The complete mitochondrial genome of invasive insect Leptinotarsa decemlineata Say 1824 (Coleoptera: Chrysomelidae).

Leptinotarsa decemlineata Say 1824, an invasive and globally devastating beetle, inflicts great damage to potato crops worldwide. The complete mitogenome of L. decemlineata is described in this study. It is a 16,741 bp long circular DNA molecule with a high A + T content of 76.9%, containing a typical 37 gene pattern. All PCGs (protein-coding genes) initiate with typical ATN codons. Most PCGs use TAN as a stop codon, whereas ND4 and COX3 use the incomplete codon TA as the stop codon. The lengths of rrnL and rrnS genes are 1,337 bp and 811 bp, respectively. All 22 tRNAs ranged from 62 to 77 bp. Phylogenetic analysis of Chrysomelidae indicated that L. decemlineata clusteres with three other Chrysomelinae species, which is consistent with previous analyses.

Comparative analysis of eight DNA extraction methods for molecular research in mealybugs.

For molecular research, the quality and integrity of DNA obtained will affect the reliability of subsequent results. Extracting quality DNA from scale insects, including mealybugs, can be difficult due to their small body size and waxy coating. In this study, we evaluate eight commonly used DNA extraction methods to determine their efficacy in PCR analysis across life stages and preservation times. We find that fresh samples, immediately upon collection or after 2 wks, resulted in the most effective DNA extraction. Methods using the DNeasy Blood & Tissue kit, NaCl, SDS-RNase A, and SDS isolated DNA of sufficient quality DNA. The SDS method gave high DNA yield, while the NaCl and SDS-RNase A methods gave lower yield. NaCl, SDS-RNase A, SDS, chloroform-isopentyl alcohol, and the salting-out methods all resulted in sufficient DNA for PCR, and performed equal to or better than that of the DNeasy Blood & Tissue kit. When time and cost per extraction were considered, the SDS method was most efficient, especially for later life stages of mealybug, regardless of preservation duration. DNA extracted from a single fresh sample of a female adult mealybug was adequate for more than 10,000 PCR reactions. For earlier stages, including the egg and 1st instar nymph samples, DNA was most effectively extracted by the Rapid method. Our results provide guidelines for the choice of effective DNA extraction method for mealybug or other small insects across different life stages and preservation status.

Thermal Discrimination and Transgenerational Temperature Response in Bemisia tabaci Mediterranean (Hemiptera: Aleyrodidae): Putative Involvement of the Thermo-Sensitive Receptor BtTRPA.

Anthropogenic climate change and global warming are expected to alter the geographic distribution and abundance of many ectothermic species, which will increase the invasion of new areas by exotic species. To survive in variable or fluctuating temperature conditions, insects require sensitive thermal sensory mechanisms to detect external thermal stimuli and induce the appropriate behavioral and physiological responses. TRPA, a thermal-activated transient receptor potential (TRP) family ion channel, is essential for thermotaxis in insects. Here, we investigated the potential role of BtTRPA in short-term and long-term thermal stress in Bemisia tabaci Mediterranean (Gennadius; Hemiptera: Aleyrodidae). We found that BtTRPA was mainly expressed in the head, where the antennae are located. Under short-term thermal stress, the BtTRPA gene was robustly expressed after exposure to acute low or high temperatures, BtTRPA expression reached the highest levels after exposure to 0°C for 3 h and 40°C for 5 h, but was relatively low after exposure to milder stimuli (12 and 35°C). These results demonstrated that BtTRPA could discriminate between innocuous and noxious temperature stimuli. Under long-term thermal stress, the highest expression level of BtTRPA occurred at G1 exposed to mild innocuous temperature of 21 and 31°C, along with BtTRPA sharply increased and peaked in adult females, implying that mild innocuous long-term thermal exposure could cause transgenerational expression effects to enhance the ability of offspring to cope with the same stress. This study demonstrates that the channel BtTRPA is important in temperature sensing and provides a molecular basis for thermosensation regulation in response to varied environmental temperature in B. tabaci Mediterranean.

The homology gene BtDnmt1 is Essential for Temperature Tolerance in Invasive Bemisia tabaci Mediterranean Cryptic Species.

The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading most parts of the world owing to its strong ecological adaptability, particularly its strong resistance to temperature stress. Epigenetic mechanisms play important roles in mediating ecological plasticity. In particular, DNA methylation has been the focus of attempts to understand the mechanism of phenotypic plasticity. The relationship between temperature and DNA methylation and how it affects the adaptability of invasive insects remain unknown. To investigate the temperature resistance role of DNA methyltransferase 1 (Dnmt1) in MED, we cloned and sequenced BtDnmt1 homology and identified its functions under various temperature conditions. The full-length cDNA of MED BtDnmt1 homology was 5,958 bp and has a 4,287 bp open reading frame that encodes a 1,428-amino-acid protein. BtDnmt1 mRNA expression levels were significantly down-regulated after feeding with dsRNA. Furthermore, after feeding with dsBtDnmt1, the MED adults exhibited significantly higher mortality under temperature stress conditions than the controls, suggesting that MED BtDnmt1 homology plays an essential role in the temperature tolerance capacity of MED. Our data improve our understanding of the temperature resistance and temperature adaptability mechanisms that have allowed the successful invasion and colonization of various environments by this alien species.

Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED.

The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (ß-act, GAPDH, ß-tub, EF1-α, GST, 18S, RPL13A and α-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that ß-tub and EF1-α were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. ß-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects.