Alberto Fereres

Intracellular ingestion and salivation by aphids may cause the acquisition and inoculation of non-persistently transmitted plant viruses

Transmission of non-persistent plant viruses is related to aphid behaviour during superficial brief probes. A widely accepted hypothesis postulates that virus acquisition occurs during ingestion of plant cell contents, and inoculation during egestion or regurgitation of previously ingested sap. Although conceptually attractive, this ingestion-egestion hypothesis has not been clearly demonstrated. Furthermore, it overlooks the anatomy of the tips of the stylets (mouthparts) and, consequently, the potential role of salivation in the inoculation process. Here, we used the electrical penetration graph (EPG) technique to investigate aphid-stylet activities associated with uptake (acquisition) and release (inoculation) of two non-persistently transmitted viruses. Our results show that acquisition occurs primarily during the last sub-phase (II-3) of intracellular stylet punctures, whereas inoculation is achieved during the first sub-phase (II-1). An alternative mechanism to the ingestion-egestion hypothesis is proposed on the basis of our findings.

Behavioural aspects influencing plant virus transmission by homopteran insects

Homopterans including aphids, whiteflies and leafhoppers are the major vectors of viruses comprising more than 80% of insect-transmitted viruses which represents close to 400 virus species within 39 different genera. Host plant recognition by homopterans requires a series of steps that are linked to plant virus transmission, including host searching or pre-alighting behaviour, probing on superficial tissues, settlement and stylet penetration to the target feeding tissues and salivation and continuous sap ingestion from the preferred feeding site. This review considers how vector behaviour influences the transmission and spread of plant viruses depending on the type of virus-vector relationship. Most studies have concentrated on aphid-transmitted viruses and particular probing and feeding behavioural processes and activities leading to the transmission of cuticula-borne and circulative viruses have been identified. The review also focuses on which are the most likely retention sites within the insects body of cuticula-borne viruses. Finally, the influences of virus infection on vector behaviour such as changes in the attractiveness, settlement or feeding preference together with changes on vector performance (development, fecundity, rate of population increase and survival) are discussed.

A protein key to plant virus transmission at the tip of the insect vector stylet

Hundreds of species of plant viruses, many of them economically important, are transmitted by noncirculative vector transmission (acquisition by attachment of virions to vector mouthparts and inoculation by subsequent release), but virus receptors within the vector remain elusive. Here we report evidence for the existence, precise location, and chemical nature of the first receptor for a noncirculative virus, cauliflower mosaic virus, in its insect vector. Electron microscopy revealed virus-like particles in a previously undescribed anatomical zone at the extreme tip of the aphid maxillary stylets. A novel in vitro interaction assay characterized binding of cauliflower mosaic virus protein P2 (which mediates virus–vector interaction) to dissected aphid stylets. A P2-GFP fusion exclusively labeled a tiny cuticular domain located in the bottom-bed of the common food/salivary duct. No binding to stylets of a non-vector species was observed, and a point mutation abolishing P2 transmission activity correlated with impaired stylet binding. The novel receptor appears to be a nonglycosylated protein deeply embedded in the chitin matrix. Insight into such insect receptor molecules will begin to open the major black box of this scientific field and might lead to new strategies to combat viral spread.

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings.

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.

A Plant Virus Manipulates the Behavior of Its Whitefly Vector to Enhance Its Transmission Efficiency and Spread

Plant viruses can produce direct and plant-mediated indirect effects on their insect vectors, modifying their life cycle, fitness and behavior. Viruses may benefit from such changes leading to enhanced transmission efficiency and spread. In our study, female adults of Bemisia tabaci were subjected to an acquisition access period of 72 h in Tomato yellow leaf curl virus (TYLCV)-infected and non-infected tomato plants to obtain viruliferous and non-viruliferous whiteflies, respectively. Insects that were exposed to virus-infected plants were checked by PCR to verify their viruliferous status. Results of the Ethovision video tracking bioassays indicated that TYLCV induced an arrestant behavior of B. tabaci, as viruliferous whitefly adults remained motionless for more time and moved slower than non-viruliferous whiteflies after their first contact with eggplant leaf discs. In fact, Electrical Penetration Graphs showed that TYLCV-viruliferous B. tabaci fed more often from phloem sieve elements and made a larger number of phloem contacts (increased number of E1, E2 and sustained E2 per insect, p<0.05) in eggplants than non-viruliferous whiteflies. Furthermore, the duration of the salivation phase in phloem sieve elements (E1) preceding sustained sap ingestion was longer in viruliferous than in non-viruliferous whiteflies (p<0.05). This particular probing behavior is known to significantly enhance the inoculation efficiency of TYLCV by B. tabaci. Our results show evidence that TYLCV directly manipulates the settling, probing and feeding behavior of its vector B. tabaci in a way that enhances virus transmission efficiency and spread. Furthermore, TYLCV-B. tabaci interactions are mutually beneficial to both the virus and its vector because B. tabaci feeds more efficiently after acquisition of TYLCV. This outcome has clear implications in the epidemiology and management of the TYLCV-B. tabaci complex.

