Mohamed Makni

Genetic Diversity of Potato Aphid, Macrosiphum euphorbiae, Populations in Tunisia Detected by RAPD

In this study, the genetic structure of 15 populations of the potato aphid, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae), from Tunisia was characterized using RAPD markers. Five decameric random primers revealed 113 polymorphic loci that were used to estimate population structure with an average of 22.6 polymorphic bands per primer. Analysis of molecular variance (AMOVA) showed significant (P < 0.001) genetic diversity among populations (78.16 %) rather than within populations (21.84 %) that has been confirmed by Fst and Nm values. A cluster analysis based on genetic distance revealed that the 15 populations were divided into three groups. Populations from the south were differentiated into one group irrespective of host plant, whereas the other populations were distributed between two groups independently of the geographic origin and host plant. These results represent the first evidence for genetic structuring in M. euphorbiae.

Genetic variability of green citrus aphid populations from Tunisia, assessed by RAPD markers and mitochondrial DNA sequences

The green citrus aphid Aphis spiraecola (Patch) is one of the major pests of several plant species including economically important crops such as citrus. In this study, we used random amplified polymorphic DNA (RAPD) markers and mitochondrial cytochrome oxidase subunit I sequences to assess the level and distribution of genetic diversity of A. spiraecola populations reared from Rutaceae and Rosaceae in different regions in Tunisia. RAPD analysis conducted on 141 individuals with 5 primers revealed only 50 polymorphic RAPD markers, indicating a low genetic diversity that might result from the lack of sexual phase for this species in Tunisia. Analysis of molecular variance (amova) showed that the genetic structure was not associated with geographic location or year of collection (P = 0.70 and 0.34, respectively); however, the host‐plant had a significant effect on the partitioning of the total genetic diversity (P < 0.01). Multidimensional scaling analysis indicated that the distribution of genetic variability was significantly influenced by the host‐plant with no evidence of spatial differentiation. Based on 20 barcode sequences of the mitochondrial cytochrome‐c oxidase subunit I (COI) gene, we revealed the occurrence of two haplotypes in association with the host‐plant. Results reported here suggest the occurrence of a limited gene flow between A. spiraecola populations from Rosaceae and Rutaceae and, therefore, a possible host‐race status that could be considered in the development of an integrated controlling strategy.

Genetic variation in target-site resistance to pyrethroids and pirimicarb in Tunisian populations of the peach-potato aphid, Myzus persicae (sulzer) (Hemiptera: Aphididae)

BACKGROUND We used molecular assays to diagnose resistance to pyrethroids and pirimicarb in samples of Myzus persicae from field crops or an insect suction trap in Tunisia. Genotypes for resistance loci were related to ones for polymorphic microsatellite loci in order to investigate breeding systems and patterns of genetic diversity, and to inform resistance management tactics. RESULTS The kdr mutation L1014F conferring pyrethroid resistance was found in all samples. The M918T s-kdr mutation also occurred in most samples, but only in conjunction with kdr. We discovered a previously unreported genotype heterozygous for L1014F but homozygous for M918T. Samples with modified acetylcholinesterase (MACE) conferring resistance to pirimicarb were less common but widespread. 16% of samples contained both the kdr and MACE mutations. Many unique microsatellite genotypes were found, suggesting that M. persicae is holocyclic in Tunisia. There were no consistent associations between resistance and microsatellite markers. CONCLUSION This first study of insecticide resistance in M. persicae in North Africa showed genetic variation in insecticide resistance within microsatellite multilocus genotypes (MLGM s) and the same resistance mechanisms to be present in different MLGM s. This contrasts with variation in northern Europe where M. persicae is fully anholocyclic. Implications for selection and control strategies are discussed.

