Beet leafhopper

Mode of transmission

The bacterium S. citri is initially acquired by the leafhopper through feeding on a plant that has already been infected.^[6] Through feeding, the prokaryote enters the gut of the beet leafhopper where most of its cells are killed, but some do survive.^[6] These surviving organisms then enter the epithelial cells of the intestine and multiply.^[6] Eventually the organisms are able to make their way into the hemocoel of the insect through which they are transferred to the insect’s salivary glands.^[6] When the insect then feeds on healthy plants, the prokaryotes enter the phloem of the plant and thus infect the new plant.^[6] One study has shown that this process of transmission also has negative effects on the leafhopper itself, where significant numbers of leafhoppers may die after being infected with S. citri.^[6] The researchers suggest that this increased mortality may be as a result of ingesting toxins that are produced either by S. citri itself or by plants defending themselves against the pathogen.^[6] In any case, these toxins have a significant on the longevity of infected beet leafhoppers.^[6]

Another study tested some of the characteristics that are important for transmission of S. citri by the beet leafhopper. Researchers tested the impacts of modes of acquisition on the latent period of S. citri within the leafhopper and found that insects that had been injected directly with the prokaryote in their gut had the lowest latency period of 10 days followed by leafhoppers who had ingested the pathogen from an infected plant (16 days).^[7] In testing the time taken for the insects to acquire S. citri, the acquisition access period, researchers found that a time period of at least 6 hours of feeding on S. citri cultures were required for insects to acquire the pathogen.^[7] However this study should be viewed critically as insects were membrane fed a culture of the organism to determine this time period and actual results may vary when insects feed on an infected plants. The study also found that the numbers of host plants that were infected with S. citri increased with the numbers of infected leafhoppers that were feeding on these plants.^[7]

Movements of Spiroplasma citri inside beet leafhopper hosts

In an experiment to understand how S. citri cells move within the beet leafhopper, researchers inoculated insects with the pathogen and then observed the locations of S. citri inside the leafhoppers using electron microscopy.^[8] They observed S. citri in the gut as well as the salivary glands of the insects which further proves that the pathogen may be transmitted by the mechanisms described in the studies above.^[8] In particular, researchers observed that S. citri were often found within gut epithelial and salivary gland cells on infected hosts within small membrane bound vesicles.^[8] This led them to believe that the S. citri pathogen may be using cell mediated endocytosis as a predominant way to enter gut and salivary cells and may be travelling directly though the cells rather than travelling in the spaces between cell membranes of individual cells through a process called diacytosis.^[8] They also observed damage to muscle cells in the gut and salivary glands of insects infected with S. citri which is evidence that S. citri causes damage to the beet leafhoppers when it uses them as vectors.^[8] However the authors also noted how this damage was not as significant as seen in other insects which may suggest that the beet leafhopper may be co-evolving to reduce harm by S. citri pathogens.^[8]

In a follow-up study to understand the exact mechanism of uptake of S. citri cells by beet leafhopper gut cells, researchers developed a line of beet leafhopper cells called the CT1 cell line.^[9] They used this cell line to study how leafhopper cells interacted with S. citri cells in vivo.^[9] After incubation of beet leafhopper cells with S. citri cells, researchers used an electron microscope to see how cell lines interact.^[9] They found that S. citri cells were pressed against the cell membranes of C. tenellus cells, and they also observed invaginations of the cell membrane.^[9] They concluded that these sighting were consistent with previous studies which suggested that S. citri cells are taken up by beet leafhopper cells by endocytosis.^[9]

Transmission

In order to understand the movement of the MBCTV inside its hosts, experiments have been conducted using PCR analysis which allow for the detection and quantification of MBCTV particles inside leafhoppers. One such study measured the amounts of MBCTV found in the gut, hemocoel and salivary glands of beet leafhoppers and found that the virus was present in all those regions suggesting that the virus may use a circulative transmission mechanism inside its host.^[11] A circulative transmission mechanism refers to a transmission mechanism in which the vector ingests a pathogen during feeding from an infected host, the pathogen is absorbed in the vector’s gut and then travels through the hemocoel into the salivary glands.^[11] When the vector then bites into an uninfected host, the pathogen enters the new host. This mechanism is similar to the transmission mechanism used by "Spiroplasma citri" in transmitting the Stubborn citrus disease which also uses the beet leafhopper as a vector.

In the same study, researchers were able to detect viral particles of MBCTV in beet leafhopper guts after as soon as an hour of feeding on infected plants.^[11] Furthermore, they found that insects that had fed on an infected plant for an hour were able to spread the disease to uninfected plants.^[11] These findings suggest that the insects become virulent very shortly after feeding on infected plants.^[11] The study also found that as time of feeding on infected plants increased, the numbers of viral particles in the gut, hemocoel and salivary glands of the insects also increased, even though there were individual differences in the amounts of BMCTV particles detected in the body.^[11] Another important finding of this study was showing that individual insects can maintain MBCTV in their bodies for up to a 30 days after getting infected by it, even though the amount of virus detected does not increase in this period.^[11] The authors propose that this indicates that MBCTV is unable to replicate within its vector. It was also found that the virus is not transmitted between generations of beet leafhoppers from adults to nymphs, proposing that the virus does not use transovarial transmission.^[11]

In another study, waveforms from electrical penetration graphs (EPG) were used to understand which waveforms coincided with the inoculation of plants with BCTV.^[12] As insects feed on different parts of the plant, they produce different EPG waves which can be measured using probes wired to the insects.^[12] Researchers found that D waveforms were most likely to coincide with successful inoculation of the plant with BCTV.^[12] This waveform is also the same waveform which is produced during phloem ingestion by beet leafhoppers. Thus, this study further lends credibility to the transmission model that suggests that beet leafhoppers transmit the BCTV when they feed on the phloem sap of healthy plants.

Transmission to non-host plants

A previous study of oviposition in the beet leafhopper has shown that the insects prefer to lay eggs on beet plants even when they prefer different host plants for feeding.^[2] In another study, researchers tried to study which plants the insects prefer to settle and lay eggs on and which plants were good for insect survival. These studies are important as factors like host preference may impact which species of plants the BCTV is transmitted to.^[10] It is also important to understand the complex interactions that take place between the beet leafhopper and non-host plants to understand how disease may be spread.^[10] Tests of settling behaviors showed that although the insects settled similarly on all plants in the beginning, the numbers of beet leafhoppers on bean and tomato plants declined significantly after a period of four hours.^[10] Instead, the insects preferred to settle on sugar beets, radish, potato and carrot plants.^[10] These results were similar to studies of insect mortality on the same plants, which showed that the majority of insects confined to bean and tomato plants had died within a week.^[10] There were also differences noticed in mortality on plants that were preferred for settling: insects were more likely to die on carrot plants as compared to beets, potato and radish.^[10] Experiments on oviposition also showed that the insects had a preference to lay eggs on beet, potato and radish plants where nymphs hatched from eggs and continued providing generations of beet leafhoppers.^[10] Carrot, bean and tomato plants were also rejected for oviposition by the insects where no eggs were laid on these plants.^[10]

Even though bean and tomato plants proved to be in-hostile hosts for the beet leafhopper, research shows that the insects still transmit the BCTV to these plants. A major implication of this study is that it shows that plants which are inappropriate hosts of the beet leafhopper insect can also be transmitted with the Beet Curly Top Virus. The authors propose that these findings may suggest that the virus can be transmitted very quickly even after only a couple of hours of exposure to the insects.^[10]