Halloween genes AhCYP307A2 and AhCYP314A1 modulate last instar larva–pupa–adult transition, ovarian development and oogenesis in Agasicles hygrophila (Coleoptera:Chrysomelidae)

In insects,ecdysteroids are synthesized by genes of the Halloween family and play important roles in several key developmental events,including molting and metamorphosis.However,the roles of these genes in Agasicles hygrophila are still largely unknown.In this study,the expression patterns of the two Halloween genes AhCYP307A2 and AhCYP314A1 were determined by quantitative PCR(qPCR)at different developmental stages.Moreover,the functions of these two genes were explored using RNA interference(RNAi),and ovarian development was observed by dissecting the ovaries of A.hygrophila females.The qPCR results showed that AhCYP307A2 and AhCYP314A1 were highly expressed in last instar larvae and in adult females.In addition,AhCYP307A2 was also highly expressed in eggs and pupae but was markedly lower than in third-instar larvae and females.The RNAi results showed that the injection of dsAhCYP307A2 or dsAhCYP314A1 markedly inhibited their expression and the transcription levels of three related AhVgs.Knockdown of AhCYP307A2 or AhCYP314A1 significantly inhibited larval molting,impaired last instar larva–pupa–adult transition,delayed ovarian development,and stopped egg production(i.e.,no eggs were laid).These results indicate that AhCYP307A2 and AhCYP314A1 play important regulatory roles in last instar larva–pupa–adult transition and reproduction in A.hygrophila.

Sequential loss of genetic variation in flea beetle Agasicles hygrophila （Coleoptera： Chrysomelidae） following introduction into China

The flea beetle （Agasicles hygrophila） was imported to Florida, USA and then introduced from Florida into China in 1987 as a biological control agent for the invasive plant alligator weed （Alternanthera philoxeroides）. The initial beetle population was subsequently used for sequential introductions in other areas of China, but little is known about the genetic consequences of the introductions. In this study, the genetic diversity and population structure of five beetle populations, the source Florida population, three intentionally introduced China populations and one accidentally dispersed China population, were examined using amplified fragment length polymorphisms. The results showed a clear pattern of decreasing genetic diversity with the sequential introductions. The diversity was highest in the Florida population followed by the first introduction to Chongqing and then in Kunming and Fuzhou. The lowest diversity was found in the accidentally dispersed Guangzhou population that was first recorded in 1996. Both loci parameters and Nei＇s genetic diversity showed a high variation among these populations. Genetic differentiation among populations was further verified by the GST statistic （0.136–0.432）. Beetles in Kunming had the highest gene flow with those in Guangzhou, and therefore lowest differentiation and closest genetic distance. These data show that sequential introduction influenced the genetic diversity of populations in China. Genetic diversity should be considered in planning introduction and long-term maintenance of populations.

Effects of physiological integration on defense strategies against herbivory by the clonal plant Alternanthera philoxeroides

Aims The plant-herbivore interaction is one of the most fundamental inter-actions in nature.Plants are sessile organisms,and consequently rely on particular strategies to avoid or reduce the negative impact of herbivory.Here,we aimed to determine the defense strategies against insect herbivores in the creeping invasive plant Alternanthera philoxeroides.Methods We tested the defense response of A.philoxeroides to herbivory by a leaf-feeding specialist insect Agasicles hygrophila and a pol-yphagous sap-feeding insect Planococcus minor.We also tested the mechanisms triggering defense responses of A.philoxeroides by including treatments of artificial leaf removal and jasmonic acid application.Furthermore,we examined the effect of physiological integration on these defense strategies.Important Findings The combination of artificial leaf removal and jasmonic acid appli-cation produced a similar effect to that of leaf-feeding by the real herbivore.Physiological integration influenced the defense strat-egies of A.philoxeroides against herbivores,and increased biomass allocation to aboveground parts in its apical ramets damaged by real herbivores.Our study highlights the importance of physio-logical integration and modular plasticity for understanding the consequences of herbivory in clonal plants.

Effects of clonal integration and nitrogen supply on responses of a clonal plant to short-term herbivory

Aims Clonal integration,i.e.resource sharing between interconnected ramets,can help clonal plants tolerate abiotic stress.However,few studies have directly examined the ecological significance of clonal integration in the ability of clonal plants to tolerate biotic stress such as herbivory.Methods We grew clonal fragments of an invasive clonal plant Alternanthera philoxeroides,each consisting of an apical part(relatively young)and a basal part(relatively old),at two levels(low and high)of soil nitrogen(N).For each fragment,the apical part was subjected to either no herbivory or herbivory for 2 weeks by a specialist insect,Agasicles hygrophila,and was either connected with or discon-nected from the basal part not subjected to herbivory.Important Findings Herbivory in the apical part severely reduced leaf growth(leaf mass,leaf number or leaf area)and ramet production of both apical and basal parts,and increased root to shoot ratio of the apical part.Irrespective of herbivory and soil N,stem connection between the apical and the basal part decreased root to shoot ratio of the ap-ical part,but did not change its growth measures.Meanwhile,con-nection increased stem N concentration of the apical part growing under high-N supply and decreased stem carbon(C)concentration of the apical part under low-N supply.By contrast,connection in-creased root to shoot ratio of the basal part,but reduced its leaf and ramet production.Connection also increased leaf and stem C concentrations of the basal part under low-N supply.Thus,clonal integration can be beneficial for the early development of young A.philoxeroides ramets,but not for their local adaptation to herbivory damage by A.hygrophila.

Effects of soil nitrogen levels on growth and defense of the native and introduced genotypes of alligator weed

Plants growing in nutrient-rich environment are predicted to be less defended than conspecifics under nutrient limitation.However,less is known about the effects of nutrient levels on tolerance and induced resistance,and whether the effects differ between native and introduced populations of invasive plants.We performed a greenhouse experiment with introduced(the USA)and native(Argentina)genotypes of Alternanthera philoxeroides in order to study the effects of soil nitrogen levels on plant growth,constitutive and herbivore(Agasicles hygrophila)-induced chemical defense,and herbivory tolerance.We measured total biomass,elongation rate(as proxy of growth rate),carbon and nitrogen,and the concentration of triterpenoid saponins(defensive chemicals)in leaves and roots.Constitutive resistance(+33%higher leaf triterpenoid saponins in control treatment at low nitrogen level)and tolerance[less decreased total biomass after herbivory treatment(−24%and−15%for high and low nitrogen levels)]were favored at lower nitrogen level,while induced resistance was favored at higher nitrogen level(+24%increased leaf triterpenoid saponins after herbivory treatment at high nitrogen level).Constitutive resistance and tolerance exhibited trade-offs with growth rate,while induced resistance positively correlated with growth rate.Additionally,the introduced genotypes had−6%lower content of leaf carbon in the presence of herbivores than the native genotypes at low nitrogen level,but such difference was absent at high nitrogen level.Our results indicate that soil nitrogen levels influence the preference of different defensive strategies of plant,and interweave with herbivory to determine the performance of introduced genotypes.