Large-scale analysis of NBS domain-encoding resistance gene analogs in Triticeae

Proteins containing nucleotide binding sites (NBS) encoded by plant resistance genes play an important role in the response of plants to a wide array of pathogens. In this paper, an in silico search was conducted in order to identify and characterize members of NBS-encoding gene family in the tribe of Triticeae. A final dataset of 199 sequences was obtained by four search methods. Motif analysis confirmed the general structural organization of the NBS domain in cereals, characterized by the presence of the six commonly conserved motifs: P-loop, RNBS-A, Kinase-2, Kinase-3a, RNBS-C and GLPL. We revealed the existence of 11 distinct distribution patterns of these motifs along the NBS domain. Four additional conserved motifs were shown to be significantly present in all 199 sequences. Phylogenetic analyses, based on genetic distance and parsimony, revealed a significant overlap between Triticeae sequences and Coiled coil-Nucleotide binding site-Leucine rich repeat (CNL)-type functional genes from monocotyledons. Furthermore, several Triticeae sequences belonged to clades containing functional homologs from non Triticeae species, which has allowed for these sequences to be functionally assigned. The findings reported, in this study, will provide a strong groundwork for the isolation of candidate R-genes in Triticeae crops and the understanding of their evolution.

Genetic Variation and Invasion Pattern of the Arabian Rhinoceros Beetle, Oryctes agamemnon arabicus (Burmeister) (Coleoptera: Scarabaeidae), in Tunisia, Deduced from Mitochondrial DNA Sequences

The Arabian rhinoceros beetle, Oryctes agamemnon arabicus (Burmeister, 1847), is an invasive species, introduced to Tunisia from the United Arab Emirates and causing serious damage to date palm trees in southern Tunisia. Considering the economic importance of this pest, it is necessary to understand the genetic diversity within and among its populations and investigate its dispersal mode. In the present study, the mitochondrial gene encoding the cytochrome oxidase subunit I (COI) was used to analyse the extent of genetic variation between samples of O. agamemnon arabicus, collected from geographically distant locations in Tunisia. A 505 bp DNA fragment, resulting from PCR amplification, was sequenced. Representative sequences were aligned, revealing nine polymorphic sites that identified eight haplotypes. Haplotype diversity (Hd) and Tajimas D neutral test were 0.707 and -0.043, respectively, indicating a low level of mtDNA variability. The calculated gene flow value was Nm = 0.47, indicating a high amount of gene flow occurring between populations from Tozeur and those from Kebili, although both departments are separated by the Chott-El-Jerid salt lake. The presence of common haplotypes between Tozeur and Kebili as well as the clustering pattern obtained by the NJ method confirmed this result. Based on these facts and considering the limited flight of O. agamemnon arabicus and its slow reproduction, it is likely that its expansion in Tunisia was facilitated mainly by human transportation of infected offshoots.

Genetic Variability of the Tomato Leaf Miner (Tuta absoluta Meirick; Lepidoptera: Gelechiidae), in Tunisia, Inferred from RAPD-PCR

El minador de hojas de tomate Tuta absoluta Meyrick ha invadido el cultivo del tomate (Solanum lycopersicum L.) en Tunez desde 2008 y actualmente representa una importante amenaza para su produccion. En este estudio usamos la tecnologia de ADN polimorfico amplificado al azar-reaccion de cadena polimerasa (RAPD-PCR) para evaluar la variabilidad genetica dentro y entre siete poblaciones de T. absoluta, colectadas desde tomate en diferentes regiones de Tunez. Usando cinco primers RAPD-PCR y 108 individuos, se registraron 140 fragmentos polimorficos. Se generaron 335 fenotipos RAPD diferentes, entre los cuales 71 fueron redundantes y 264 unicos para una poblacion especifica. La estructura genetica de T. absoluta se investigo usando analisis de varianza molecular (AMOVA), distancias geneticas (Fst) y escalamiento multidimensional (MDS). Detectamos una alta diversidad genetica dentro y entre poblaciones en conjunto con una diferenciacion significativa entre poblaciones, sugiriendo que los genotipos fundadores podrian haber sido responsables de la introduccion de T. absoluta en Tunez. La presencia de fenotipos superpuestos probablemente indica eventos de migracion entre poblaciones, principalmente a traves de material vegetal infestado transportado por humanos.

