Identification and molecular characterization of groundnut leaf miner in Uganda

Although the leaf miner attacking groundnut in Africa has been widely reported as Aproaerema modicella (Deventer), a common groundnut (Arachis hypogaea L.) and soya bean (Glycine max (L.) Merr.), a Pest in Indo-Asian countries, a proper taxonomic identification of the pest has not been completed. A survey for species diversity of the pest was conducted on groundnut, the common host crops for leafminer species in Uganda, across 100 groundnuts farmers’ fields in four agro ecological zones during the 2016-2017 growing season. 80 specimens comprising 40 larvae, 25 pupae and 15 moths of what was thought to be A. modicella (all from groundnut) were collected from the ten survey sites, and their mitochondrial DNA (mtDNA) COI were sequenced and compared with those from the BOLD gene bank. Infestation by GLM was observed on all groundnut fields sampled with Eastern Uganda being the hot ecological spot especially Namutumba district. The mtDNA COI from all specimens of the pest, matched 100% with the sequences in BOLD belonging to Aproaerema simplexella PS1, a species occurring in Australia, and known as the soya bean moth in that country. There was very little genetic diversity between and within the populations from the ten sites, which suggested that the populations were maternally of the same origin. Conclusively, this study like other studies elsewhere in Africa confirm to the fact that the leafminer attacking groundnuts and other crops such as soya bean was A. simplexella PS1 (100% match on the BOLD system), native to Australia, which suggested that Australia may be the origin of the pest not Indo-Asian countries. Keywords— Arachis hypogaea, Aproaerema modicella, mitochondrial DNA.


INTRODUCTION
Groundnut (Arachis hypogaea L.) is the second most important legume after beans (Phaseolus vulgaris) in both nutritional and economic empowerment of communities in Uganda. It's grown primarily for high quality edible oil (36-54% on dry matter basis) and easily digestible proteins (20-50%) in its seeds (D. okello et al, 2010). It is cultivated worldwide in tropical, sub-tropical and warm temperature areas located between 400N to 400S with world production of 36.9 million tonnes from an area of 25.2 million ha. Africa accounts for 40% of the global area planted to groundnuts, with only 26% production. The highest average yields were observed in Southern Africa and the lowest in East Africa (ICRISAT 2012). In 2006, the average groundnut yield recorded in Sub Saharan Africa was 980 kg/ha, considerably less than the world average of 1,690 kg/ha (Bucheyeki et al. 2008). The production of these crops on the continent is seriously threatened by a leaf miner, widely reported as Aproaerema modicella (Deventer) (Lepidoptera: Gelechiidae) (Kenis and Cugala, 2006). First noticed as a serious pest on groundnut in 1998 in Uganda 4 25 leaf miner (GLM), the pest has raised considerable alarm and concern in the groundnut production zones of Uganda (Epieru, 2004).
In Uganda, GLM is known to be present in all groundnut producing areas of the country (Epieru 2004). However, the epidemic of GLM on groundnut in Uganda is sporadic and its severity appears to vary from place to place and from year to year, making it extremely difficult to predict. As a new pest, not much information is available on its ecology and ecophysiology that might help to predict its incidence and outbreaks.  Shanower et al. (1993), which reinforced the assumption that the pest was A. modicella. Because of its sudden appearance, the GLM occurring in Africa is thought to be a recent invasion from the IndoAsian continent (Kenis and Cugala, 2006) where A. modicella is native and seriously infests groundnut and soybean (Shanower et al., 1993). Whilst this is possible, the pest may have evolved and spread within Africa.
Whereas morphological studies have been the keystone of insect pest identification in the past, and continue to be in the present, modern molecular techniques offer complementary, faster and more precise options for species identification (Scheffer, 2000), and is especially useful in differentiating related species that share similar morphological characteristics.
In addition, molecular techniques, e.g. DNA finger printing, especially those involving the mitochondrial DNA (mtDNA), are more reliable in pinpointing or tracing the geographical origin/links of pests and their paths of spread, (Scheffer, 2000;Simmons and Scheffer, 2004). There were two objectives in the present study. The first objective was to identify the species of leafminer attacking groundnuts in Uganda.t and the second objective was to determine its inter and intra-population genetic diversity by analysing in both cases the mtDNA CO1 gene of specimens collected from widely separated sites.

