First records of the tomato leaf miner Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) in South Africa

Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is an invasive and extremely damaging leaf-mining moth of South American origin. It is a serious pest of tomatoes. This species was first recorded outside of its native range in Spain (2006), and has subsequently spread into Asia and Africa. Here we report the first records of this species for the Republic of South Africa, where moths were first trapped in August and October 2016. The species was identified using both morphological and molecular approaches. Monitoring of the spread of tomato leaf miner in South Africa and the implementation of control measures are managed by the Department of Agriculture, Fisheries and Forestry (DAFF), South Africa.

Tuta absoluta is native to South America and was first reported outside of its native range in 2006, from Spain (Desneux et al. 2010).It has already spread widely through Europe (Desneux et al. 2010), and is currently spreading eastwards into Asia and southwards into Africa.It has invaded numerous sub-Saharan countries, including Nigeria, Niger and Senegal in West Africa (Guimapi et al. 2016) and Tanzania (Chidege et al. 2016) Kenya and Uganda (Tumuhaise et al. 2016) in East Africa.Through simulations inferred by the moth's ability to cover long distances, the model proposed by Guimapi et al. (2016) accurately predicted T. absoluta reaching South Africa in 2016, ten years after its initial detection in Spain; Tuta absoluta was first confirmed from South Africa in August 2016.
TLM larvae make blotch leaf mines (Figure 1) and superficial mines on tomato fruit, resulting in the death of plants (Figure 2) or in unmarketable, rotten fruit (Figure 3).Crop losses of 80-100% have been reported from countries in northern and western Africa invaded by TLM (Chidege et al. 2016).
The importance of the tomato leaf miner to tomato production in sub-Saharan Africa was discussed by Brévault et al. (2014).In South Africa, more than 45 pests are known to attack tomato (Visser 2009).Prior to the arrival of the tomato leaf miner, the most important tomato pests in South Africa were spider mites (Acari: Tetranychidae), American leaf miner Liriomyza trifolii (Burgess, 1880) and potato leaf miner L. huidobrensis (Blanchard, 1926) (Diptera: Agromyzidae), African bollworm Helicoverpa armigera (Hübner, [1809]) (Lepidoptera: Noctuidae), whitefly (Hemiptera: Aleyrodidae) as virus vectors, and semiloopers (Noctuidae) (Visser 2015).Using the CLIMEX modelling platform, Tonnang et al. (2015) predicted a very high likelihood of long-term survival of T. absoluta along the southern and eastern coastal areas, and the northern and eastern regions of the Mpumalanga and Limpopo Provinces of South Africa.The newly established presence of the tomato leaf miner will undoubtedly have an impact on the composition of pest complexes, and consequently on current pest control strategies, in particular insecticidal control.
The potato tuber moth, Phthorimaea operculella (Zeller, 1873), also a member of the family Gelechiidae, is a major pest of potato (also Solanaceae) and a minor pest of tomato in South Africa (Visser 2009).It inflicts the same type of damage on tomato as the tomato leaf miner, but to a much lesser extent.Although potato is a host of the tomato leaf miner, the risk to potato appears to be limited (EPPO 2005).However, reports indicate that severe simultaneous attacks by the tomato leaf miner and potato tuber moth are possible (Sannino and Espinosa 2010), although differences in cultivar and environment may lead to a higher or lower pest status on potato in South Africa.
Here we report on the first records of Tuta absoluta in the Republic of South Africa.Total genomic DNA from 20 moths from sample AcP 9557 was extracted at the laboratory of the Plant Quarantine Station, DAFF, Stellenbosch.DNA was extracted with the QIAmp ® DNA Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol.Extracted DNA concentrations were measured with a NanoDrop ® ND-1000 spectrophotometer (NanoDrop Technologies, Inc.).One nanogram of DNA was used in subsequent PCR amplifications.PCR products of ~658 bp in length were amplified for a fragment of the COI gene using the primer pair LCO1490 (5'-GGTCAACAAATCA TAAAGATATTGG-3') and HCO2198 (5'-TAAAC TTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994).This primer pair consistently amplifies a 710 bp fragment of the COI gene in a wide range of invertebrate taxa (Folmer et al. 1994).The TopTaq MasterMix kit (QIAGEN) was used in all reactions.Thermocycling conditions consisted of denaturation at 95 °C for 1 minute, followed by 35 cycles of 95 °C for 45 seconds, 51 °C for 45 seconds and 72 °C for 1 minute, with a final extension at 72 °C for 3 minutes.The PCR product was visualized on a 1.2% agarose gel and purified with the Wizard ® Genomic DNA Purification Kit (Promega Corporation).Purified products were sequenced in both directions using BigDye ® Terminator v3.1 chemistry (Applied Biosystems) and the same primer pair used for the PCR reactions.Sequences were edited and aligned using CLC Main Workbench 6.9.

