INVERTEBRATE MICROBIOLOGY
crossm
A Plant Bacterial Pathogen Manipulates
Its Insect Vector’s Energy Metabolism
Nabil Killiny,a Faraj Hijaz,a Timothy A. Ebert,b Michael E. Rogersb
ABSTRACT Insect-transmitted plant-pathogenic bacteria may alter their vectors’ fit-
ness, survival, behavior, and metabolism. Because these pathogens interact with
their vectors on the cellular and organismal levels, potential changes at the biochemical level might occur. “Candidatus Liberibacter asiaticus” (CLas) is transmitted
in a persistent, circulative, and propagative manner. The genome of CLas revealed
the presence of an ATP translocase that mediates the uptake of ATP and other nucleotides from medium to achieve its biological processes, such as growth and multiplication. Here, we showed that the levels of ATP and many other nucleotides were
significantly higher in CLas-infected than healthy psyllids. Gene expression analysis
showed upregulation for ATP synthase subunits, while ATPase enzyme activity
showed a decrease in ATPase activity. These results indicated that CLas stimulated
Diaphorina citri to produce more ATP and many other energetic nucleotides, while it
may inhibit their consumption by the insect. As a result of ATP accumulation, the
adenylated energy charge (AEC) increased and the AMP/ATP and ADP/ATP ratios decreased in CLas-infected D. citri psyllids. Survival analysis confirmed a shorter life
span for CLas-infected D. citri psyllids. In addition, electropenetrography showed a
significant reduction in total nonprobing time, salivation time, and time from the
last E2 (phloem ingestion) to the end of recording, indicating that CLas-infected
psyllids were at a higher hunger level and they tended to forage more often. This
increased feeding activity reflects the CLas-induced energetic stress. In conclusion,
CLas alters the energy metabolism of its psyllid vector, D. citri, in order to secure its
need for energetic nucleotides.
Received 30 October 2016 Accepted 19
December 2016
Accepted manuscript posted online 30
December 2016
Citation Killiny N, Hijaz F, Ebert TA, Rogers ME.
2017. A plant bacterial pathogen manipulates
its insect vector's energy metabolism. Appl
Environ Microbiol 83:e03005-16. https://
doi.org/10.1128/AEM.03005-16.
Editor Harold L. Drake, University of Bayreuth
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Nabil Killiny,
nabilkilliny@ufl.edu.
IMPORTANCE Insect transmission of plant-pathogenic bacteria involves propaga-
tion and circulation of the bacteria within their vectors. The transmission process
is complex and requires specific interactions at the molecular and biochemical
levels. The growth of the plant-pathogenic bacteria in the hemolymph of their
vectors indicated that the hemolymph contains all the necessary nutrients for
their growth. In addition to nutrients, “Candidatus Liberibacter asiaticus” (CLas)
can take up energetic nucleotides, such as ATP, from its vector, Diaphorina citri,
using ATP translocase. In this study, we found that the CLas pathogen manipulates the energy metabolism of its insect vector. The accumulation of ATP in
CLas-infected D. citri psyllids indicated that CLas induces ATP production to fulfill
its need for this energetic compound. As a result of ATP accumulation, a shorter
life span and altered feeding behavior were observed. These findings increase
our knowledge of insect transmission of the persistent-circulative-propagative
type of plant pathogens vectored by insects.
KEYWORDS huanglongbing, Diaphorina citri, “Candidatus Liberibacter asiaticus,”
energy metabolism, ATP, adenylated energy charge, HPLC, electric penetration
graph, electropenetrography
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Plant Pathology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, Florida,
USAa; Entomology and Nematology Department, Citrus Research and Education Center, University of Florida,
Lake Alfred, Florida, USAb
Killiny et al.
Applied and Environmental Microbiology
nsect vectors transmit a variety of pathogens to a wide range of animal and plant
hosts (1). Human vector-borne diseases account for more than 17% of the total
infectious diseases every year (2). Malaria, the most important vector-borne disease,
causes 1 to 3 million deaths per year (2). In addition, vector-borne diseases can cause
huge economic losses when they attack livestock and crops (2). Huanglongbing, citrus
tristeza, and citrus stubborn disease are examples of economically important plant
vector-borne diseases in the citrus agroecosystem (3). Studying vector-pathogen-host
relationships is essential to understanding the epidemiology of many important plant
diseases (2).
The interaction between bacteria and their insect vectors can be mutualistic,
parasitic, or commensal, depending on the relative effects of the bacteria on the fitness
of the insect (4). Most of the previous studies about the interactions between the
bacteria and their insect vectors showed that animal pathogens were virulent to their
vectors whereas plant pathogens were beneficial to their vectors (4). Because most of
the studies that demonstrated positive effects did not focus on the direct consequences of the plant pathogens to vector fitness, the results obtained from these
studies could be biased (4). On the other hand, a number of studies demonstrated that
some plant pathogens were virulent to their vector (5, 6). Nachappa et al. (4) showed
that there was a negative relationship between “Candidatus Liberibacter solanacearum”
level and the fecundity of its psyllid vector (Bactericera cockerelli [Šulc] [Hemiptera:
Triozidae]). Interestingly, “Ca. Liberibacter solanacearum” did not affect the mortality
index of adult psyllids (6).
Studying the effect of plant pathogens on the fitness of their vector is difficult,
because many of these pathogens cannot be cultured in vitro (5). The number of
studies that focused on the effects of phytopathogens on the fitness of their insect
vectors is limited, and only a few systems have been studied (5, 6). Furthermore,
information about the effect of “Candidatus Liberibacter asiaticus” (CLas), the causal
agent of citrus greening, on its insect vector (Diaphorina citri) is limited.
Citrus greening, also known as huanglongbing (HLB), is currently threatening the
citrus industry worldwide. HLB was first identified in China in the beginning of the 20th
century and has been recently identified in Brazil and Florida (7). The CLas bacterium,
which is associated with HLB, is a phloem-restricted Gram-negative bacterium that has
not yet been cultured (8). Three species of the causal agent have been associated with
HLB: “Candidatus Liberibacter asiaticus” (Asia, North America, and Brazil), “Candidatus
Liberibacter africanus” (Africa), and “Candidatus Liberibacter americanus” (Brazil) (9).
CLas and “Candidatus Liberibacter americanus” are transmitted by the Asian citrus
psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), whereas “Ca. Liberibacter
africanus” is transmitted by the African citrus psyllid, Trioza erytreae (Del Guercio)
(Hemiptera: Triozidae) (10). D. citri has received great attention in the past few years,
and its transcriptomes (egg, nymph, and adult) were characterized (11).