Sampling
Groundnut leaf miner samples (both adult and larvae) were collected from ten agro ecological zones in Uganda. All larvae were preserved in 70% ethanol then brought to the icipe Molecular Pathology Lab in the Arthropod Pathology Unit for processing. Morphological characteristics were documented using a Leica EZD stereomicroscope (Leica Microsystems (UK) Ltd) then the samples were immediately preserved in 95% ethanol and stored at -20 °C for DNA extraction later.

DNA extraction, PCR and sequencing
Each individual insect sample was surface-sterilized using 3% NaOCl and rinsed three times with distilled water. Genomic DNA was extracted using the Isolate II genomic DNA Kit (Bioline, UK), following the manufacturer's instructions. The purity and concentration of the resultant extracted DNA was determined using Nanodrop 2000/2000c Spectrophotometer (Thermo Scientific). Polymerase chain reaction (PCR) was done to amplify the mitochondrial region and 2 sets of markers were used (  (Hall, 1999) to create consensus sequences which were saved in the fasta format in MEGA5 (Hall, 1999).
Additionally, all consensus sequences were entered in BOLD to positively identify species. All specimen were identified to be from the same species, except two sample, which was identified to be from a different species, and was therefore used as an outgroup in the analysis. Additionally, the sequences were also exposed to Multiple Sequence Alignment by ClustalW (http://www.genome.jp/tools/clustalw/) to verify level of similarity between samples.

Summary of the identities of the processed samples
For conclusive identification of the species, similarity analysis was done. Similarity searches were conducted by querying the consensus sequences via BLAST at the GenBank database hosted by National Centre of Biotechnology Information (NCBI). BLAST (Basic Local Alignment Search Tool) algorithm finds regions of local similarity between sequences, in which consensus sequences were compared to reference sequences in the GenBank database. In addition to this, query was also done in BOLD (Barcode of Life Database). within and between the specimens from the ten surveyed sites (Table 1).

IV. DISCUSSION
There has been a general assumption that the GLM occurring on groundnut in Africa has its origins in Asia,  (Bailey, 2007). This particular strain of A. simplexella is thought to be native to Australia where it is reported to be a pest for soybean (Common, 1990;Bailey, 2007). The evidence obtained from DNA analysis in the present study therefore suggests that the GLM in Uganda may have links with Australia. This is further supported by the fact that all GLM specimens taken from the ten widely separated sites in Uganda were identified as A. simplexella PS1, with A. modicella not listed in the most closely related species (Table 1). This infers that all infestations of GLM in Africa may be caused by the former, and not the latter species. Based on morphological characteristics, Shanower et al. (1993) suggested that the species found in Africa may be different from that found in India or Indonesia, describing the GLM in India as Anacampsis nerteria (Meyr.) (Meyrick, 1906), the one in Africa as Stomopteryx subsecivella and another in India-Indonesia as A. modicella (Deventer). It is thus clear that a large degree of uncertainty has always existed as to the correct classification of GLM in Africa. No attempt has however been made to discriminate between the species genetically. Previous to our DNA analysis, A. simplexella PS1 was known to be present only in Australia with recent identification in South Africa.

V. CONCLUSION
Mitochondrial DNA COI analysis identified GLM in Uganda as A. simplexella PS1 (100% match on the BOLD system), native to Australia, which suggested that Australia may be the origin of the pest. It is most likely that GLM being reported on groundnut in other parts of Africa is also A. simplexella PS1. Secondly, the 100% match on the BOLD system indicated that there was very little genetic diversity between and within the populations, suggesting that the pest might be from the same origin and could be a recent introduction to Uganda. Given that the sequences of GLM in Uganda matched those of A. simplexella PS1 and that the damage symptoms of the pest on groundnut are similar to those of A. modicella found in Asia, there is a need to determine if the two species are indeed genetically different. This has a bearing on the development and use of groundnut lines that are resistant to GLM, in countries where it is a problem. For the purpose of formulating strategies for managing the pest, there is also a need to determine its correct identity, its host range as well as it's in between season survival tactics in Africa.