Persistency of Indigenous and Exotic Entomopathogenic Fungi Isolates under Ultraviolet B (UV-B) Irradiation to Enhance Field Application Efficacy and Obtain Sustainable Control of the Red Palm Weevil.

The red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae) has become a key invasive pest and major threat to the palm tree worldwide. Several entomopathogenic fungi are used in insect biological control programs. In the present study, persistency of different local and exotic fungal isolates of Beauveria bassiana and Metarhizium anisopliae was evaluated under UV-B irradiation with different exposure intervals. Several factors, including ultraviolet (UV) light, significantly decrease germination rate of fungi, as UV penetrates and damages their DNA. Several studies have investigated that UV-resistant conidia germinate better under harsh environmental conditions. Seven local and exotic fungi isolates ("BbSA-1", "BbSA-2", "BbSA-3", "MaSA-1", "BbIDN-1", "MaIDN-1", and "MaIDN-2") were tested in the current study under UV-B irradiation having different UV exposure times (i.e., 15, 30, 60, 120, 180, 240, and 300 min). The colony-forming unit (CFU) in each isolate was used to calculate the survival rate. Results indicated that survival rate of all the isolates decreased under UV-B irradiation for all exposure times compared to no exposure to UV-B irradiation. The CFU number decreased as the exposure time increased. Fungi isolates "MaSA-1", "BbSA-1", "BbSA-2", "MaIDN-1", and "MaIDN-2" could persist after 300 min exposure to UV-B, while the remaining isolates, such as "BbIDN-1", and "BbSA-3", could not persist after 300 min exposure to UV-B. The ultimate objective of the present research was to explore an ultraviolet-tolerant fungal isolate that might be useful in the field application for the sustainable management of the red palm weevil, which has become a key invasive pest in many regions rather than its native range. Most of the fungus isolates studied in the present work were collected from Saudi Arabia's Al-Qatif region, where the red palm weevil has infested more than ten thousand trees, worth millions of riyals.

Taxonomy and distribution of termite fauna (Isoptera) in Riyadh Province, the Kingdom of Saudi Arabia, with an updated list of termite species on the Arabian Peninsula.

The present study shows an updated synoptic list of the 30 known Isoptera of the Arabian Peninsula which are classified under four families and nine genera. Twenty-seven species are hitherto known from the Kingdom of Saudi Arabia (KSA). The present inventory of the termites of Riyadh Province (KSA) indicated three species, Anacanthotermes ochraceous (Burmeister 1839), Psammotermes hypostoma Desneux, 1902 and a rare species, Coptotermes heimi (Wasmann 1902). We present an illustrated key to species based on the soldier caste. Anacanthotermes ochraceous, and P. hypostoma are widely distributed Palearctic species whereas C. heimi seems rare and is a new record for KSA. Distribution maps for the three species are provided based on recently collected material and literature records and remarks on species habitat preference are given.

Pathogenicity of local and exotic entomopathogenic fungi isolates against different life stages of red palm weevil (Rhynchophorus ferrugineus).

Entomopathogenic fungi are regarded as effective biocontrol agents in pest management. Different fungi isolates exhibit varying degree of pathogenicity against red palm weevil [Rhynchophorus ferrugineus (Olivier)]. The pathogenicity of four native isolate from Saudi Arabia (three Beauveria bassiana named as BbSA-1, BbSA-2, BbSA-3 and one Metarhizium anisopliae regarded as MaSA-1) and three exotic isolates from Indonesia (B. bassiana coded as BbIDN-1 and M. anisopliae named as MaIDN-1 and MaIDN-2) was evaluated against red palm weevil under laboratory conditions. The isolates were applied to eggs (1 day old), larvae (3 and 35 days old), pupae (5 days old) and adults (10 days old). The average mortality rate of eggs and hatched larvae was 100% in all of the isolates except BbSA-2 and BbIDN-1, where mortality was 93.3 and 90%, respectively. The lowest mortality rate (73.3%) was recorded for BbSA-3 against 3-days-old larvae; however, all other isolates caused >80% larval mortality. Meanwhile, 93.3% mortality of 35-day-old larvae was noted for MaSA-1 isolate. The highest pupa mortality (80%) was observed for MaSA-1, while remaining isolates caused >60% mortality. The isolates BbSA-1 and MaSA-1 caused 61 and 74.3% mortality in adults, respectively. The tested fungi isolates exhibited high virulence against all life stages of red palm weevil. Local isolates had higher pathogenicity than exotic isolates. The findings of the current study suggest that entomopathogenic fungi could be used as biological control agents for the management of red palm weevil. However, field studies are needed to reach the sound conclusions and practical applications.