Molecular Evolution of piggyBac Superfamily: From Selfishness to Domestication

The piggyBac transposable element was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and specificity compared to the other Class II elements, the diversity and evolution of this superfamily have been only partially analyzed. Two main types of elements can be distinguished: the piggyBac-like elements (PBLE) with terminal inverted repeats, untranslated region, and an open reading frame encoding a transposase, and the piggyBac-derived sequences (PGBD), containing a sequence derived from a piggyBac transposase, and which correspond to domesticated elements. To define the distribution, their structural diversity and phylogenetic relationships, analyses were conducted using known PBLE and PGBD sequences to scan databases. From this data mining, numerous new sequences were characterized (50 for PBLE and 396 for PGBD). Structural analyses suggest that four groups of PBLE can be defined according to the presence/absence of sub-terminal repeats. The transposase is characterized by highly variable catalytic domain and C-terminal region. There is no relationship between the structural groups and the phylogeny of these PBLE elements. The PGBD are clearly structured into nine main groups. A new group of domesticated elements is suspected in Neopterygii and the remaining eight previously described elements have been investigated in more detail. In all cases, these sequences are no longer transposable elements, the catalytic domain of the ancestral transposase is not always conserved, but they are under strong purifying selection. The phylogeny of both PBLE and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors.

A rapid diagnostic technique of Bactrocera cucurbitae and Bactrocera zonata (Diptera: Tephritidae) for quarantine application

Backround The melon fruit fly Bactrocera cucurbitae and the peach fruit fly Bactrocera zonata are serious pests, native to Asia, that have recently invaded Egypt. In Tunisia, no report of these species has yet been made, but pest risk data suggest that both Bactrocera species are likely to establish in other countries of the Mediterranean region. Results A quick method, based on a PCR-RFLP of the mitochondrial COI gene, has been developed to enhance species identification for quarantine purposes. The restriction enzymes DdeI and XmnI were used to digest COI PCR products, enabling a rapid diagnosis of B. cucurbitae and B. zonata and their distinction from Tunisias most devastating fruit pest, Ceratitis capitata. Conclusion The simplicity and relatively low cost of this molecular approach will replace the need to rear immature stages through to adults for identification and will facilitate rapid quarantine decisions providing greater plant protection.

Occurrence of target-site resistance to neonicotinoids in the aphid Myzus persicae in Tunisia, and its status on different host plants: Resistance to neonicotinoids in Myzus persicae

BACKGROUND The R81T mutation conferring target-site resistance to neonicotinoid insecticides in Myzus persicae was first detected in France and has since spread across much of southern Europe. In response to recent claims of control failure with neonicotinoids in Tunisia, we have used a molecular assay to investigate the presence and distribution of this target-site mutation in samples collected from six locations and six crops attacked by M. persicae. RESULTS The resistance allele containing R81T was present at substantial frequencies (32-55%) in aphids collected between 2014 and 2016 from northern Tunisia but was much rarer further south. It occurred in aphids collected from the aphids primary host (peach) and four secondary crop hosts (potato, pepper, tomato and melon). Its absence in aphids from tobacco highlights complexities in the systematics of M. persicae that require further investigation. CONCLUSION This first report of R81T from North Africa reflects a continuing expansion of its range around the Mediterranean Basin, although it remains unrecorded elsewhere in the world. Loss of efficacy of neonicotinoids presents a serious threat to the sustainability of aphid control.

Development of polymorphic microsatellite loci for the tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae)

The tomato leaf miner Tuta absoluta (Lepidoptera: Gelechiidae) is a devastating pest of tomato originating from South America (García and Espul 1982). Since its initial detection in tomato glasshouses in Spain at the end of 2006, T. absoluta has been spreading rapidly across Europe, northern Africa and Middle East, causing severe damage to tomato crops (Desneux et al. 2010; Roditakis et al. 2010; Abbes et al. 2012). The main host plant of T. absoluta is tomato (Lycopersicon esculentum). Nevertheless this pest also attacks various plants as secondary hosts such as potato (Solanum tuberosum L.), sweet pepper (S. muricatum L.), eggplant (S. melongena L.) and various wild solanaceous plants (Siqueira et al. 2000). Variable molecular markers were used to estimate genetic diversity and population structure of this invasive species. AFLP markers were used to assess genetic diversity of T. absoluta populations from Brazil (Suinaga et al. 2004). Mitochondrial and nuclear markers were also used to estimate genetic diversity of T. absoluta populations from Spain (Cifuentes et al. 2011) and, more recently, RAPD markers have been applied to understand the structure of T. absoluta populations from Tunisia to furnish information about genetic variation during the colonization process (Bettaibi et al. 2012). In recent years, microsatellites markers have proven to be very powerful in studying genetic structure of many insect populations such as Myzus persicae (MingHua et al. 2009); Aphis spiraecola (Cao et al. 2012) and Bactrocera dorsalis (Isasawin et al. 2012).