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.

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.