Infection density pattern of Cardinium affects the responses of bacterial communities in an invasive whitefly under heat conditions

Communities of bacteria,especially symbionts,are vital for the growth and development of insects and other arthropods,including Bemisia tabaci Mediterranean(MED),a destructive and invasive insect pest.However,the infection density patterns and influence factors of bacteria in whiteflies,which mainly include symbionts,remain largely unclear.To reveal the different density patterns of Cardinium in B.tabaci MED popula-tions and the impacts of high temperatures on whiteflies with different Cardinium density infection patterns,2 isofemale lines isolated from B.tabaci MED from the same geo-graphical population of China and from B.tabaci MED collected from other countries and locations were examined using several techniques and methods,including fluorescence in situ hybridization(FISH),quantitative real-time polymerase chain reaction(qPCR),16S rRNA gene sequencing,and 2b-RAD sequencing.The results showed that there were 2 different infection density patterns of Cardinium in B.tabaci MED(including 1 high-density pattern and 1 low-density pattern).For whiteflies with low-density Cardinium,conventional PCR could not detect Cardinium,but the other techniques confirmed that there was a low level of Cardinium within hosts.High temperature significantly decreased the diversity of bacterial communities:the relative titer of Cardinium increased but the density of Rickettsia decreased in the isofemale line with high-density Cardinium.How-ever,high temperature did not influence the diversity and symbiont density in the line with low-density Cardinium.Moreover,high temperature influenced the functions of bacterial communities in whiteflies with high-density Cardinium but did not affect the bacterial functions in whiteflies with low-density Cardinium.Our results provide novel insights into the complex associations between symbionts and host insects.

Expression profile of CYP402C1 and its role in resistance to imidacloprid in the whitefly, Bemisia tabaci

Bemisia tabaci(Gennadius)(Hemiptera:Aleyrodidae)is a cosmopolitan insect pest causing serious damage to crop production.Cytochromes P450(CYPs)of B.tabaci are widely known to be involved in the metabolic resistance to a variety of insecticides,continuously increasing the difficulty in controlling this pest.In this study,four P450 genes(CYP6CMI,CYP6CXl,CYP6CX3,and CYP402CI)in B.tabaci exhibited correlations with the resistance to imidacloprid.We have focused on trying to understand the function and metabolism capacity of CYP402C1.The expression profiles of CYP402C1 were examined by reverse transcription quantitative real-time PCR and fluorescence in situ hybridizations.Its role in resistance to imidacloprid was investigated by RNA interference,transgenic Drosophila melanogaster,and heterologous expression.The results showed that CYP402CI was highly expressed in the active feeding stages of B.tabaci,such as nymphs and female adults.CYP402C1 was mainly expressed in midguts of nymphs and adults,especially in the filter chamber.Knockdown of CYP402CI significantly decreased the resistance of B.tabaci to imidacloprid by 3.96-fold(50%lethal concentration:186.46 versus 47.08 mg/L).Overexpression of CYP402CI in a transgenic D.melanogaster line(Gal4>UAS-CYP402CI)significantly increased the resistance to imidacloprid from 12.68-to 14.92-fold(129.01 and 151.80 mg/L versus 1925.14 mg/L).The heterologous expression of CYP402CI showed a metabolism ability of imidacloprid(imidacloprid decreased by 12.51%within 2 h).This study provides new insights for CYP402C1 function in B.tabaci and will help develop new strategies in B.tabaci control and its insecticide resistance.

Heterogeneous distribution of Cardinium in whitefly populations is associated with host nuclear genetic background

Inherited bacterial symbionts are very common in arthropods, but infection frequency can vary widely among populations. Experiments and interpopulation comparisons suggest that host genetic background might be important in explaining this variation. Our extensive field investigation showed that the infection pattern of the facultative symbiont Cardinium was heterogeneous across geographical populations of the invasive whitefly Bemisia tabaci Mediterranean (MED) in China, with genetic nuclear differences evident in 2 of the populations: 1 with a low infection rate (SD line) and 1 with a high infection rate (HaN line). However, whether the heterogeneous frequency of Cardinium is associated with the host genetic background remains poorly understood. Here, we compared the fitness of the Cardinium-infected and uninfected sublines with similar nuclear genetic backgrounds from SD and HaN lines, respectively, and further determine whether host extranuclear or nuclear genotype influenced the Cardinium–host phenotype by performing 2 new introgression series of 6 generations between SD and HaN lines (i.e., Cardinium-infected females of SD were backcrossed with uninfected males of HaN, and vice versa). The results showed that Cardinium provides marginal fitness benefits in the SD line, whereas Cardinium provides strong fitness benefits in the HaN line. Further, both Cardinium and the Cardinium–host nuclear interaction influence the fecundity and pre-adult survival rate of B. tabaci, whereas the extranuclear genotype does not. In conclusion, our results provide evidence that Cardinium-mediated fitness effects were closely associated with the host genetic background, which provides a fundamental basis for understanding the mechanism underlying the heterogeneous distribution of Cardinium in B. tabaci MED populations across China.