CLas has a relatively small genome (⬃1.2 Mbp), and it is an obligate intracellular
pathogen (12). Genome sequencing of CLas revealed that CLas is not able to synthesize
tryptophan, tyrosine, leucine, isoleucine, and valine from metabolic intermediates (13).
Consequently, the CLas bacterium counters these deficiencies by importing these
amino acids from its host (14). The genome sequencing of CLas also revealed that it
cannot synthesize fumarate, malate, succinate, and aspartate because it lacks an
isocitrate lyase and malate synthase (13). CLas needs to acquire these intermediates
from its host (14).
CLas encodes ⬎100 proteins, with 92 genes that are involved in active transport,
and 40 of these genes are ATP binding cassette (ABC) transporter genes (14). Analysis
of these ABC transporter-related proteins by Li et al. (15) showed that CLas can use
them to import metabolites and enzyme cofactors (14). It is also thought that the
presence of this large number of transporter proteins might play an important role in
providing CLas with the nutrients necessary for growth and reproduction (14).
CLas encodes ATP synthase, ATPase, and ATP/ADP translocase, which means that
CLas can synthesize its own ATP or utilize it directly from its host (13). To test this
I
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hypothesis, Vahling et al. (16) expressed the ATP translocase nucleotide transport
protein (NttA) gene contained in CLas in Escherichia coli. E. coli harboring the NttA gene
was able to import exogenous ATP directly into the cell. Vahling et al. (16) concluded
that some intracellular bacteria of plants have the potential to import ATP from their
environment.
Interestingly, it has been shown that the antigenic membrane protein of “Candidatus Phytoplasma asteris” interacts with the ATP synthase of its leafhopper vectors (17).
Because the phytoplasmas lack the ATP synthetic pathway and depend partly on their
host for energy, it has been suggested that host extracellular ATP in the gut lumen and
hemocoel may be required for the survival of phytoplasmas (17). Vahling et al. (16) also
suggested that the addition of external ATP to the culture medium might facilitate the
growth of the CLas bacterium in vitro.
In the current study, we hypothesize that the bacterial pathogen CLas alters the
energy metabolism of its insect vector, the Asian citrus psyllid, in order to meet its
needs for energetic nucleotides, mainly ATP. We also hypothesize that the bacterium
may stimulate the insect to overproduce and/or inhibit its utilization of the energetic
nucleotides. The accumulated ATP in the insect would be translocated to the bacterial
cell by ATP/ADP translocase.
RESULTS
Infection with CLas increased the ATP level in D. citri. Enzymatic quantification
showed that the level of ATP in CLas-infected D. citri psyllids was significantly higher
than that in the controls (Fig. 1A). The quantity of ATP in control psyllids was between
2 to 5 ng/insect, whereas it ranged from 4 to 14 ng/insect in CLas-infected psyllids. This
result indicates that the amount of ATP was affected by the presence of the CLas
bacterium.
Level of ATP was dependent on the CLas population. The linear regression of the
levels of ATP versus the cycle threshold (CT) value for healthy psyllids did not show any
correlation between ATP level and CT value (data not shown). On the other hand, the
linear regression of the levels of ATP versus the CT value showed a significant negative
relationship between ATP level and CT value (Fig. 1B). In other words; there was a
significant positive density-dependent relationship between CLas cells and ATP content
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FIG 1 (A) Enzymatic quantification of ATP. (B) Simple linear regression plot of the ATP level versus the cycle threshold (CT) of real-time PCR for the detection
of CLas. (C) Total ATPase/GTPase activity in CLas-infected (CLas⫹) and healthy (CLas⫺) Diaphorina citri psyllids. For panels A and C, horizontal thick lines indicate
the medians, boxes show the interquartile ranges including 25 to 75% of the values, and whiskers show the highest and the lowest values in each set. The circles
represent outliers. Letters on the bars indicate significant differences (P ⱕ 0.05).
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FIG 2 Mirror high-performance anion-exchange chromatograms of nucleotides and sugar nucleotides detected in healthy (CLas⫺)
and CLas-infected (CLas⫹) Diaphorina citri adults. (B to D) AMP/ATP ratios (B), ADP/ATP ratios (C), and adenylated energy charge (AEC)
values (D) in healthy and CLas-infected Diaphorina citri adults. For panels B to D, horizontal thick lines indicate the medians, boxes
show the interquartile ranges including 25 to 75% of the values, and whiskers show the highest and the lowest values in each set.
Letters on the bars indicate significant differences (P ⱕ 0.05).
in CLas-infected D. citri psyllids. This correlation confirmed that the increase in ATP level
was due to the presence of CLas.
Infection with CLas decreased ATPase/GTPase activity in D. citri. ATPase/GTPase
activity was significantly reduced (P value ⫽ 0.0008) in CLas-infected psyllids compared
to that in the control (Fig. 1C). The ATPase/GTPase level (mean ⫾ SD) in control psyllids
was 93 ⫾ 32 U/insect, whereas it was 67 ⫾ 23 U/insect in CLas-infected psyllids.
Infection by CLas altered the nucleotide profile of its vector. Eighteen nucleotides, four sugar nucleotides, and five unknowns were detected in the perchloric acid
extracts of D. citri (Fig. 2A). In agreement with the Enliten ATP kit’s result, the highperformance liquid chromatography (HPLC) results (Table 1) showed that the ATP level
in CLas-infected psyllids was significantly higher than in the controls. In addition to ATP,
the levels of AMP, UMP, ADP, UDP-Glc, GMP, UDP, ATP, GDP, unknown 2 (UK2), UK3, and
UK4 in CLas-infected psyllids were significantly higher than those in the controls. The
levels of CMP, UDP-GalNa, UDP-Gal, GDP-F, and UK1 were also higher, but not significantly so. On the other hand, the level of GDP-Man in CLas-infected psyllids was
significantly lower than in the controls. The reduction in GDP-Man in CLas-infected
psyllids indicated that GDP-Man could be essential for the survival of the CLas bacterium. Slight reductions in NADP and IMP levels were also observed.
