three Asian nations (China, India and Pakistan) as the likely origin of invading FAW populations. Upon comparison of both mitochondrial and Tpi marker results, we observed a substantial disagreement between markers. Although the disagreement between Tpi and CO1 markers was noted, a concordant result between CO1A and CO1B was obtained and this enabled the elucidation of genetic identity, diversity, distribution, predominance, and haplotypes determination. To some extent, the use of a combination of both Tpi and CO1 markers in this study increased the detection power that revealed the likely sources of FAW invasions in Uganda and Africa.

This study has enhanced knowledge in the genetics of pest invasion in Uganda, necessary to confirm and monitor pathways of introductions and the spread of pest infestation. The information in this study is therefore beneficial in enhancing integrated pest management strategies which may be aimed at sustainable development and deployment of genetically modified environmentally friendly Bt. corn and in choosing effective insecticides for control, coupled with conventional host plant resistance breeding and other biological control methods. Effective management will then result in crop protection, increased maize production, food security, and better income for stakeholders, especially the smallholder farmers in Africa and beyond.

Data availability

All data analyzed or generated in this study are included in or referenced in this article.

Acknowledgments

We gratefully acknowledge the financial support for this research by the government of the Republic of Uganda under the Agricultural Technology and Agribusiness Advisory Services (ATAAS) Project (Project ID No. P109224). The Authors thank Moses Opio and Rwot Onen Bob Innocent for their support in sample collection. We are grateful to Geoffrey Okao-Okuja and J. F Osingada for technical assistance. We are also grateful to farmers for allowing us access to their fields and extension officers who guided the team during sample collection.

Author contributions

G. O. Conceptualized, performed formal analysis, Investigation, Methodology, Visualization, Data curation. Wrote original draft, reviewed and edited; H. G. M. Visualized, Wrote Original draft, reviewed and edited; D. K. Performed formal analysis, Investigation, Methodology, Visualization, Data curation. Wrote original draft, reviewed and edited; F. A. carried out formal analysis, Visualization, Writing - review and editing; S. A. Investigated, curated Data, Methodology and Supervision; H. W. Visualization, Supervision, Writing - review and editing; M. H. O. Conceptualization, Methodology, funding acquisition, Investigation, Project administration, Resources, project Supervision, Validation, Visualization, Writing – original draft, writing-review and editing.

Competing interests

The authors declare no competing interests.

Additional information

Correspondence and requests for materials should be addressed to M.H.O.

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No competing interests reported.

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Genomic analyses of fall armyworm (Spodoptera frugiperda) populations from Uganda using mitochondrial cytochrome oxidase subunit 1 and nuclear triose-phosphate isomerase (Tpi) genes

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