Morphological identification
The adult of Tuta absoluta is diagnosed by the following characters: External characters: moth small, body length ca.6 mm (Figure 5).Forewings narrow, with brown, grey and black mottling; hindwings lanceolate, dark grey with long cilia.Antennae, labial palpi and legs with dark brown and grey banded appearance; antennae long and filiform, labial palpi prominent and curved upward.
The original samples submitted to Dr Martin Krüger were positively identified as Tuta absoluta.The internal and external morphology of all samples were examined by (VMU) and were found to conform entirely to the above-mentioned list of characters, thus confirming their identity as T. absoluta.
Tuta absoluta moths are similar in appearance to P. operculella, which presently is the only other pest of tomato in South Africa with which the tomato leaf miner could be confused.However, in addition to subtle differences in colouration and markings (Figure 5), the potato tuber moth is larger (ca.8 mm in length) and the male genitalia are distinct, with the valvae being slender and curved apically.

Molecular analysis
The consensus sequence was used in a BLASTN (basic local alignment search tool) search (Boratyn et al. 2013) to find matching sequences (Zhang et al. 2000).This sequence was deposited in GenBank with accession number KY212128.This COI sequence (KY212128) matched the Tuta absoluta sequences KX443111 and KX443108 (Sint et al. 2016) and JQ749676 from Tunisia (Bettaïbi et al. 2012) with 100% identity.
The molecular data support the morphological studies and confirm the present specimens as T. absoluta.

Implications and discussion
Tuta absoluta and P. operculella can easily be confused, especially when they co-exist in single cropping systems, as the moths and the larvae are very similar  in appearance and behaviour.Although moth pheromone traps are fairly species-specific, the identity of initial trap catches should be verified by examination of the male genitalia.
The tomato leaf miner is undoubtedly a threat to tomato production in South Africa.The development of resistance of T. absoluta populations of diverse geographic origin to diamide insecticides, a relatively new introduction to the market for the control of T. absoluta and other Lepidoptera (Roditakis et al. 2017), as well as varying efficacy of conventional pesticides (Campos et al. 2014), could result in complete destruction of tomato plots by TLM in the future.Smallscale farmers and home gardeners will necessarily be most vulnerable, as many of them may lack the resources required to combat this pest.Urgent research, including investigations into alternative control strategies is therefore imperative.These strategies may include natural control by indigenous predators and parasitoids, the use of pheromone traps for monitoring and mass trapping, and the role of sanitation in reducing infestation levels.
Tuta absoluta were trapped in August 2016 from the Komatipoort vicinity and the southern Kruger National Park (KNP) (all Mpumalanga province) (Figure 4) following extensive surveillance by Crop Watch Africa of the entire eastern border of South Africa with Mozambique, using Delta pheromone traps (voucher specimens AcP 9555 -AcP 9558); the traps contained the Tuta absoluta pheromone: (E,Z,Z) -3,8,11 -Tetradecatrienyl 0,76 mg / lure and (E,Z) -3,8 -Tetradecadienyl 0,04 mg / lure.These original samples were submitted to Dr Martin Krüger (MK), Ditsong National Museum of Natural History, Pretoria, on request of the Department of Agriculture, Fisheries and Forestry (DAFF), South Africa, for verification.This first detection of T. absoluta in South Africa was subsequently announced by the International Plant Protection Convention (IPPC 2016).Subsequent material (voucher specimens: AcP 9559) was collected from trap catches from Pretoria.All samples were

Table 1 .
Initial sampling sites of Tuta absoluta in South Africa (KNP: Kruger National Park).