De novo transcriptome analysis and identification of reproduction control genes from the red palm weevil Rhynchophorus ferrugineus.

Recent attacks by the red palm weevil, Rhynchophorus ferrugineus (Olivier), have become a severe problem for palm species. In present work, fat body transcriptome of adult female red palm weevil was analyzed, focusing on the identification of reproduction control genes. Transcriptome study was completed by means of next-generation sequencing (NGS) using Illumina Hiseq 2000 sequencing system. A total of 105,938,182 raw reads, 102,645,544 clean reads, and 9,238,098,960 clean nucleotides with a guanine-cytosine content of 40.31%, were produced. The processed transcriptome data resulted in 43,789 unique transcripts (with mean lengths of 1,172 bp). It was found that 20% of total unique transcripts shared up to 80%-100% sequence identity with homologous species, mainly the mountain pine beetle Dendroctonus ponderosae (59.9%) and red flour beetle Tribolium castaneum (26.9%). Nearly 25 annotated genes were predicted to be involved in red palm weevil reproduction, including five vitellogenin (Vg) transcripts. Among the five Vg gene transcripts, one was highly expressed compared with the other four (FPKM values of 1.963, 1.471, 1.028, and 1.017, respectively), and the five Vg gene transcripts were designated as RfVg, RfVg-equivalent1, RfVg-equivalent2, RfVg-equivalent3, and RfVg-equivalent4, respectively. The high expression level of RfVg verified by RT-polymerase chain reaction analysis suggested that RfVg is the primary functional Vg gene in red palm weevil. A high similarity of RfVg with other Coleopterans was also reflected in a phylogenetic tree, where RfVg was placed within the clade of the order Coleoptera. Awareness of the major genes that play critical roles in reproduction and proliferation of red palm weevil is valuable to understand their reproduction mechanism at a molecular level. In addition, for future molecular studies, the NGS dataset obtained will be useful and will promote the exploration of biotech-based control strategies against red palm weevil, a primary pest of palm trees.

RNAi-mediated silencing of vitellogenin gene curtails oogenesis in the almond moth Cadra cautella.

Vitellogenins, major yolk protein precursors, play an essential role in the reproduction and spread of all oviparous species, including insects. To investigate reproductive strategies of the warehouse moth Cadra cautella at the molecular level, a partial transcript of the C. cautella vitellogenin (CcVg) gene was extended through the rapid amplification of cDNA ends PCR and sequenced. The complete CcVg mRNA transcript was 5,334 bp long, which encoded a protein of 1,778 amino acids, including the first 14 amino acids of the signal peptide. The deduced CcVg protein contained a putative cleavage site (RTRR) at the amino-terminal side, similar to several other insect species. DGQR and GI/LCG motifs were present at the CcVg gene C-terminus, followed by nine cysteine residues. CcVg harbored 131 putative phosphorylation sites, numbering 84, 19, and 28 sites for serine, threonine, and tyrosine, respectively. The transcript showed a great resemblance with other lepidopteran Vgs. CcVg protein analysis revealed three conserved regions: 1) vitellogenin-N domain, 2) DUF 1943 (domain of unknown function), and 3) a von Willebrand factor type D domain. Additionally, sex, stage-specific, and developmental expression profiles of the CcVg gene were determined through RT-PCR. The Vg was first expressed in 22-day-old female larvae, and its expression increased with growth. The phylogenetic analysis based on different insect Vgs revealed that the CcVg exhibited close ancestry with lepidopterans. The CcVg-based RNAi experiments were performed, and the effects were critically evaluated. The qRT-PCR results showed that CcVg-based dsRNA suppressed the Vg gene expression up to 90% at 48 h post-injection. Moreover, CcVg-based RNAi effects resulted in low fecundity and egg hatchability in the CcVg-based dsRNA-treated females. The females laid eggs, but because of insufficient yolk protein availability the eggs could not succeed to hatch. The significant difference in the fecundity and hatchability unveils the importance of CcVg gene silencing and confirmed that the Vg gene plays a key role in C. cautella reproduction and it has the potential to be used as a target for RNAi-mediated control of this warehouse pest.

