A multi‐year dormancy strategy in a cabbage beetle population in southeastern China

The life cycle of the cabbage beetle Colaphellus bowringi in southeastern China is complex due to four options for adult development: summer diapause, winter diapause, prolonged diapsuse, and nondiapause. However, detailed information on the multi‐year emergence patterns of diapausing individuals in this beetle has not been documented. In this study, we monitored the adult emergence patterns of diapausing individuals and estimated the influence of the diapause‐inducing temperature and photoperiod on the incidence of prolonged diapause under seminatural conditions for several years. The duration of diapause for adults collected from the vegetable fields in different years varied from several months to 5 years. Approximately 25.9%–29.2% of individuals showed prolonged diapause (emergence more than 1 year after entering diapause) over the 5 years of observation. Furthermore, regardless of insect age, the emergence of diapausing adults from the soil always occurred between mid‐February and March in spring and between late August and mid‐October in autumn, when the host plants were available. The influence of diapause‐inducing temperatures (22, 25, and 28°C) combined with different photoperiods (L:D 12:12 h and L:D 14:10 h) on diapause duration was tested under seminatural conditions. Pairwise comparisons of diapause duration performed by the log‐rank test revealed that the low temperature of 22°C combined with the long photoperiod of L:D 14:10 h induced the longest diapause duration, whereas the low temperature of 22°C combined with the short photoperiod of L:D 12:12 h induced the highest proportion of prolonged diapause. This study indicates that C. bowringi adopts a multi‐year dormancy strategy to survive local environmental conditions and unpredictable risks.     

Inheritance of the photoperiodic response controlling imaginal summer diapause in the cabbage beetle, Colaphellus bowringi

Adults of the cabbage beetle Colaphellus bowringi display a summer diapause in response to the exposure of their larvae to long photoperiods. In the present study, the inheritance of the photoperiodic response controlling summer diapause in C. bowringi by crossing a high diapause strain (D strain) with a laboratory selected nondiapause strain (N strain) was investigated under different photoperiods at 22, 25 and 28 °C. The beetles in both reciprocal crosses and backcrosses showed a clear short-day response for the induction of diapause at all temperatures, similar to that of the D strain, suggesting that photoperiodic response of this beetle is heritable. The diapause incidences in the progeny from all the crosses under LD 15:9 or LD 12:12 at 25 °C suggest that genetic and genetic-environmental interactions are involved in diapause induction. The incidence of diapauses in F₁ progeny was significantly lower than that in the D × D strain but significantly higher than that in the N × N strain, indicating that the diapause capability is inherited in an incomplete dominant manner. The incidence of diapause was greater among the offspring of hybrid females when those females had a D strain mother or grandmother rather than a N strain mother or grandmother, indicating that maternal effects on diapause induction are stronger than paternal effects. The laboratory selected nondiapause strain also showed a short-day photoperiodic response at a low temperature of 22 °C, indicating that the photoperiodic photoreceptor and photoperiodic clock still function in the nondiapause strain.     

Functional identification of an FMRFamide-related peptide gene on diapause induction of the migratory locust,Locusta migratoria L

FMRFamide-related peptides (FaRPs) are a type of neuropeptide, which participate in a variety of physiological processes in insects. Previous study showed that myosuppressin, being a member of FaRPs, initiated pupal diapause in Mamestra brassicae. We presumed that FaRPs genes might play a critical role in photoperiodic diapause induction of L. migratoria. To verify our hypothesis, flrf, a precursor gene of FaRP from L. migratoria, was initially cloned under long and short photoperiods that encoded by flrf gene identified from central nervous system (CNS). Phylogenetic analysis showed that the protein encoded by L. migratoria flrf gene, clustered together with Nilaparvata lugens (Hemiptera: Delphacidae) with 100% bootstrap support, was basically an FMRFamide precursor homologue. We noticed the availability of -RFamide peptides (GSERNFLRFa, DRNFIRFa) under short photoperiod only, which suggested their functions related to photoperiodic diapause induction. RNAi and quantitative real-time PCR (qRT-PCR) results further confirmed that the flrf gene promoted locust's diapause.     

大猿叶虫夏滞育的诱导：基于定量的光周期反应

为了探明大猿叶虫Colaphellus bowringi Baly夏滞育诱导的光周期时间测量特性, 我们通过室内实验系统比较了该虫在25℃、 不同长光照条件下,夏滞育的发生以及诱导50%个体进入夏滞育所需求的光 暗循环数。结果表明：不同长光照诱导的夏滞育比率有显著差异, 其中15 h或16 h光照诱导的滞育比率最高, 短于或长于这两个光照其滞育率均明显下降。在不同光 暗循环实验中, 14 h诱导的滞育比率均低于50%, 诱导50%个体进入夏滞育所需求的光 暗循环数在L15∶D9, L16∶D8, L17∶ D7和L18∶D6分别为2.61, 3.72, 4.64和5.92 d, 处理间存在显著差异。这些结果提示该虫夏滞育的诱导是基于定量的光周期反应。     

Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq

qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is necessary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently cannot be accurately analyzed due to a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera Abel seeds and specific PCR Primers were designed for each. Cycle threshold (C _t) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and Best-Keeper were applied to calculate the expression stability of the candidate reference genes according to the C _t values. The results were similar between the three software packages, and indicated that the traditional genes TUBα-3, ACT7α and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from 2 to 6. Finally, the relative expression of squalene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, for the different tissues of six oil-tea camellia species different optimal numbers of suitable reference genes were found.     

