Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid(Aphis citricidus)to facilitate its transmission

Sap-sucking insects often transmit plant viruses but also carry insect viruses,which infect insects but not plants.The impact of such insect viruses on insect host biology and ecology is largely unknown.Here,we identified a novel insect-specific virus carried by brown citrus aphid(Aphis citricidus),which we tentatively named Aphis citricidus picornavirus(AcPV).Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses,suggesting that these viruses represent a new family in order Picornavirales.Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference,resulting in asymptomatic tolerance.Importantly,we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants.AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration,thus promoting its transmission among aphids with plants as an intermediate site.The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling.Together,our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors,thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

An electropenetrography waveform library for the probing and ingestion behaviors of Culex tarsalis on human hands

Culex tarsalis Coquillett(Diptera:Culicidae)mosquitoes are capable of vectoring numerous pathogens affecting public and animal health.Unfortunately,the probing behaviors of mosquitoes are poorly understood because they occur in opaque tissues.Electropenetrography(EPG)has the potential to elucidate these behaviors by recording the electrical signals generated during probing.We used an AC–DC EPG with variable input resistors(Ri levels)to construct a waveform library for Cx.tarsalis feeding on human hands.Biological events associated with mosquito probing were used to characterize waveforms at four Ri levels and with two electrical current types.The optimal settings for EPG recordings of Cx.tarsalis probing on human hands was an Ri level of 10^(7)Ohms using an applied signal of 150 millivolts alternating current.Waveforms for Cx.tarsalis included those previously observed and associated with probing behaviors in Aedes aegypti L.(Diptera:Culicidae):waveform families J(surface salivation),K(stylet penetration through the skin),L(types 1 and 2,search for a blood vessel/ingestion site),M(types 1 and 2,ingestion),N(type 1,an unknown behavior which may be a resting and digestion phase),and W(withdrawal).However,we also observed variations in the waveforms not described in Ae.aegypti,which we named types L3,M3,M4,and N2.This investigation enhances our understanding of mosquito probing behaviors.It also provides a new tool for the automated calculation of peak frequency.This work will facilitate future pathogen acquisition and transmission studies and help identify new pest and disease management targets.

EPG Comparison of Sitobion avenae (Fab.) Feeding Behavior on Three Wheat Varieties

This article was to study the potential resistance mechanism of three different wheat varieties （Ww2730, Xiaoyan 22 and Batis） in the seedling stage to Sitobion avenae. The aphid feeding behavior was ascertained by stylet penetration activities monitoring using the electrical penetration graph （EPG） technique. When the aphids feed on Ww2730 seedlings, the time for the 1st duration probing was later than that on Xiaoyan 22 and Batis significantly, and the number of interrupted probing before the 1st duration probing was more than that on Xiaoyan 22 and Batis significantly, and the 1st duration probing was shorter than that on Xiaoyan 22 and Batis significantly. The durations of the stylet pierce from the extra- to the intra-cellular （pd Ⅱ-1） on Ww2730 and Xiaoyan 22 were longer than those on Batis significantly. The duration of the potential drop （pd） in C wave on Ww2730 was longer than that on Xiaoyan 22 and Batis significantly. The times and duration of the G wave （ingestion in xylem） on Ww2730 were more and longer than those on Xiaoyan 22 and Batis significantly. The times of spot G wave on Batis was more than that on Ww2730 significantly, but the duration of spot G was shorter than that on Xiaoyan 22 and Ww2730 significantly. The total time of E1 wave, the longest duration of E1 fractions, and the mean duration time of E1 fractions that followed E2 wave on Xiaoyan 22 and Batis were all significantly longer than those on Ww2730. There was no difference of the mean duration of the 1st E1 wave on the 3 varieties, but the mean durations of other E1 wave on Ww2730 and Xiaoyan 22 were shorter than those on Batis significantly. The other wave parameters, including times and durations of F and E2 were all not different on the 3 wheat varieties. It is suggested that the resistance mechanism of wheat variety Ww2730 to S. avenae is a restriction factor of feeding in epidermis, the thicker cell wall in mesophyll, and secondary metabolites or nutrition unbalance in phloem in the seedling phase. The resistance mechanism of Xiaoyan 22 is thicker cell wall and more cell layer in mesophyll in the seedling phase. The feeding strategy of S. avenae against the resistance of Ww2730 was to shorten the second duration of secrete watery salivation to xylem than the first.