Neonicotinoid's resistance monitoring, diagnostic mechanisms and cytochrome P450 expression in green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)].

Green peach aphid [Myzus persicae (Sulzer) (Hemiptera: Aphididae)] is a significant pest with a known history of insecticide resistance. Neonicotinoids could manage this pest; however, their frequent use led to the evolution of resistance in field populations of M. persicae. Toxicity data for neonicotinoid insecticides synergized with pipernyl butoxide (PBO) in a field population (FP) were collected and compared to a laboratory susceptible clone (SC) of aphids. The enhanced expression of metabolic resistance-related cytochrome P450 gene CYP6CY3 and an arginine-threonine substitution were detected in FP, causing a single point mutation (R81T) at ß1 subunit of nicotinic acetylcholine receptor (nAChR) within D loop. High level of resistance to imidacloprid was developed in FP with 101-fold resistance ratio and moderate resistance level (10.9-fold) to acetamiprid. The results of PBO synergized bioassay suggested that cytochrome P450 enzymes were involved in the resistance to neonicotinoids. The mRNA transcriptional level of CYP6CY3 gene was significantly higher (3.74 fold) in FP compared to SC. The R81T mutation associated with neonicotinoid resistance had 26% resistant allele frequency in FP. Both P450 enzymes and R81T mutation of nAChR were found in field-evolved neonicotinoid resistance. It is concluded that field-evolved resistance in green peach aphid could be managed by using appropriate synergists such as PBO.

DNA barcoding of the fire ant genus Solenopsis Westwood (Hymenoptera: Formicidae) from the Riyadh region, the Kingdom of Saudi Arabia.

The ant genus Solenopsis Westwood, 1840 is the largest in Myrmicinae subfamily having almost 200 described species worldwide. They are commonly distributed in the tropics and temperate areas of the world. Some invasive Solenopsis species are very dreadful. We have already reported a fire ant species, Solenopsis saudiensis Sharaf & Aldawood, 2011, identified using traditional morphometric approaches of species identification. Present study was carried out to develop DNA Barcoding to identify Solenopsis saudiensis and to elucidate genetic structure of the various S. saudiensis populations across their distribution range in Riyadh, Saudi Arabia. The comparison of DNA barcodes showed no genetic diversity among six populations and a queen from S. saudiensis analyzed from the Riyadh region. This genetic resemblance probably reflects their adaptation toward a specific habitat, thus constituting a single and strong gene pool. Our comprehensive field survey did not provide any evidence of Solenopsis species except S. saudiensis in the Riyadh region. Solenopsis saudiensis populations were only found around date palm trees indicating their strong association with date palm groves. Moreover, S. saudiensis has 83-86% sequence identity to other Solenopsis spp. from other parts of the world. Interestingly, the highest sequence identity of (86%) was with that of Solenopsis molesta Say, 1836, the thief ant, from the USA. This study provides a working laboratory procedure and a reference library for the identification of Solenopsis saudiensis.

Transcriptome analysis of the almond moth, Cadra cautella, female abdominal tissues and identification of reproduction control genes.