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TABLE 1 Nucleotide and sugar nucleotide concentrations in healthy and CLas-infected Asian citrus psyllids
Concn (mean ⴞ SD) (g/insect)a
aACP,
Abbreviation
UK1
NAD
UK2
UK3
CMP
UK4
UK5
AMP
UK6
NADP
CDP
UMP
CTP
UDP-GalNAc
ADP
UDP-GlcNAc
UDP-Gal
UDP-Glc
GMP
IMP
UDP
ATP
GDP-Fuc
GDP-Man
UTP
GDP
IDP
FAD
GTP
ITP
Healthy ACP
0.055 ⫾ 0.032 A
0.095 ⫾ 0.046 A
0.020 ⫾ 0.012 B
0.008 ⫾ 0.002 B
0.009 ⫾ 0.003 A
0.012 ⫾ 0.001 B
0.069 ⫾ 0.020 A
0.549 ⫾ 0.116 B
0.268 ⫾ 0.049 A
0.068 ⫾ 0.025 A
ND
0.036 ⫾ 0.010 B
0.019 ⫾ 0.006 B
0.029 ⫾ 0.016 A
0.141 ⫾ 0.038 B
ND
0.002 ⫾ 0.001 A
0.018 ⫾ 0.012 B
0.033 ⫾ 0.017 B
0.009 ⫾ 0.003 A
0.005 ⫾ 0.001 B
0.029 ⫾ 0.008 B
0.014 ⫾ 0.002 A
0.332 ⫾ 0.083 A
ND
0.051 ⫾ 0.011 B
ND
ND
0.278 ⫾ 0.04 A
0.181 ⫾ 0.04 A
CLas-infected ACP
0.113 ⫾ 0.049 A
0.155 ⫾ 0.067 A
0.074 ⫾ 0.013 A
0.025 ⫾ 0.008 A
0.048 ⫾ 0.036 A
0.032 ⫾ 0.013 A
0.084 ⫾ 0.004 A
0.970 ⫾ 0.082 A
0.389 ⫾ 0.059 A
0.065 ⫾ 0.004 A
ND
0.079 ⫾ 0.017 A
0.024 ⫾ 0.003 A
0.077 ⫾ 0.032 A
0.252 ⫾ 0.028 A
ND
0.009 ⫾ 0.007 A
0.057 ⫾ 0.009 A
0.090 ⫾ 0.025 A
0.008 ⫾ 0.004 A
0.019 ⫾ 0.004 A
0.108 ⫾ 0.017 A
0.085 ⫾ 0.045 A
0.063 ⫾ 0.011 B
ND
0.079 ⫾ 0.021 A
ND
ND
0.304 ⫾ 0.06 A
0.184 ⫾ 0.01 A
Asian citrus psyllids; ND, nondetected compound. Numbers that are followed by the same letters do not show significant differences (P ⬍ 0.05).
CLas altered the energy homeostasis of its insect vector. CLas infection changed
the level of many nucleotides in D. citri, thereby altering the nucleotide ratios used to
measure cellular energy balance. CLas infection significantly decreased the ratios of
AMP/ATP (Fig. 2B) and ADP/AMP (Fig. 2C). The ratios of AMP/ATP and ADP/ATP in
CLas-infected psyllids were about half of those of the controls. On the other hand, the
adenylated energy charge (AEC) of CLas-infected psyllids was significantly higher than
that of the control (Fig. 2D). These results showed that CLas significantly alters the
energy homeostasis of its vector.
CLas increased ATP synthesis and decreased its breakdown in its insect vector.
The gene expression analysis showed that the levels of expression of ATP synthase
mitochondrion-like alpha/beta subunits (ATP synthase ␣/-subunits) were upregulated
in CLas-infected psyllids (Fig. 3). On the other hand, the levels of expression of the
V-type proton ATPase catalytic subunit A (V-ATPase-V1a) and transitional endoplasmic
reticulum ATPase (TER94) genes in CLas-infected psyllids were reduced compared to
those in the controls (Fig. 3). In addition, the level of expression of the nucleotide
diphosphate kinase (NDPK) gene in CLas-infected D. citri psyllids was increased,
whereas expression of the AMP-activated protein kinase ␣ subunit (AMPK-A) gene was
significantly reduced in CLas-infected psyllids (Fig. 3).
CLas shortened the life span of its vector. Survival analysis using the Kaplan-Meier
method showed that CLas decreased the survival probability (Fig. 4A) and the life span
(Fig. 4B) of its vector, D. citri. Since the survival assay was carried out using an artificial
diet system, this result suggested that CLas was directly responsible for the reduced life
span.
CLas-infected psyllids were more susceptible to hunger than healthy psyllids.
Characteristic electrical penetration graph (EPG) waveforms produced by D. citri on
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Nucleotide or sugar nucleotide
Unknown 1
Beta-NAD hydrate
Unknown 2
Unknown 3
Cytidine 5=-monophosphate
Unknown 4
Unknown 5
Adenosine 5=-monophosphate
Unknown 6
B-NADP, oxidized form
Cytidine 5=-diphosphate
Uridine 5=-monophosphate
Cytidine 5=-triphosphate
Uridine 5=-diphospho-N-acetyl-D-galactosamine
Adenosine 5=-diphosphate
Uridine 5=-diphospho-N-acetyl-D-glucosamine
Uridine 5=-diphospho-D-galactose
Uridine 5=-diphospho-D-glucose
Guanosine 5=-monophosphate
Inosine 5=-monophosphate
Uridine 5=-diphosphate
Adenosine 5=-triphosphate
Guanosine 5=-diphosphate-beta-L-fucose
Guanosine 5=-diphosphate-D-mannose
Uridine 5=-triphosphate
Guanosine 5=-diphosphate
Inosine 5=-diphosphate
Flavin adenine dinucleotide
Guanosine 5=-triphosphate
Inosine 5=-triphosphate
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FIG 3 Expression of genes encoding ATP synthase mitochondrion-like alpha/beta subunits (ATP synthase ␣/subunits), V-type proton ATPase catalytic subunit A (V-ATPase-V1a), transitional endoplasmic reticulum ATPase
(TER94), nucleotide diphosphate kinase (NDPK), AMP-activated protein kinase ␣-subunit (AMPK-A) in CLas-infected
(CLas⫹) and healthy (CLas⫺) Diaphorina citri psyllids. Horizontal thick lines indicate the medians, boxes show
the interquartile ranges including 25 to 75% of the values, and whiskers show the highest and the lowest
values in each set.
leaves of Midsweet orange with enlargements and a detailed view of each waveform
are shown in Fig. 5A and B. The EPG data showed that there was no significant
difference in the phloem phase (E2) or xylem phase (G) between CLas-infected and
healthy psyllids (Table 2). The numbers of phloem probing phases and xylem probing
phases and the proportions of time spent on each phase were also similar (Table 2). On
the other hand, the durations of the total nonprobing period and the time from the last
E2 to the end of recording (TmLstE2EndRcrd) in CLas-infected psyllids were significantly
shorter than those of the controls (27,606 ⫾ 2,675 and 12,416 ⫾ 1,158 [CLas-infected
psyllids], respectively, and 32,696 ⫾ 1,985 and 39,170 ⫾ 1,637 [healthy psyllids]
respectively). In addition, the salivation time in CLas-infected psyllids was significantly
lower than that of the controls (Table 2). These observations together suggested that
CLas-infected psyllids were at a higher hunger level, and they tended to look for more
food.
