The infection of its insect vector by bacterial plant pathogen "Candidatus Liberibacter solanacearum" is associated with altered vector physiology

Highlights

• The vector’s physiology is altered by the presence of the pathogen.

• Infected psyllids had lower hemolymph oxygen tension (33.99% ± 1.35%).

• Healthy psyllids had higher hemolymph oxygen tension (67.83% ± 2.03%).

• A linear relationship was observed between pH and GE/insect in infected psyllids.

Abstract

Many bacterial and viral plant pathogens are transmitted by insect vectors, and pathogen-mediated alterations of plant physiology often influence insect vector behavior and fitness. It remains largely unknown for most plant pathogens whether, and how, they might directly alter the physiology of their insect vectors in ways that promote pathogen transmission. Here we examined whether the presence of “Candidatus Liberibacter solanacearum” (“Ca. L. solanacearum”), an obligate bacterial pathogen of plants and of its psyllid vector alters the physiochemical environment within its insect vector, the potato psyllid (Bactericera cockerelli). Microelectrodes were used to measure the local pH and oxygen tension within the abdomen of “Ca. L. solanacearum”-free psyllids and those infected with “Ca. L. solanacearum”. The hemolymph of infected psyllids had higher pH at 9.09 ± 0.12, compared to “Ca. L. solanacearum”-free psyllids (8.32 ± 0.11) and a lower oxygen tension of 33.99% vs. 67.83%, respectively. The physicochemical conditions inside “Ca. L. solanacearum”-free and –infected psyllids body differed significantly with the infected psyllids having a higher hemolymph pH and lower oxygen tension than “Ca. L. solanacearum”-free psyllids. Notably, the bacterial titer increased under conditions of higher pH and lower oxygen tension values. This suggests that the vector’s physiology is altered by the presence of the pathogen, potentially, resulting in a more conducive environment for “Ca. L. solanacearum” survival and subsequent transmission.

Introduction

Plant pathogens transmitted by insect vectors are a considerable threat to global crop production. Losses due to diseases caused by insect vector-transmitted pathogens amount to ˜10% decreased productivity [1], with some losses in excess of 1 billion dollars annually [2]. Many vector-borne plant pathogens are known to alter the physiology of the host plant in ways that indirectly increase vector fitness [3,4]. Some scenarios involve pathogen-mediated increased palatability to the insect vector [4], or increased nutrient availability due to pathogen infection of the plant host [5]. Pathogens can also indirectly benefit their insect vector because they often have silencing suppressor proteins that counter plant defenses [[6], [7], [8]]. Thus, many insect vector-plant pathogen relationships are considered to be mutualistic, for example, the vector population may increases more rapidly on infected, compared to non-infected plants due to increased palatability of infected plants [3,9].

Plant pathogens might also promote their own spread by altering the physiology of their arthropod vectors. In the case of the Asian citrus psyllid (ACP), Diaphorina citri (L.) (Triozidae, Hemiptera), which is both a vector and host of “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) (Class Alphaproteobacteria, Order Rhizobiales), “Ca. L. asiaticus”-infected psyllids have increased levels of adenosine triphosphate (ATP) in hemolymp, which has been associated with enhancement of the tricarboxylic acid (TCA) cycle (10). Infected psyllids with elevated ATP levels also show increased expression of ATP synthase, the key enzyme involved in ATP production, while at the same time exhibiting decreased ATPase and GTPase activity are essential for utilizing ATP as a substrate [10]. At the same time, the mitochondrial enzymes related to TCA cycle is downregulated in gut tissue [11]. Since there is no significant changes in the expression of the whole insect body, it is hypothesized that the upregulation of the expression of the enzymes in other organs is compensating the changes in the gut [11]. Moreover, the infected psyllids exhibit accelerated feeding activity, during which time they probe plants with greater frequency, a phenomenon has been shown to result in increased frequency of “Ca. L. asiaticus” transmission [10]. Moreover, the proteins involved in energy storage and utilization, endocytosis, defense and immunity are expressed differently in ACP infected with “Ca. L. asiaticus” [12]. However, it is expected that plant pathogens may directly alter the physiology of their insect vector through additional mechanisms.

Several species of psyllids are known to transmit bacterial pathogens in the genus, “Ca. Liberibacter” [13]. In the western United States, zebra chip of potato [[14], [15], [16], [17], [18]] and vein-greening of tomato [19] are economically-important diseases caused by “Ca. L. solanacearum”, transmitted by the potato psyllid Bactericera cockerelli (Sulc.) in a circulative, propagative manner [20,21]. In this type of transmission pathway, the bacteria are ingested by the psyllid vector during phloem-feeding and following intracellular invasion, they multiply in the gut. The bacteria exit the gut through an exocytosis-like mechanism and enters the hemolymph [21,22]. From here, bacterial cells are translocated in hemolymph to the salivary glands, the site of transmission specificity, where acquisition occurs. During psyllid feeding, the bacteria are transmitted to phloem cells in salivary secretions [[23], [24], [25], [26]].

The abundance of “Ca. L. solanacearum” biofilms observed on the external midgut surface of the psyllid host by scanning electron microscopy was an early step in beginning to understand the route by which Ca. L. asiaticus” circulates in its vector, which is now known to involve crossing the midgut epithelial cell barrier to access the hemolymph [20]. Once in the hemolymph, specific nutritional and physiochemical conditions are expected to be required to achieve conditions favorable to “Ca. L. solanacearum” survival while en route to the salivary glands [20,26,27].

Studies have shown that “Ca. L. solanacearum” infection of the psyllid affects host fitness (15–18). Based on gene expression profiling of the different (immature and adult) life stages of “Ca. L. solanacearum” infected psyllid host, gene ontology identifiers suggest that gene expression profiles are significantly altered, compared to “Ca. L. solanacearum”-free psyllids. in particular, proteins with predicted roles in adherence and invasion, multiplication, biofilm formation, immune system modulation, and nutritional parasitism, among other physiological effects [27]. These insights strongly indicate that “Ca. L. solanacearum” may directly influence psyllid physiology, however, most of the specific mechanisms involved have not been elucidated, making our understanding of this pathosystem somewhat limited.

The objectives of this study were to compare the pH and oxygen tension, respectively, in the hemolymph of adult potato psyllids post-“Ca. L. solanacearum” infection, compared to “Ca. L. solanacearum”-free psyllids, to determine if infection by the bacterium directly affects the physiology of the psyllid host. The pH and oxygen tension were considered key parameters for assessment, because although infection by “Ca. L. solanacearum” has been associated with metabolic and other changes [[27], [28], [29], [30]], its effects on these two key internal environmental ‘indicators’ occurring within infected psyllids have not been studied. Moreover, changes in metabolic rate and energy metabolism in some other insects has been shown to be accompanied with changes in oxygen tension and pH in the insects [31]. Because the metabolism of “Ca. L. solanacearum”-infected psyllids is altered within the body [10,27], we reasoned that pH and oxygen levels may likewise be altered inside the hemolymph, accessible by non-destructive penetration to the psyllid abdomen. To accomplish this, the local oxygen tension and pH were measured in psyllid hemolymph using microelectrodes with tip diameters smaller than 20 μm to penetrate the abdomen, pass through the hemolymph and gut, and finally into the hemolymph beyond, where measurements were made. Also, the average oxygen tension and average pH in potato psyllid hemolymph were determined, respectively, in relation to “Ca. L. solanacearum” infection status and infection levels (genome equivalents).