Correlation Between Whitefly (Homoptera: Aleyrodidae) Feeding Behavior and Transmission of Tomato Yellow Leaf Curl Virus

Abstract The feeding behavior of the whitefly Bemisia tabaci (Gennadius) was monitored using the electrical penetration graph (EPG) technique during the transmission process of tomato yellow leaf curl geminivirus (TYLCV). The behavior of individual viruliferous whiteflies was recorded on two-leaf stage tomato test plants (Lycopersicon esculentum Mill ‘Riofuego’). A total of 213 whitefly individuals was recorded on single test plants during an inoculation access period that ranged from 3.5 to 14 h. Recordings were classified into 4 categories depending of the waveforms observed: group I, including only waveform C (pathway), was associated with a residual 2.4% TYLCV transmission efficiency; group II, showing stylet pathway and a single E(pd)1 waveform, was associated with a 7.4% transmission efficiency; group III, showing stylet pathway and a single E(pd)1 + E(pd)2 waveform, achieved a 23.4% transmission efficiency; and finally group IV, showing pathway followed by several E(pd)1 + E(pd)2 waveforms, was associated with the highest transmission efficiency (37.5%). A total of 16 different behavioral variables was introduced into a stepwise-backward logistic regression model to determine the variables most related to TYLCV inoculation. Among them, the total duration of E(pd)1 was the most significant variable associated with virus inoculation by B. tabaci (P = 0.002, positive relationship). In addition, the regression analysis indicated a strong positive relationship (P = 0.005) between transmission efficiency and total number of E(pd)1 (t = 4.39, β = 0.45 ± 0.10, r = 0.87), and total duration of waveform E(pd)1 (P = 0.05) (t = 4.00, β = 0.02 ± 0.004, r = 0.94). The minimum phloem contact E(pd)1 threshold period observed for successful inoculation of TYLCV by B. tabaci on tomato plants was 1.8 min. Implications of these results for persistent virus transmission by whiteflies are discussed.

Estimation of the Effective Number of Founders That Initiate an Infection after Aphid Transmission of a Multipartite Plant Virus

ABSTRACT The fecundity of RNA viruses can be very high. Thus, it is often assumed that viruses have large populations, and RNA virus evolution has been mostly explained using purely deterministic models. However, population bottlenecks during the virus life cycle could result in effective population numbers being much smaller than reported censuses, and random genetic drift could be important in virus evolution. A step at which population bottlenecks may be severe is host-to-host transmission. We report here an estimate of the size of the population that starts a new infection when Cucumber mosaic virus (CMV) is transmitted by the aphid Aphis gossypii, based on the segregation of two CMV genotypes in plants infected by aphids that acquired the virus from plants infected by both genotypes. Results show very small effective numbers of founders, between one and two, both in experiments in which the three-partite genome of CMV was aphid transmitted and in experiments in which a fourth RNA, CMV satellite RNA, was also transmitted. These numbers are very similar to those published for Potato virus Y, which has a monopartite genome and is transmitted by aphids according to a different mechanism than CMV. Thus, the number of genomic segments seems not to be a major determinant of the effective number of founders. Also, our results suggest that the occurrence of severe bottlenecks during horizontal transmission is general for viruses nonpersistently transmitted by aphids, indicating that random genetic drift should be considered when modeling virus evolution.

Cauliflower mosaic virus is preferentially acquired from the phloem by its aphid vectors

Cauliflower mosaic virus (CaMV) is transmitted in a non-circulative manner by aphids following the helper strategy. Helper proteins P2 and P3 act as a bridge between virions and the aphid cuticle. Electronic monitoring of aphid stylet activities (EPG technique), transmission tests and electron microscopy showed that CaMV is preferentially acquired from the phloem by its most common aphid vectors, Brevycorine brassicae and Myzus persicae. We also found that CaMV is semipersistently transmitted and that the rate of acquisition does not follow a typical bimodal curve. Instead, the virus could be acquired from non-phloem tissues at a low and fairly constant rate after one or more intracellular punctures within a few minutes, but the probability of acquisition rose significantly when aphids reached the phase of committed ingestion from the phloem. The acquisition rate of CaMV did not increase with increasing number of intracellular punctures, but the total duration of intracellular puncture was one of the variables selected by the stepwise logistic regression model used to fit the data that best explained acquisition of CaMV. Furthermore, aphids reaching the phloem faster had a higher probability of acquiring the virus. Our results support the hypothesis that multiple intracellular punctures of epidermal and mesophyll cells result in loading aphids with the CaMV-encoded aphid transmission factor (P2), and that aphids, in most cases, subsequently acquire CaMV particles during phloem sap ingestion. Consistently, immunoelectron microscopy showed that P3-virions are frequently found in the sieve element lumen, whereas P2 could not be detected.

Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.

Feeding Behavior of Aphis gossypii on Resistant Accessions of Different Melon Genotypes (Cucumis melo)

The feeding behavior of the melon aphidAphis gossypii Glover (Homoptera: Aphididae) was monitored using the electrical penetration graph (EPG) technique on different melon (Cucumis melo L.) genotypes showing resistance to the aphid. The aphid-resistant genotypes used were PI-161375 and PI-414723, sources of theVat andAgr genes, respectively. TGR-1551, a newC. melo accession from Zimbabwe, was also tested. Our goal was to localize the tissues where the resistance factors are expressed and to determine if the resistance mechanisms operating in the three aphid-resistant accessions were the same. Our results indicated that the three selected lines have resistant factors located at the epidermis, mesophyll and vascular tissues. However, the behavior ofA. gossypii on TGR-1551 was different from the two other resistant accessions, as indicated by a longer phloem salivation phase (E1 phase). Many of the E1 phases observed for aphids feeding on TGR-1551 were not followed by phloem ingestion (E2 phase). These results suggest that TGR-1551 has a resistance mechanism that preventsA. gossypii from initiating ingestion from the phloem. Preference tests under free choice conditions also showed that aphids rejected accessions TGR-1551 or PI-414723 faster than PI-161375. Our results support the hypothesis thatAgr andVat are coding for different kinds of resistance strategies. Comparisons of aphid life history parameters also indicated that TGR-1551 is a very promising new source to breed for resistance againstA. gossypii.