FIG 4 (A) Kaplan-Meier analysis of survival of healthy (CLas⫺) and CLas-infected (CLas⫹) Diaphorina citri adults
carried out with a 20% sucrose solution. The log-rank and Wilcoxon values were used to compare survival curves.
(B) The average life spans for CLas-infected and healthy Diaphorina citri psyllids. Bars represent standard errors.
Letters on the bars indicate significant differences (P ⱕ 0.05).
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DISCUSSION
Our results showed that CLas infection shortened the life span of Asian citrus
psyllids, and this was accompanied by a decrease in AMP/ATP and ADP/ATP ratios. In
agreement with the current results, we have recently found that adult D. citri survival
under different temperatures was positively correlated with AMP/ATP and ADP/ATP
TABLE 2 Selected EPG variables associated with ingesting xylem or phloem along with
three variables that showed a difference in behavior between healthy and CLas-infected
psyllids on plantsa
Variable name
NumG
MeanG
PrcntPrbG
NumE2
NumLngE2
MnDurE2
MaxE2
PrcntPrbE2
DurNnprbBfrFrstD
DurE1FlwdFrstSusE2
DurE1FlldFrstE2
CLas-infected psyllids
n
25
25
25
25
25
14
14
25
15
14
14
Mean
5.52
1,757.7
18.53
2.08
1.68
5,463.2
8,912.4
15.72
9,659.6
22.07
25.65
SD
4.86
1,681.0
20.01
3.00
2.30
2,242.7
3,702.3
17.88
6,619.3
21.85
22.69
Healthy psyllids
n
27
27
27
27
27
16
16
27
18
16
16
Mean
6.19
1,383.5
16.58
1.59
1.41
9,402.7
10,976.2
20.89
19,327.4
72.92
72.47
SD
4.04
675.5
10.10
1.91
1.67
9,888.7
9,155.3
22.08
10,892.2
76.53
76.88
F
0.29
1.14
0.01
0.1
0.03
1.77
0.43
0.37
9.69
6.3
4.55
Pr > F
0.5926
0.2907
0.9232
0.7581
0.8596
0.1944
0.5157
0.5435
0.004
0.0181
0.0418
aNumG,
number of xylem ingestion events; MeanG, mean duration of xylem ingestion; PrcntPrbG,
percentage of probing duration spent in xylem ingestion; NumE2, number of phloem ingestion events;
NumLngE2, number of phloem ingestion events longer than 600 s; MnDurE2, mean duration of E2; MaxE2,
longest recorded E2 for each insect; PrcntPrbE2, percentage of probing duration spent ingesting phloem;
DurNnprbBfrFrstD, duration of nonprobing period before first phloem contact; DurE1FlwdFrstSusE2, duration
of the phloem salivation event before first sustained (600⫹ s) phloem ingestion event; DurE1FlldFrstE2,
duration of the phloem salivation event immediately before the first phloem ingestion event. All variables
were calculated by insect, and the mean values are reported. Pr ⬎ F, the probability of a greater F statistic
value under the null hypothesis.
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FIG 5 (A) General (24-h) scheme of characteristic electrical penetration graph (EPG) waveforms produced by Diaphorina citri on leaves of Midsweet orange,
Citrus sinensis, with enlargements of specific waveforms showing nonprobing (NP) pathway (C), xylem ingestion (G), contact with phloem (D), salivation into
the phloem (E1), and phloem ingestion (E2). (B) Detailed view of each waveform showing characteristic patterns of each waveform.
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ratios (18). Colinet (19) also found that the higher level of ATP was observed in lesser
mealworm beetles (Alphitobius diaperinus) during chronic cold stress. Stenesen et al.
(20) showed that heterozygous mutations of AMP biosynthetic enzymes extend Drosophila life span by increasing AMP/ATP and ADP/ATP ratios and the activity of AMPK.
Apfeld et al. (21) and Curtis et al. (22) showed that increased AMPK activity due to
increased AMP/ATP ratio also elongated the life span of the nematode Caenorhabditis
elegans.
The increase in the adenylated energy charge (AEC) in CLas-infected psyllids was
also correlated with a reduction in the life span and survival of D. citri. Interestingly, our
recent study also showed that the AEC of D. citri was negatively correlated with adult
psyllid survival (23). In agreement with our results, AEC was also negatively correlated
with the survival of other species. Marazza et al. (24) showed that the AEC of shrimp
(Palaemonetes varians) was increased upon exposure to lethal levels (3 mg/liter) of
ammonia. An increase in AEC was also observed in other inhabitants of polluted
seawater (25, 26).
Adenine nucleotides play important roles in metabolic regulation, and the mechanism of life span extension involves an increase in AMP/ATP and ADP/ATP ratios (20).
Hydrolysis of ATP in tissues produce ADP, while AMP is produced by the reaction
catalyzed by adenylate kinase (2 ADP ↔ ATP ⫹ AMP) (27). The activity of many
metabolic enzymes, including glycogen phosphorylase and 6-phosphofructo-1-kinase
in muscle, responds to the AMP/ATP ratio (27). An increase in AMP/ATP ratios activates
both glycogen phosphorylase and 6-phosphofructo-1-kinase and consequently
switches on glycogenolysis and glycolysis (3). High AMP/ATP ratios inhibit fructose-1,6bisphosphatase activity in the liver and switch off the anabolic pathway, gluconeogenesis (27). In addition, the increase in ADP/ATP and AMP/ATP ratios activates the AMPK
energy sensor, which regulates all aspects of cell function, including energy homeostasis (27).
CLas increased ATP levels in D. citri, and this increase was accompanied by an
increase in the gene expression of NDPK and a decrease in AMPK. Onyenwoke et al. (28)
demonstrated that AMPK and NDPK genetically antagonize each other (Fig. 6). Under
nutrient-rich conditions (high ATP level), AMPK is inactive, while NDPK is active and
consumes ATP to produce other nucleotides in order to maintain cellular homeostasis
(Fig. 6) (28). On the other hand, during starvation (low ATP level), AMPK is active and
inhibits NDPK activity (Fig. 6) (28). The increase in NDPK expression in CLas-infected D.
citri psyllids could be an attempt to maintain cellular homeostasis (Fig. 6). Failure to
maintain cellular homeostasis and the decrease in AMPK activity disrupts many aspects
of cellular function and results in cell death (28). In addition, overexpression of NDPK
in Drosophila spp. led to a decrease in their survival under starvation conditions (29).