Bacterial plant pathogens affect the locomotor behavior of the insect vector:a case study of Citrus volkameriana-Triozae erytreae-Candidatus Liberibacter asiaticus system

Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction.The African psyllid,Trioza erytreae,is one of the vectors of Huanglongbing,a citrus disease caused mainly by"Candidatus Liberibacter asiaticus"(CLas).The purpose of this study was to characterize the effects of CLas on the psyllid,T.erytreae using Citrus volkamerina plants as the study system.The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T.erytreae.Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition;few differences were observed between uninfected T.erytrea feeding on CLas-infected versus control plants.On the other hand,compared to psyllids that had completed their development on control plants,the ones that had completed their development on a CLas-infected plant exhibited changes in their behavior(greater velocity),physiology(smaller mass)and biochemistry(lower water and lipid content).Altogether,our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior,which can be partially explained by physiological and biochemical changes.

Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid,Diaphorina citri

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid,Diaphorina citri Kuwayama,which serves as vector of phytopathogens causing citrus greening.However,development of resistance to neonicotinoids among populations of D.citri has coincided with occasional control failures in the field.The objectives of this research were to(1)survey current levels of imidacloprid resistance in Florida citrus;(2)compare feeding behavior between imidacloprid-resistant and susceptible D.citri using electrical penetration graph recordings,and(3)investigate the possible amplification of insecticide hormoligosis associated with resistance.Field surveys confirmed that the susceptibility of D.citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population.Following 12 generations of selection,resistance to imidacloprid increased by 438 fold compared with the susceptible strain.Imidacloprid-susceptible D.citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway(C),phloem penetration(D),phloem salivation(E1),and nonprobing(Np)activities than imidacloprid-resistant counterparts.However,there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D.citri.There was no statistical difference in fecundity between resistant and susceptible strains.However,the fecundity of imidacloprid-susceptible female D.citri treated with a sublethal concentration of imidacloprid(LC_(25))increased significantly compared with controls,while such hormoligosis was less pronounced among imidacloprid-resistant psyllids.Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide,indicative of a behavioral resistance mechanism.

Impacts of transgenic Bt cotton on the stylet penetration behaviors of Bemisia tabaci biotype B Evidence from laboratory experiments

Stylet penetration behaviors of Bemisia tabaci biotype B on two transgenic cotton lines ＂GK12＂ and ＂GK19＂ expressing Bt toxic protein CrylA （Bt cotton） and a non-Bt conventional cotton line ＂Simian-3＂ （CK cotton） were recorded with the direct current electrical penetration graph （DC-EPG） technique. Our results suggested that EPG waveform patterns, types and characteristics [non-probe （NP）, pathway （C）, potential drops （pd） and phloem phase （E（pd））] ofBemisia tabaci biotype B were very similar on the three cotton lines. There were no obvious differences of pathway variables among whiteflies on the three cotton lines. Some phloem variables related to E（pd）l differed. Duration of 1st E（pd）l and mean duration of E（pd）l on both GK12 and GK19 were significantly shorter than that on CK cotton （P 〈 0.05）. Fewer whiteflies on GK have long E（pd）l. Other phloem variables including total duration of E（pd） summed, mean E（pd） duration and percentage of whiteflies reaching the phloem phase were similar among the three cotton lines.

Stylet penetration behaviors of the cotton aphid Aphis gossypfi on transgenic Bt cotton

Stylet penetration behaviors of cotton aphids Aphis gossypii Glover on a transgenic cotton line ＂GK-12＂ expressing Bt toxic protein of Cry 1A （Bt cotton） and a non- Bt conventional cotton line ＂Simian-3＂ （CK cotton） were recorded with the direct current electrical penetration graph （DC-EPG） technique. Cotton aphids reared on Bt cotton （abbreviated as Bt-aphids） and its parental non-Bt control line （CK-aphids） for more than 20 generations each, were used for recordings on two cotton lines. Among 47 selected parameters reflecting the activities of aphid stylets within plant tissues, there were eight parameters of CK-aphids showing significant differences between the performances of CK- aphids on Bt cotton and CK cotton, while for Bt-aphids, all the parameters were statistically equal between the performances on the two cotton lines. All parameters with significant differences indicated that CK-aphids could penetrate into Bt cotton more easily, but the phloem saps of Bt cotton were not as good as those of regular cotton for CK-aphids. Based on the present results, we concluded that there were some factors in Bt cotton affecting penetration behaviors of CK-aphids, but it just took several generations for CK-aphids to completely adapt Bt cotton, and Bt-aphids could feed on two cotton lines without difficulty.