Cardinium infection alters cotton defense and detoxification metabolism of its whitefly host

Field monitoring revealed that the infection ratio of the bacterial symbiont Cardinium in the whitefly(Bemisia tabaci MED)was relatively low in northern China.However,the role of this symbiont and the symbiont-whitefly-host plant interaction mechanism are poorly understood.We investigated the influence of Cardinium on the competitiveness of the host whitefly and the physiological interaction between the host plants and host whiteflies.Cardinium-infected whiteflies were displaced by uninfected whiteflies after 5 generations,which showed that Cardinium infection reduced whitefly competitiveness.The defense response genes of cotton significantly decreased under infestation by infected whiteflies compared to uninfected whiteflies.The expression of detoxification metabolism genes,especially the uridine 5ʹ-diphospho-glucuronyltransferase and P450 genes,in infected whiteflies significantly decreased.These results demonstrated that Cardinium could inhibit the defense response of the host plant and decrease the detoxification metabolism ability of the host whitefly.The reduced competitiveness of infected whiteflies may be associated with the inhibition of the whitefly detoxification metabolism by Cardinium,resulting in the reduced performance of infected whiteflies.However,Cardinium infection can suppress plant defenses,which may benefit both infected and uninfected whiteflies when they coexist.This research illustrates the symbiont–whitefly–host plant interaction mechanism and the population dynamics of the whitefly.

Genetic differentiation of Bemisia tabaci （Gennadius） （Hemiptera： Aleyrodidae） biotype Q based on mitochondrial DNA markers

In the present study, genetic differentiation of Bemisia tabaci （Gennadius） biotype Q was analyzed based on mitochondrial cytochrome oxidase I （mt CO1） gene sequence. The results showed that B. tabaci biotype Q could be separated into two subclades, which were labeled as subclades Q1 and Q2. Subclade Q1 was probably indigenous to the regions around the Mediterranean area and subclade Q2 to Israel or Cyprus. It was because B. tabaci was composed of several genetically distinct groups with a strong geographical association between more closely related biotypes. Not all of the B. tabaci biotype Q in the non-Mediterranean countries come from the same regions. Until now, all B. tabaci biotype Q in China were grouped into subclade Q 1. The B. tabaci biotype Q introduced into the US included both subclades Q 1 and Q2. The genetic structure analysis showed higher genetic variation of subclade Q 1 than that of subclade Q2.

Effects of host, temperature and relative humidity on competitive displacement of two invasive Bemisia tabaci biotypes [Q and B]

Bemisia tabaci shifted unexpectedly in China from a predominance of B biotype to Q biotype during 2005-2008. This observation stimulated an interest in inves- tigating whether environmental factors, including host, temperature and relative humidity （RH） could possibly explain the observed shift in biotypes distribution. Results indicated that all three parameters examined influenced biotype survivability. The percentage of B biotype, when reared together on pepper plants with the Q biotype, decreased significantly from 66.7% in the founder population, to 13.6% and 3.7% in the first and second genera- tions, respectively. When the B （founder at 66.7%） and Q （founder at 33.3%） biotypes were reared together on eggplant alone, or on pepper-plus-eggplant combination, the population size of the B biotype either remained constant, or increased somewhat in the first and sec- ond generations. On eggplant, the effects of RH and temperature on the competitiveness between the Q and B biotypes （3 pairs of Q and 6 pairs of B） were not significant.

Analysis of expressed sequence tags （ESTs） from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags （ESTs） were analyzed from a normalized complementary DNA library of/？. solenopsis. In addition, EST-derived microsatellite loci （also known as simple sequence repeats or SSRs） were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.

Up-regulation of calmodulin involved in the stress response to cyantraniliprole in the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)

Cyantraniliprole is the first diamide insecticide to have cross-spectrum activity against a broad range of insect orders.The insecticide,like other diamides,selectively acts on ryanodine receptor,destroys Ca^(2+) homeostasis,and ultimately causes insect death.Although expression regulations of genes associated with calcium signaling pathways are known to be involved in the response to diamides,little is known regarding the function of calmodulin(CaM),a typical Ca^(2+)sensor central in regulating Ca^(2+) homeostasis,in the stress response of insects to the insecticide.In this study,we cloned and identified the full-length complementary DNA of CaM in the whitefly,Bemisia tabaci(Gennadius),named BtCaM.Quantitative real-time reverse transcription polymerase chain reaction-based analyses showed that the messenger RNA level of BtCaM was rapidly induced from l.5l-to 2.43-fold by cyantraniliprole during 24 h.Knockdown of BtCaM by RNA interference increased the toxicity of cyantraniliprole in whiteflies by 42.85%.In contrast,BtCaM expression in Sf9 cells significantly increased the cells,tolerance to cyantraniliprole as much as 2.91-fold.In addition,the expression of BtCaM in Sf9 cells suppressed the rapid increase of intracellular Ca^(2+) after exposure to cyantraniliprole,and the maximum amplitude in the Sf9-BtCaM cells was only 34.9%of that in control cells(Sf9-PIZ/V5).These results demonstrate that overexpression of BtCaM is involved in the stress response of B.tabaci to cyantraniliprole through regulation of Ca^(2+)concentration.As CaM is one of the most evolutionarily conserved Ca^(2+) sensors in insects,outcomes of this study may provide the first details of a universal insect response to diamide insecticides.