The increase in NDPK activity and the decrease in AMPK may explain the shorter life
span observed in CLas-infected psyllids.
Taken together, the current results suggested that CLas manipulates the energy
metabolism of its insect vector to fulfill its need for energetic nucleotides. In other
words, CLas increases the levels of ATP in its host insect in order to increase its
availability and utilize it. The presence of ATP/ADP translocase in CLas (13) and the
ability of E. coli, which encodes the CLas ATP translocase, to import exogenous ATP (16)
support this hypothesis. CLas encodes both ATP synthase and ATP/ADP translocase,
which means that CLas can synthesize its own ATP or utilize it directly from its host (13).
However, it is still not clear which of them (ATP synthase or ATP/ADP translocase)
contributes more to CLas energy. CLas could be like phytoplasmas, as it depends partly
on its host for energy (17). Consequently, extracellular ATP in the gut lumen and
hemocoel may also be required for CLas survival (17).
Parasites usually acquire nutrients from their host, and this results in energetic
stresses in their host. Immunological responses of the host to the infectious agent are
also energetically expensive, further increasing energetic stress (30). Mayack and Naug
(31) showed that Nosema ceranae decreases the survival of its honeybee host (Apis
mellifera) by inducing energetic stress. Feeding and extension experiments showed that
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FIG 6 A hypothetical model for the decrease in life span in CLas-infected psyllids as a result of
accumulation of ATP. Infection with CLas enhances ATP accumulation and consequently increases
adenylated energy charge (AEC) and decreases AMP/ATP and ADP/ATP ratios. As a result of the increase
in AEC and the decrease in AMP/ATP and ADP/ATP ratios, the activity of AMPK will decrease and the
activity of NDPK will increase in order to maintain energy homeostasis. Failure to maintain cellular
homeostasis and the decrease in AMPK activity will disrupt many aspects of cellular functions and result
in cell death and shorter life span in D. citri.
hunger levels of infected honeybees were higher than in uninfected honeybees (31).
The metabolic profile of N. ceranae-infected honeybees showed a decline in most of the
carbohydrates and amino acids (32). Because mitochondria are absent in Nosema
species, Martín-Hernández et al. (33) suggested that they either consume ATP from
their host or synthesize it by metabolizing carbohydrates present in their host. The
accumulation of ATP in CLas-infected psyllids and the decrease in its life span indicated
that CLas induces energetic stress in its vector. Since the CLas does not produce any
known toxin and does not have any specialized secretion system or extracellular
degrading enzymes, the pathogenicity of CLas could be due to metabolic imbalances
caused by nutrient depletion and energy parasitism (13, 14, 16).
Recently, Martini et al. (34) found that CLas infection affected the behavior of D. citri
by increasing dispersal and flight initiation; however, it did not affect duration and
speed of flight. However, the reason behind the increase in dispersal and flight
initiation in CLas-infected psyllids was not investigated in that study. We believe that
the energetic stress and increased hunger level in CLas-infected psyllids could explain
the observed increase in their dispersal and flight initiation. Energetic stress and hunger
in N. ceranae-infected honeybees increased their foraging rates and consequently
resulted in the disappearance of honeybee colonies (31). Our EPG data (the significant
reduction in total nonprobing time, salivation time, and time from the last E2 to the end
of recording) indicated that CLas-infected psyllids were at a higher hunger level.
Enhancement of ATP and glutamate synthesis was also observed in Chlamydia
psittaci-infected HeLa cells (35). The increase in the expression of the glucose transporter (GLUT-1) on HeLa cells indicated that the stimulation of ATP was attributed to
enhancement of glucose consumption by infected cells (35). Enhanced synthesis of ATP
in infected cells could benefit both Chlamydia spp. and its host (35). In fact, C. psittaci
can also take up external ATP via ADP/ATP translocase, and it was concluded that C.
psittaci compensates for the energy load it imposes on infected cells by increasing the
production of ATP and other high-energy metabolites (35).
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The accumulation of ATP was accompanied by an increase in the gene expression
of ATP synthase subunits and a decrease in ATPase/GTPase enzyme activity. Our
comparative proteomic analyses of CLas-infected and healthy D. citri psyllids showed
that the amounts of ATP synthase ␣/-subunits were higher in CLas-infected psyllids (N.
Killiny, unpublished data). On the other hand, our proteomic analyses showed that the
level of V-ATPase-V1A was downregulated (unpublished data). In agreement with our
results, proteins involved in metabolism and cellular energy storage and utilization
were upregulated in CLas-infected D. citri psyllids (36). The acyl-coenzyme A (acyl-CoA)
dehydrogenase and enoyl-CoA hydrolase proteins, the enzymes catalyzing the first and
the second steps in fatty acid -oxidation and the production of acetyl-CoA, which
feeds into the citric acid cycle, were highly upregulated (2-fold and 12-fold, respectively) in CLas-infected D. citri psyllids (36). A number of enzymes involved in the citric
acid cycle were also upregulated in CLas-infected D. citri psyllids, including
2-oxoglutarate dehydrogenase, L-2-hydroxyglutarate dehydrogenase, phosphoglycerate mutase, succinate dehydrogenase, and succinate semialdehyde dehydrogenase
(36). In addition, glycerol kinase, which is involved in triglyceride breakdown, and
aldose-L-epimerase, which is involved in glycolysis, were upregulated in CLas-infected
D. citri psyllids (36). In addition, the comparative transcriptomic analysis of CLas
infection altered the expression of many genes involved in nutrient reservoir activity in
D. citri (37). The gene expression results indicated that CLas alters its host environment
to make the nutrients more available (37). Although our results showed that ATP
synthesis was increased and its consumption was decreased in CLas-infected D. citri
psyllids, the results indicated that the increased ATP synthesis may contribute more to
the accumulation of ATP level than the decrease in consumption. Since CLas encodes
ATP synthase, synthesis of ATP by CLas may also contribute to ATP accumulation in
D. citri.
Our current study showed that CLas shortened the life span of its insect vector by
exerting energy stress. By rearing CLas-infected psyllids on healthy plants, Pelz-Stelinski
and Killiny (38) showed that the life span of CLas-infected D. citri psyllids was less than
that of healthy psyllids. However, CLas infection also increased the fitness of adult
psyllids, resulting in increased egg laying and faster development time (38). PelzStelinski and Killiny (38) assumed that there was a physiological trade-off between
reproduction and life span and concluded that that CLas developed a relationship with
D. citri before it moved to plants. In contrast to CLas, “Ca. Liberibacter solanacearum”
reduces the fitness of its psyllid vector but does not affect its mortality index (6).