Lethal, sublethal and transgenerational effects of the novel chiral neonicotinoid pesticide cycloxaprid on demographic and behavioral traits of Aphis gossypii （Hemiptera： Aphididae）

Aphis gossypii Glover （Hemiptera： Aphididae） is a key pest in cotton crops, notably owing to its increasing resistance to commonly used pesticides. Such resistance prompts for the development of integrated pest management （IPM） programs that include novel pesticides being effective against the aphid. In the present study, we assessed lethal and sublethal effects of cycloxaprid, a novel chiral neonicotinoid pesticide developed in China, on A. gossypii. The lethal concentration at 50% （LCs0） value of cycloxaprid on A. gossypii was estimated, using the dipping method, at 7.73 mg/L. The impact of a sublethal concentration （LC10） and a lethal concentration （LC40） of cycloxaprid on A. gossypii population growth and feeding behavior （using electrical penetration graph technique [EPG]）, and its transgenerational effect were further assessed. Adult longevity and fecundity significantly decreased after exposure to LEa0 or LC10 of cycloxaprid. Cycloxaprid with sublethal concentrations （especially LC40） had negative effects on phloem ingestion by A. gossypii. Additionally, the offspring of the adults exposed to LC40 of cycloxaprid had shorter nymphal development duration and adult longevity than the control, and those from LC10 and LC40 treatments had lower adult fecundity and net productive rate. We demonstrated that cycloxaprid is a pesticide showing both lethal and sublethal activities, and transgenerational effects on,4. gossypii; it may be useful for implementation in IPM programs against this aphid pest.

Comparative analysis of Solanum stoloniferum responses to probing by the green peach aphid Myzus persicae and the potato aphid Macrosiphum euphorbiae

Plants protect themselves against aphid attacks by species-specific de- fense mechanisms. Previously, we have shown that Solanum stoloniferum Schlechtd has resistance factors to Myzus persicae Sulzer （Homoptera： Aphididae） at the epidermal/mesophyll level that are not effective against Macrosiphum euphorbiae Thomas （Homoptera： Aphididae）. Here, we compare the nymphal mortality, the pre-reproductive development time, and the probing behavior of M. persicae and M. euphorbiae on S. stoloniferum and Solanum tuberosum L. Furthermore, we analyze the changes in gene expression in S. stoloniferum 96 hours post infestation by either aphid species. Although the M. euphorbiae probing behavior shows that aphids encounter more probing constrains on phloem activities-longer probing and salivation time- on S. stoloniferum than on S. tuberosum, the aphids succeeded in reaching a sustained ingestion of phloem sap on both plants. Probing by M. persicae on S. stoloniferum plants resulted in limited feeding only. Survival of M. euphorbiae and M. persicae was affected on young leaves, but not on senescent leaves of S. stoloniferum. Infestation by M. euphorbiae changed the expression of more genes than M. persicae did. At the systemic level both aphids elicited a weak response. Infestation orS. stoloniferum plants with a large number ofM. persicae induced morphological changes in the leaves, leading to the development of pustules that were caused by disrupted vascular parenchyma and surrounding tissue. In contrast, an infesta- tion by M. euphorbiae had no morphological effects. Both plant species can be regarded as good host for M. euphorbiae, whereas only S. tuberosum is a good host for M. persicae and S. stoloniferum is not. Infestation ofS. stoloniferum by M. persicae or M. euphorbiae changed the expression of a set of plant genes specific for each of the aphids as well as a set of common genes.