BACKGROUND: The almond moth, Cadra cautella is a destructive pest of stored food commodities including dates that causes severe economic losses for the farming community worldwide. To date, no genetic information related to the molecular mechanism/strategies of its reproduction is available. Thus, transcriptome analysis of C. cautella female abdominal tissues was performed via next-generation sequencing (NGS) to recognize the genes responsible for reproduction. RESULTS: The NGS was performed with an Illumina Hiseq 2000 sequencer (Beijing Genomics Institute: BGI). From the transcriptome data, 9,804,804,120 nucleotides were generated and their assemblage resulted in 62,687 unigenes. The functional annotation analyses done by different databases, annotated, 27,836 unigenes in total. The transcriptome data of C. cautella female abdominal tissue was submitted to the National Center for Biotechnology Information (accession no: PRJNA484692). The transcriptome analysis yielded several genes responsible for C. cautella reproduction including six Vg gene transcripts. Among the six Vg gene transcripts, only one was highly expressed with 3234.95 FPKM value (fragments per kilobase per million mapped reads) that was much higher than that of the other five transcripts. Higher differences in the expression level of the six Vg transcripts were confirmed by running the RT-PCR using gene specific primers, where the expression was observed only in one transcript it was named as the CcVg. CONCLUSIONS: This is the first study to explore C. cautella reproduction control genes and it might be supportive to explore the reproduction mechanism in this pest at the molecular level. The NGS based transcriptome pool is valuable to study the functional genomics and will support to design biotech-based management strategies for C. cautella.

Biological Traits of Cadra cautella (Lepidoptera: Pyralidae) Reared on Khodari Date Fruits Under Different Temperature Regimes.

The almond moth, Cadra cautella (Walker) (Lepidoptera: Pyralidae), is a serious worldwide pest of dates and other crops, both in the field and in storage. Infestation by this pest significantly limits the sale and export of dates. Modified atmospheres, the temperature manipulations, are the best eco-friendly alternatives in stored product protection. We examined temperature regulation as an ecologically friendly method of pest management in stored food commodities, in particular, its effects on characteristics of C. cautella biology, including development time, oviposition period, fecundity, fertility, and longevity of adults reared on date cultivar 'Khodari,' at temperatures of 15, 25, and 35 ± 1 °C, with 65 ± 5% relative humidity and a photoperiod of 15:9 (L:D) h. Six larval instars were recorded at each temperature. Larval development was sluggish at 15 °C; therefore, daily observations were terminated after 180 d of the larval span for this temperature. Total larval spans of 48.95 ± 0.76 and 32.12 ± 1.08 d were recorded at 25 °C and 35 °C, respectively. A mean ovipositional period of 5.80 ± 0.44 d yielding 213.60 ± 13.41 eggs per female, with hatchability of 85.94 ± 2.97%, was recorded at 25 °C, whereas at 35 °C, a mean ovipositional period of 1.60 ± 0.26 d yielded 19.80 ± 5.11 eggs per female, with no hatchability. These differences were highly significant. Our observations of poor development at 15 °C, lower numbers of eggs and absence of hatchability at 35 °C, together with head capsule measurements and growth ratios, can be of benefit when planning management strategies to reduce C. cautella infestation.

Effectiveness of carbon dioxide against different developmental stages of Cadra cautella and Tribolium castaneum.

Methyl bromide is an excellent fumigant but has been banned because it has high potential for depleting the ozone layer which leads to many environmental and human health hazard issues. In this connection, effectiveness of carbon dioxide (CO2, 99.9%) was studied as an alternative to methyl bromide under various exposure timings, 25 ± 1 °C, against different developmental stages of the almond moth, Cadra cautella, and red flour beetle, Tribolium castaneum. In case of C. cautella, the LT99 against adult, pupa, and larval stages was achieved after 37.5, 78.1, and 99.9 h of CO2 application, respectively. While for T. castaneum, the LT99 values were obtained after exposure timings of 29.3, 153.9, and 78.4 h against adult, pupa, and larval stages, respectively. Adults were very susceptible; in contrast, pupae and larvae were more tolerant. The susceptibility order was observed as follows: T. castaneum adult > C. cautella adult > C. cautella pupae > T. castaneum larvae > C. cautella larvae > T. castaneum pupae. This study could be useful in developing the management strategies to prevent stored dates from C. cautella and T. castaneum infestation.

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry.

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.