Although the genomes of “Ca. Liberibacter solanacearum” and CLas are similar, these
two pathogens are not identical at the molecular level (39). Recently, Gahnim et al. (39)
showed observed apoptotic responses in the midgut of CLas-infected psyllids. Although the cause of this apoptosis response was not fully understood, it was hypothesized that it served to limit the acquisition and transmission efficiency of CLas (39).
Better understanding of the interactions between CLas and its vector, D. citri, may help
in creating new approaches for controlling HLB.
MATERIALS AND METHODS
Diaphorina citri colonies. Colonies of the Asian citrus psyllid D. citri were maintained on ‘Valencia’
sweet orange, Citrus sinensis L. Osbeck, inside 400-mesh rearing and observation cages (BioQuip, Rancho
Dominguez, CA). Colonies were kept in temperature-controlled growth rooms set at 25 ⫾ 3°C, 60% ⫾ 5%
relative humidity (RH), and with a 16:8 (light/dark) photoperiod. Originally, insects were collected in 2000
from citrus groves in Polk City, FL. For CLas-infected colonies, CLas-infected ‘Valencia’ sweet orange trees
(symptomatic and PCR positive) were used to rear and maintain D. citri colonies. The infection rates were
assessed using conventional PCR, as described below. When the infection rate reached 80 to 90%, insects
were used for the survival analysis or collected and kept at ⫺80°C for gene expression, enzymatic activity
assays, and nucleotide analysis.
DNA extraction and conventional PCR for CLas. DNA from individual adult psyllids was extracted
using potassium acetate buffer and a TissueLyser II (Qiagen, Valencia, CA). Briefly, an individual psyllid
was placed in a 2-ml safe-lock microcentrifuge tube with a 5-mm-diameter stainless steel bead (Qiagen).
The tubes were immersed in liquid nitrogen for up to 5 min and then were placed into the TissueLyser
adaptor blocks (kept in the freezer) and fixed into the block clamps. Immediately, the samples were
processed for 30 s at 30 Hz. The vibration was repeated three times with rotation of the adaptor blocks.
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Eleven hundred microliters of extraction buffer (100 mM NaCl, 10 mM EDTA, 50 mM Tris [pH 9.0], 10 mM
dithiothreitol [DTT]) was added to each tube, and brief centrifugation at 5,000 rpm for 1 min was
performed to remove the debris. One milliliter of supernatant was recovered into a new 2-ml tube, and
20 l of 10% SDS was added. The mixture was incubated at 65°C in a water bath for 45 min; 500 l of
5 M potassium acetate was then added. After vortexing, the tubes were incubated in ice for 20 min and
then were subjected to centrifugation at 16,595 ⫻ g for 10 min at 4°C. One milliliter of supernatant was
recovered and mixed with 1 ml of ice-cold isopropanol. The tubes were centrifuged again at high speed
for 20 min at 4°C. After discarding the supernatant, 1 ml of ice-cold 70% ethanol was added to the pellet,
and the tubes were well vortexed and centrifuged for 10 min at 4°C. Supernatant was pipetted carefully
and discarded. DNA pellets were dried under a N2 stream and were resuspended in 20 l of RNase-free
water. The DNA concentration of each sample was measured using a NanoDrop ND1000 (Thermo
Scientific). Extracted DNA was used for conventional PCR amplification using 16S rRNA gene primers OI1
and I2C and Taq PCR master mix kit (Qiagen), as well as the protocol described by Jagoueix et al. (8).
ATP quantification by enzymatic assay. ATP was extracted using perchloric acid, as described by
Tomiya et al. (40), with slight modification. In brief, one psyllid was placed in a 1-ml tube with 50 l of
ice-cold 5% perchloric acid and crushed for 5 min in an ice bath using a Kontes mortar and pestle (Fisher
Scientific, Pittsburgh, PA). The samples were centrifuged at 10,621 ⫻ g for 10 min, and 4 l of the
supernatant was diluted to 200 l using ATP-free water. The ATP assay was performed using the Enliten
ATP kit (Promega, Madison, WI). Briefly, a 100-l aliquot of the diluted sample was mixed with 100 l of
luciferase reagent, provided with the kit, in 12 by 75-mm polypropylene test tubes (Fisher Scientific), and
the intensity of the emitted light was measured for 10 s using an Optocomp I luminometer (MGM
Instruments). A set of ATP standards (1 ⫻ 10⫺8 to 1 ⫻ 10⫺12 M) were also prepared in 0.1% perchloric
acid to correct for possible inhibition of light output and were used to construct the standard curve. A
“blank” containing 100 l of luciferase reagent and 100 l of 0.1% perchloric acid was run in the assay
to determine the amount of background relative luminescence units (RLU) to subtract from the sample
RLU. Fifty healthy and 50 CLas-infected psyllids were analyzed, and each sample was measured in
duplicate.
Bacterial cell quantification by real-time PCR for CLas. We carried out real-time quantitative PCR
(RT-qPCR) in order to correlate the quantity of ATP and determine the cycle threshold (CT) values, which
reflect the bacterial population (CLas) within the insect. The DNA extraction was performed on the same
perchloric acid extract used for ATP quantification. A 45-l aliquot of the perchloric acid extract was
diluted to 1,100 l with extraction buffer (100 mM NaCl, 10 mM EDTA, 50 mM Tris [pH 9.0], 10 mM DTT),
and the samples were centrifuged at 5,000 rpm for 1 min to remove the debris. A 20-l aliquot of 10%
SDS was added to the recovered supernatant (1 ml), and the DNA was extracted as described above.
Extracted DNA was used for RT-qPCR amplification using 16S rRNA primers HLBasf and HLBr, the probe
HLBp, TaqMan PCR master mix, SYBR green PCR master mix, and the protocol described by Li et al. (41)
and Zhao et al. (42). Amplifications were performed in a 7500 RT-qPCR system, and supplies were from
Applied Biosystems (Foster City, CA).