Feeding behavior explains the different effects of cabbage on MEAM1 and MED cryptic species of Bemisia tabaci

MEAMI(Middle East-Asia Minor 1."B"biotype)anpl MED(Mediterranean,"Q"biotype)are the two most destructive ery pie species of the Bemisia tabaci complex on the planet.Our previous studies have shown that MEAMI outcompets MED on cabbage;the underlying mechanism is unknown.In the Brassicaceae family,the glucosinolate-myrosinase defense system plays a crucial role im deterring feeding,inhibiting growth,and eausing acute toxicity against a wide range of generalist herbivores.In the present study.we first compared the surv ival of MEAMI and MED exposed to sinigin(a glucosinolate)and myosinase(an enzyme that degrades glucosinolates);we found that survival of both species was high in response 10 sinigrin alone bul was near zer0 in response to sinigrin+myrosinase.We then used electropenetrography(lectrical penetration graphs,EPG)to assess the feeding bchaviors of MEAMI and MED whiteflies on cabbage.The EPG results revealed that the me an dunution of each potential drop(pd,indicating an intreellular puncture)was subsantialy longer for MED than MEAMI on eabbage,indicating that the exposure to the toxic hydrolysates of glucosinolate and myrosinase is greater for MED than for MEAMI.We therefore conclude that dffreces in penetrating bchaviors may help explain the dfferent fiects of cabbuge on MEAMI and MED whitefly species.

High levels of arbuscular mycorrhizal fungus colonization on Medicago truncatula reduces plant suitability as a host for pea aphids (Acyrthosiphon pisum)

This study sheds light on a poorly understood area in insect-plant-microbe interactions,focusing on aphid probing and feeding behavior on plants with varying levels of arbuscular mycorrhizal(AM)fungus root colonization.It investigates a commonly occurring interaction of three species:pea aphid Acyrthosiphon pisum,barrel medic Medicago truncatula,and the AM fungus Rhizophagus irregularis,examining whether aphid-feeding behavior changes when insects feed on plants at different levels of AM fungus colonization(42% and 84% root length colonized).Aphid probing and feeding behavior was monitored throughout 8 h of recording using the electrical penetration graph(EPG)technique,also,foliar nutrient content and plant growth were measured.Summarizing,aphids took longer to reach their 1st sustained phloem ingestion on the 84% AM plants than on the 42% AM plants or on controls.Less aphids showed phloem ingestion on the 84% AM plants relative to the 42% AM plants.Shoots of the 84% AM plants had higher percent carbon(43.7%)relative to controls(40.5%),and the 84% AM plants had reduced percent nitrogen(5.3%)relative to the 42% AM plants(6%).In conclusion,EPG and foliar nutrient data support the hypothesis that modifications in plant anatomy(e.g.,thicker leaves),and poor food quality(reduced nitrogen)in the 84% AM plants contribute to reduced aphid success in locating phloem and ultimately to differences in phloem sap ingestion.This work suggests that M.truncatula plants benefit from AM symbiosis not only because of increased nutrient uptake but also because of reduced susceptibility to aphids.

Diurnal feeding as a potential mechanism of osmoregulation in aphids

Diurnal variation in phloem sap composition has a strong infuence on aphid performance.The sugar-rich phloem sap serves as the sole diet for aphids and a suite of physiological mechanisms and behaviors allowv them to tolerate the high osmotic stress.Here,we tested the hypothesis that night-time feeding by aphids is a behavior that takes advantage of the low sugar diet in the night to compensate for osmotic stress incurred while feeding on high sugar diet during the day.Using the electrical penetration graph(EPG)technique.we examined the eiects of diurmal rhythm on feeding behaviors of bird cherry-oat aphid(Rhopalosiphurm padi L.)on wheat.A strong diurmal rhythm in aphids as indicated by the presence of a cyclical pattern of expression in a core clock gene did not impact aphid feeding and similar feeding behaviors were observed during day and night.The major difference observed between day and night feeding was that aphids spent significantly longer time in phloem salivation during the night compared to the day.In contrast,aphid hydration was reduced at the end of the day-time feeding compared to end of the night-time fepding.Gene expression analysis of R.padi osmoregulatory genes indicated that sugar break down and water transport into the aphid gut was reduced at night.These data suggest that while diumal variation occurs in phloem sap composition,aphids use night time feeding to overcome the high osmotic stress incurred while feeding on sugar-rich phloem sap during the day.