Novel program for automatic calculation of EPG variables.

The electrical penetration graph (EPG) technique is the most powerful tool for studying the feeding behavior of pierce-sucking insects. However, calculating EPG variables is often very time-consuming, and consequently, several software programs have been developed for the automatic calculation of EPG variables. Here we present a new user-friendly Excel Workbook that uses a standardized list of EPG variables and follows expert guidelines for calculating them. The program developed in Visual Basic for Applications (VBA) is a step up from the existing software and allows easy data analysis and interpretation. It also includes a novel option for dealing with the common problem of "truncated"-waveforms artificially terminated by the end of recording. The only requirement to run the program is Microsoft Excel software running under a PC environment. The Workbook was validated by calculating variables from EPG recordings of aphids and psyllids and the results obtained were compared with those of existing software such as the Sarria Workbook. Our EPG Workbook provides researchers with a reliable and standardized tool for the automatic calculation of up to 127 EPG variables from phloem-sap-sucking insects.

Faba bean forisomes can function in defence against generalist aphids.

Phloem sieve elements have shut-off mechanisms that prevent loss of nutrient-rich phloem sap when the phloem is damaged. Some phloem proteins such as the proteins that form forisomes in legume sieve elements are one such mechanism and in response to damage, they instantly form occlusions that stop the flow of sap. It has long been hypothesized that one function of phloem proteins is defence against phloem sap-feeding insects such as aphids. This study provides the first experimental evidence that aphid feeding can induce phloem protein occlusion and that the aphid-induced occlusions inhibit phloem sap ingestion. The great majority of phloem penetrations in Vicia faba by the generalist aphids Myzus persicae and Macrosiphum euphorbiae triggered forisome occlusion and the aphids eventually withdrew their stylets without ingesting phloem sap. This contrasts starkly with a previous study on the legume-specialist aphid, Acyrthosiphon pisum, where penetration of faba bean sieve elements did not trigger forisome occlusion and the aphids readily ingested phloem sap. Next, forisome occlusion was demonstrated to be the cause of failed phloem ingestion attempts by M. persicae: when occlusion was inhibited by the calcium channel blocker lanthanum, M. persicae readily ingested faba bean phloem sap.

Infection with a plant virus modifies vector feeding behavior.

Vector infection by some animal-infecting parasites results in altered feeding that enhances transmission. Modification of vector behavior is of broad adaptive significance, as parasite fitness relies on passage to a new host, and vector feeding is nearly always essential for transmission. Although several plant viruses infect their insect vectors, we have shown that vector infection by a plant virus alters feeding behavior. Here we show that infection with Tomato spotted wilt virus (TSWV), type member of the only plant-infecting genus in the Bunyaviridae, alters the feeding behavior of its thrips vector, Frankliniella occidentalis (Pergande). Male thrips infected with TSWV fed more than uninfected males, with the frequency of all feeding behaviors increasing by up to threefold, thus increasing the probability of virus inoculation. Importantly, infected males made almost three times more noningestion probes (probes in which they salivate, but leave cells largely undamaged) compared with uninfected males. A functional cell is requisite for TSWV infection and cell-to-cell movement; thus, this behavior is most likely to establish virus infection. Some animal-infecting members of the Bunyaviridae (La Crosse virus and Rift Valley fever virus) also cause increased biting rates in infected vectors. Concomitantly, these data support the hypothesis that capacity to modify vector feeding behavior is a conserved trait among plant- and animal-infecting members of the Bunyaviridae that evolved as a mechanism to enhance virus transmission. Our results underscore the evolutionary importance of vector behavioral modification to diverse parasites with host ranges spanning both plant and animal kingdoms.

A virus capsid component mediates virion retention and transmission by its insect vector.

Numerous pathogens of humans, animals, and plants are transmitted by specific arthropod vectors. However, understanding the mechanisms governing these pathogen-vector interactions is hampered, in part, by the lack of easy-to-use analytical tools. We investigated whitefly transmission of Lettuce infectious yellows virus (LIYV) by using a unique immunofluorescent localization approach in which we fed virions or recombinant virus capsid components to whiteflies, followed by feeding them antibodies to the virions or capsid components, respectively. Fluorescent signals, indicating the retention of virions, were localized in the anterior foregut or cibarium of a whitefly vector biotype but not within those of a whitefly nonvector biotype. Retention of virions in these locations strongly corresponded with the whitefly vector transmission of LIYV. When four recombinant LIYV capsid components were individually fed to whitefly vectors, significantly more whiteflies retained the recombinant minor coat protein (CPm). As demonstrated previously and in the present study, whitefly vectors failed to transmit virions preincubated with anti-CPm antibodies but transmitted virions preincubated with antibodies recognizing the major coat protein (CP). Correspondingly, the number of insects that specifically retained virions preincubated with anti-CPm antibodies were significantly reduced compared with those that specifically retained virions preincubated with anti-CP antibodies. Notably, a transmission-defective CPm mutant was deficient in specific virion retention, whereas the CPm-restored virus showed WT levels of specific virion retention and transmission. These data provide strong evidence that transmission of LIYV is determined by a CPm-mediated virion retention mechanism in the anterior foregut or cibarium of whitefly vectors.

Sieve element occlusion provides resistance against Aphis gossypii in TGR-1551 melons.

Feeding behavior and plant response to feeding were studied for the aphid Aphis gossypii Glover on susceptible and resistant melons (cv. Iroquois and TGR-1551, respectively). Average phloem phase bout duration on TGR-1551 was <7% of the duration on Iroquois. Sixty-seven percent of aphids on TGR-1551 never produced a phloem phase that attained ingestion (EPG waveform E2) in contrast to only 7% of aphids on Iroquois. Average bout duration of waveform E2 (scored as zero if phloem phase did not attain E2) on TGR-1551 was <3% of the duration on Iroquois. Conversely, average bout duration of EPG waveform E1 (sieve element salivation) was 2.8 times greater on TGR-1551 than on Iroquois. In a second experiment, liquid nitrogen was used to rapidly cryofix leaves and aphids within a few minutes after the aphids penetrated a sieve element. Phloem near the penetration site was then examined by confocal laser scanning microscopy. Ninety-six percent of penetrated sieve elements were occluded by protein in TGR-1551 in contrast to only 28% in Iroquois. Usually in TGR-1551, occlusion was also observed in nearby nonpenetrated sieve elements. Next, a calcium channel blocker, trivalent lanthanum, was used to prevent phloem occlusion in TGR-1551, and A. gossypii feeding behavior and the plant's phloem response were compared between lanthanum-treated and control TGR-1551. Lanthanum treatment eliminated the sieve element protein occlusion response and the aphids readily ingested phloem sap from treated plants. This study provides strong evidence that phloem occlusion is a mechanism for resistance against A. gossypii in TGR-1551.

Hitching a ride: Vector feeding and virus transmission.

The majority of plant viruses rely on insect vectors for transmission. Insects with piercing-sucking mouthparts are the most common and efficient vectors because, they are able to inject viruses into specific plant tissues. Acquisition and inoculation of viruses occurs during specific vector feeding behaviors, and feeding behavior varies greatly among insects with piercing-sucking mouthparts. In this review we provide an overview of the feeding behavior of the major insect vectors with piercing sucking mouthparts: aphids, whiteflies, mealybugs, hoppers, and thrips. We briefly review the different mechanisms of plant virus transmission by these insects, and discuss how each mechanism requires a vector that engages in specific feeding behaviors, and how differences in feeding behavior among these insects can determine which viruses they are capable of transmitting. We also discuss recent findings indicating that plant viruses can directly modify their vector's behavior in a way that enhances transmission to a host plant.