Total ATPase and GTPase activity. Five adult psyllids were placed in a 1-ml tube, 100 l of Tris
buffer (10 mM Tris-HCl, 150 mM NaCl [pH 7.4]) was added, and the psyllids were crushed for 2 min on
ice, as described above. The samples were centrifuged at 10,621 ⫻ g for 5 min, and the supernatant was
further filtered through a 10,000-molecular-weight-cutoff membrane (Millipore, Bedford, MA). Another
100 l of Tris buffer was added to the retentate, and the sample was centrifuged again to wash off the
phosphate. The washing step was repeated, and the retentate was recovered in 50 l of Tris buffer. An
aliquot of 20 l of the final sample was mixed with 20 l of assay buffer and 10 l of 4 mM ATP standard
provided with the QuantiChrom ATPase/GTPase assay kit (Bioassay Systems, Hayward, CA) and placed in
96-well plate. The reaction mixture was incubated for 30 min at room temperature. A blank containing
a 20 l of the enzyme extract was also mixed with 20 l of assay buffer and 10 l of Tris buffer and was
run in the assay to determine the amount of phosphate background to be subtracted from the sample.
At the end of the reaction time, 200 l of malachite green reagent was added to the reaction mixture
and incubated for 30 min to terminate the enzyme reaction and generate the dark green color by
reacting with free phosphate produced by ATPase/GTPase. The intensity of the stable dark green color
was measured at 639 nm using a Synergy HT multimode microplate reader (Winooski, VT). The standard
curve was constructed by incubating 50 l of phosphate standard (0, 15, 30, and 50 mM) with 200 l of
malachite green reagent for 30 min and measured as mentioned above. All samples and standards were
run in duplicate. Twenty-five healthy and 25 CLas-infected samples were analyzed, and each sample
represented 5 psyllids. ATPase/GTPase activity was calculated according to the following formula:
enzyme activity (in units per liter) ⫽ ([Pi] ⫻ [RV])/([EV] ⫻ [t]), where Pi is the free phosphate produced
from ATP and calculated from the standard curve (in micromolar concentration), RV is the reaction
volume (50 l), EV is the enzyme volume used in the assay (20 l), and t is the reaction time (30 min).
According to the ATPase/GTPase kit’s manufacturer, one unit of activity is defined as the amount of
enzyme that catalyzes the production of 1 M free phosphate per minute under the assay conditions.
Nucleotides and sugar nucleotide extraction from Asian citrus psyllid for HPLC analysis.
Nucleotides and sugar nucleotides were extracted using perchloric acid, as described by Tomiya et al.
(40), with slight modification. In brief, 60 Asian citrus psyllid adults were mixed with 200 l of ice-cold
5% perchloric acid and were crushed for 5 min in an ice bath using Kontes pestle. The samples were
neutralized with potassium hydroxide and centrifuged at 10,621 ⫻ g for 10 min. The samples were
centrifuged again, and the supernatants were filtered through 10,000-molecular-weight-cutoff membranes. After adjusting to pH 7, the samples were centrifuged again, and the supernatant was kept at
⫺20°C until analysis. Five replicates of each treatment were performed.
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Nucleotide analysis by ion-pair reverse phase-HPLC. High-performance anion-exchange chromatography (HPAEC) was carried out using an Agilent 1200 series. HPLC was coupled to a diode array
detector (HPLC-DAD) and a CarboPac PA100 column (Dionex, Sunnyvale, CA).
The following solvents were used as eluents: 1 mM sodium hydroxide (eluent 1 [E1]) and 1 M sodium
acetate in 1 mM sodium hydroxide (eluent 2 [E2]). An aliquot of the D. citri extract (25 l) or a standard
mixture was injected into the column equilibrated with a mixture (80:20 [vol/vol]) of E1 and E2. Elution
was performed using E1 and E2, as described by Tomiya et al. (40) with slight modification, using the
following gradient: elution time point T0 ⫽ 20% (vol/vol) E2, T10 ⫽ 20% (vol/vol) E2, T25 ⫽ 30% (vol/vol)
E2, T35 ⫽ 40% (vol/vol) E2, T40 ⫽ 50% (vol/vol) E2, T45 ⫽ 60% (vol/vol) E2, T50 ⫽ 70% (vol/vol) E2,
T55 ⫽ 70% (vol/vol) E2, T65 ⫽ 20% (vol/vol) E2, and T70 ⫽ 20% (vol/vol) E2. The flow rate for HPLC
elution was 1 ml/min, and the column was kept at 30°C. Nucleotides and sugar nucleotides were
detected by absorbance at 260 nm. Nucleotides and sugar nucleotides were identified by matching their
retention times and UV-visible light spectra with the known standards. All of the nucleotides were
quantified relative to their standards, and the unknown compounds were quantified relative to AMP. The
adenylate energy charge was calculated using the following equation: AEC ⫽ ([ATP] ⫹ 1/2[ADP])/([ATP] ⫹
[ADP] ⫹ [AMP]) (43).
Survival assay. A 3-mm-thick blot paper (Bio-Rad, Hercules, CA) was placed in the bottom of each
Corning Snap-Seal no. 1730 polypropylene container (height, 112 mm; outer diameter, 63 mm) (Fisher
Scientific). The filter paper was saturated with 5 ml of 20% sucrose. Ten adult psyllids were introduced
into each container, and the container was closed with clear plastic wrap. The psyllids were kept in
temperature-controlled growth rooms (25 ⫾ 3°C, 60% ⫾ 5% RH, and a 16:8 [light/dark] photoperiod). The
numbers of dead insects were recorded daily. The experiment contained five replicates each of healthy
and CLas-infected psyllids. The experiment was repeated three times.
EPG. We used EPG methods to monitor D. citri feeding using two 4-channel monitors (described
below). Each day, four psyllids from a noninfected (healthy) colony and four psyllids from a CLas-infected
colony were monitored. After EPG, the four psyllids from the infected colony were tested for “Candidatus
Liberibacter asiaticus” (CLas) using a 16S rRNA gene probe and primers (41). Only EPG recordings from
psyllids that tested positive were used for the positive data, and only EPG recordings from the healthy
colony were used for the CLas-negative data. The recordings from psyllids that tested negative for CLas
(from the infected colony) were not analyzed. There were 28 recordings from healthy psyllids and 25
recordings from CLas-infected psyllids.
The equipment consisted of two alternating current/direct current (AC/DC) monitors (44), custombuilt by William H. Bennett (EPG Equipment Co., Otterville, MO), operating in DC mode with 150 mV
substrate voltage and 160⫻ amplification. Data acquisition was through a DI710 AD converter (Dataq).