The role of photoperiod and temperature in determination of summer and winter diapause in the cabbage beetle,Colaphellus bowringi (Coleoptera: Chrysomelidae)

The cabbage beetle, Colaphellus bowringi, is a short-day species undergoing an imaginal summer and winter diapause. Its photoperiodic response highly depends on temperature. All adults entered diapause at ≤ 20 °C regardless of photoperiods. High temperatures strongly weakened the diapause-inducing effects of long daylengths. The diapause-averting influence of short daylengths was expressed only at high temperatures (above 20 °C). This indicates that the beetle has a cryptic ability to reproduce in summer. In fact, summer and winter diapause were induced principally by relatively low temperatures in the field, whereas photoperiod had less influence on diapause induction. The critical daylength for the autumnal population was between 12 h and 13 h. By transferring from a long day to a short day or vice versa at different times after hatching, it was shown that the sensitive stage with regard to photoperiod was the larva, whereas a long day was photoperiodically more potent than a short day. The sensitive stage to temperature encompassed the larval, pupal and adult stages. This different response pattern serves to ensure that the beetle enters summer and winter diapause in time. The selections for non-diapause trait under laboratory (at 25 °C) and natural conditions (at >24 °C) showed that the beetle could lose its sensitivity to photoperiod very rapidly.     

Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.     

Comparative Validation of Conventional and RNA-Seq Data-Derived Reference Genes for qPCR Expression Studies of Colletotrichum kahawae

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica—C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the model-based method used by NormFinder software. For C. arabica—C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.     

Demographic History,Population Structure,and Local Adaptation in Alpine Populations of Cardamine impatiens and Cardamine resedifolia

Species evolution depends on numerous and distinct forces, including demography and natural selection. For example, local adaptation and population structure affect the evolutionary history of species living along environmental clines. This is particularly relevant in plants, which are often characterized by limited dispersal ability and the need to respond to abiotic and biotic stress factors specific to the local environment. Here we study the demographic history and the possible existence of local adaptation in two related species of Brassicaceae, Cardamine impatiens and Cardamine resedifolia, which occupy separate habitats along the elevation gradient. Previous genome-wide analyses revealed the occurrence of distinct selective pressures in the two species, with genes involved in cold response evolving particularly fast in C. resedifolia. In this study we surveyed patterns of molecular evolution and genetic variability in a set of 19 genes, including neutral and candidate genes involved in cold response, across 10 populations each of C. resedifolia and C. impatiens from the Italian Alps (Trentino). We inferred the population structure and demographic history of the two species, and tested the occurrence of signatures of local adaptation in these genes. The results indicate that, despite a slightly higher population differentiation in C. resedifolia than in C. impatiens, both species are only weakly structured and that populations sampled at high altitude experience less gene flow than low-altitude ones. None of the genes showed signatures of positive selection, suggesting that they do not seem to play relevant roles in the current evolutionary processes of adaptation to alpine environments of these species.     

Identification and evaluation of reference genes for gene expression analysis in the weevil pest Pagiophloeus tsushimanus using RT-qPCR

Pagiophloeus tsushimanus is a newly and specialist wood-boring beetle of Cinnamomum camphora in China. RT-qPCR is an accurate quantitative method to quantify target genes expression, which relies on suitable reference genes for data normalization. Reference genes must to be stably expressed under specific experimental conditions. No suitable reference genes of P. tsushimanus have been reported so far. Therefore, it is necessary to identify and evaluate suitable reference genes for the study of functional genes of this pest. In this research, the expression stability of eight candidate reference genes (RPS3, 18S rRNA, GAPDH, TBP, RPL10, UBQ, GST, and RPS27A) were systematically evaluated in P. tsushimanus by five algorithms (geNorm, BestKeeper, NormFinder, delta C_q, and RefFinder) under different developmental stages, various tissues, and insects reared on different plants, and validated by the olfactory key gene odorant binding protein 33 (PtsuOBP33). The results showed that three stable reference genes combination were necessary for quantitative analysis of target gene. RPS3, RPL10, and UBQ were the optimal reference genes combination for gene expression analysis of developmental stages, while RPL10, RPS3, and 18S rRNA were recommended for different tissues, and 18S rRNA, TBP, and RPS3 were recommended for insects reared on different plants. The results indicated that suitable reference genes should be screened out for gene expression analysis under different conditions. This paper systematically analyzed and obtained suitable reference genes in P. tsushimanus for the first time, which would contribute to the functional analysis of genes and the in-depth mining of genetic resources in it.