A 2-cm-long, 24.5-m-diameter gold wire was attached to thoracic tergites of D. citri using silver glue
(1:1:1 [wt/wt/wt] white glue-water-silver flake [8 to 10 m; Inframat Advanced Materials]). The other end
of the gold wire was attached to a 23-mm-long, 0.48-mm-diameter copper wire using the same glue. This
wire was soldered to a 20-mm-long, 1.14-mm-diameter brass nail that was inserted into the unit’s head
amp that was set to a resistance of 109 ⍀. Recordings were 24-h long. Midsweet orange scion on
Kuharsky rootstock trees were obtained as resets from a commercial nursery, repotted, pruned to a
height of 51 cm from the soil surface, and kept in a greenhouse in 3.92-liter black plastic pots measuring
18 cm at the rim and 18 cm deep filled with Fafard professional custom mix soil. Greenhouse lighting was
supplemented using high-pressure sodium lighting to give 16 h:8 h photophase/scotophase. Plants were
moved indoors to a Faraday cage constructed of a pure copper screen (0.15-mm-diameter wire, with 1
wire every 1.58 mm) attached to an aluminum frame. Illumination was provided by fluorescent bulbs,
with room temperature maintained at 26.6°C. Humidity was not controlled. A psyllid colony was
maintained in the same greenhouse using the same cultivar. All psyllids originated from a long-term
colony that has been used in previous research (18, 45). The colony used in these tests required periodic
infusions of psyllids from the original colony, but at least 1 week elapsed between colony augmentation
and the removal of psyllids for experiments.
Gene expression analysis. The effects of CLas infection on psyllid genes, including ATP synthase ␣and -subunits mitochondrion-like (ATP synthase A and B), V-type proton ATPase catalytic subunit A
(V-ATPase-V1a), the transitional endoplasmic reticulum ATPase (TER94), nucleotide diphosphate kinase
(NDPK), and AMP-activated protein kinase ␣-subunit (AMPK-A) genes in D. citri adults, were evaluated
using RT-qPCR as described by El-Shesheny et al. (23). The actin gene was used as a reference
(endogenous gene) for comparing the relative levels of gene expression among treatments (46). Table
3 contains the primers used for gene expression.
Statistical analysis. Data were analyzed using JMP version 9.0 (SAS Institute, Inc.). Survival analysis
was carried out using the Kaplan-Meier method. P values of log-rank tests were used for comparisons
among the survival curves. A two-tailed t test was used to compare ATP levels, levels of ATPase activity,
relative gene expression levels, and the mean concentrations of nucleotides and sugar nucleotides
between healthy and CLas-infected D. citri colonies.
EPG results were analyzed using an SAS program that mimics the output of the Sarria Workbook, and
25 additional variables were added that focus on xylem feeding and the D waveform (Ebert 2.0
[http://www.crec.ifas.ufl.edu/extension/epg/sas.shtml]). The D waveform indicates that a psyllid has
made contact with the phloem. There were 84 variables examined in total. These values were then
transformed to mitigate issues with departures from normality. Durations were log transformed, counts
were square root transformed, and percentages were arcsine square root transformed. Data were
analyzed by mixed-model analysis of variance (ANOVA) using restricted maximum likelihood estimation
(REML) (Proc Glimmix; SAS Institute, 2001).
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TABLE 3 Primers used in gene expression for the selected genes in current study
Gene
hsp70
Accession no.
XM_008482897
ATP synthase A
XP_008482039
V-ATPase-V1A
XP_008470205
TER-94
XM_017447919.1
NDPK (awd)
ABG81980.1
AMPK
XM_008486820.2
Actin
XP-008468690
Primer sequence
CGGTTATTACTGTCCCCGC
TTGAATCACCCCCAACAGAT
GGTATTCGTCCCGCTATCAA
GGCAGATCCTACACGGGATA
CGAACTGGTACGAGTGGGAT
GGATACCAGGACCAAGCTCA
TGGAAACGGAAGACGAAGAC
CCACCGGATTGACTCTGATT
AGAGGACTTGTGGGAAACATC
TGACAAGACCAGGGAAGAAAG
CCCCTAGTACAGGCAAACCA
TGGAGAAGGACGAGGAGAGA
CCCTGGACTTTGAACAGGAA
CTCGTGGATACCGCAAGATT
Reference or source
This study
This study
This study
This study
El-Shesheny et al. (23)
This study
Tiwari et al. 2011 (46)
The new variables that were added to the Sarria Workbook involving phloem contact were as follows:
number of probes to first D, number of D, total duration of D, duration of nonprobing before first D,
mean duration of D, average number of D per probe, time from first probe to first D, time from start of
probe with first D to first D, number of probes after first D, number of probes ⬍3 min after first D, number
of D ⬎10 min, time to first D ⬎10 min, duration of longest D, and percent probing spent in D. The same
set of variables was also calculated for G (xylem sap feeding), but three variables overlap existing Sarria
variables. All EPG waveforms were interpreted by an experienced practitioner of EPG.
ACKNOWLEDGMENTS
We acknowledge the assistance of S. Jones and L. Lindsey with the psyllid colonies.
This work was supported by a grant from the Citrus Research and Development
Foundation, Lake Alfred, FL, USA (grant 769-14).
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Applied and Environmental Microbiology
ERRATUM
crossm
Erratum for Killiny et al., “A Plant
Bacterial Pathogen Manipulates Its
Insect Vector’s Energy Metabolism”
Nabil Killiny,a Faraj Hijaz,a Timothy A. Ebert,b Michael E. Rogersb
Plant Pathology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, Florida,
USAa; Entomology and Nematology Department, Citrus Research and Education Center, University of Florida,
Lake Alfred, Florida, USAb
Volume 83, no. 5, e03005-16, 2017, https://doi.org/10.1128/AEM.03005-16. Page 14:
Reference 39 should read as follows.
39. Ghanim M, Fattah-Hosseini S, Levy A, Cilia
M. 2016. Morphological abnormalities and cell
death in the Asian citrus psyllid (Diaphorina
citri) midgut associated with Candidatus Liberibacter asiaticus. Sci Rep 6:33418. https://
doi.org/10.1038/srep33418.
June 2017 Volume 83 Issue 12 e00910-17
Applied and Environmental Microbiology
Citation Killiny N, Hijaz F, Ebert TA, Rogers ME.
2017. Erratum for Killiny et al., “A plant bacterial
pathogen manipulates its insect vector's
energy metabolism.” Appl Environ Microbiol
83:e00910-17. https://doi.org/10.1128/AEM
.00910-17.
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Microbiology. All Rights Reserved.
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