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OPEN
Received: 19 July 2018
Accepted: 11 January 2019
Published: xx xx xxxx
Laser-induced breakdown
spectroscopy (LIBS) as a novel
technique for detecting bacterial
infection in insects
Nabil Killiny1, Ed Etxeberria1, Alejandro Ponce Flores2, Pedro Gonzalez Blanco1,
Teresa Flores Reyes1,3 & Luis Ponce Cabrera1,3
To prevent the spread of diseases in humans, animals or plants, determining whether potential
vectors are infected is crucial. For example, early detection of the citrus disease Huanglongbing,
which has been a scourge on the citrus industries around the world, is a critical need. This vectorborne disease is transmitted by Diaphorina citri, the Asian citrus psyllid, which carries the putative
bacterial phytopathogen, Candidatus Liberibacter asiaticus (CLas). In this investigation, we introduced
Laser-Induced Breakdown Spectroscopy (LIBS) to reveal key biochemical differences between CLasinfected and non-infected psyllids. The emission spectra captured from laser ablation of CLas-infected
and healthy psyllids were processed through the principal component analysis (PCA) method and
compared. Thirteen peaks from seven different elements were detected in D. citri. The t-test showed
that CLas-infected D. citri were deficients in zinc, iron, copper, magnesium, calcium, and nitrogen.
The PCA showed that LIBS can successfully differentiate between CLas-infected and healthy D. citri by
comparing their elemental profile. In this work, we demonstrated a method that allows for a fast and
precise compositional microanalysis of an insect vector which can contribute to the early detection of
citrus huanglongbing
Huanglongbing (HLB), or citrus greening disease, has been a scourge on citrus industries around the world.
The vector-borne disease is transmitted by two species of psyllids (Hemiptera: Liviidae): the Asian citrus psyllid, Diaphorina citri, and the African psyllid, Trioza erytrea1,2. Like other Hemiptera, psyllids are phloem sap
feeders. These two species feed and reproduce on all members of the family Rutacea, which includes all citrus
species of commercial importance. During the feeding process, the psyllid transmits the putative phytopathogen
Candidatus Liberibacter asiaticus (CLas), a Gram-negative alpha-proteobacterium3,4. Currently, CLas remains
unculturable complicating research efforts. In addition to CLas, psyllids host several other bacterial endosymbionts in their gut5.
In Florida, HLB is widespread, already affecting almost 100% of citrus groves6–8. However, in states such
as Texas, Arizona, California, Mediterranean area and Australia, disease presence is much lower, making early
detection a priority. For several reasons, detecting HLB in citrus trees is difficult at best. First, there is a six- to
nine-month asymptomatic period after inoculation9,10. During this time, the trees appear healthy, whilst feeding
psyllids can acquire and spread the bacterium from tree to tree. Foliar symptoms include blotchy mottling (uneven distribution of chlorophyll in the leaves), vein corking, starch accumulation, and leaf chlorosis, which resembles zinc deficiency11–13. Symptoms in fruit include small size, lopsided fruit, aborted seeds, bitter off-flavors, early
fruit drop, and uneven coloration during ripening. Once trees are heavily symptomatic, tree death usually occurs
within five years, depending on their variety and tolerance to HLB. Second, some citrus cultivars are more tolerant to HLB than others14–16. In Florida, sweet oranges and grapefruits, which make up the majority of production,
are particularly sensitive to HLB, while those of mandarin heritage are slightly more tolerant. Finally, the bacterial
1
University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL, USA.
Universidad Nacional Autonoma de Mexico, Fac. De Ciencias, Universidad 3000, Circuito Exterior S/N, Distrito
Federal, 04510, Mexico. 3Instituto Politecnico Nacional, CICATA, Carretera Tampico-Puerto Industrial Altamira Km
14.5, Industrial Altamira, 89600, Altamira, Tampico, Mexico. Correspondence and requests for materials should be
addressed to N.K. (email: nabilkilliny@ufl.edu)
2
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titer (a measurement of bacterial concentration) is not uniform within the tree, so a random sampling of leaves
using conventional or quantitative polymerase chain reaction (PCR) methods may not reveal a presence of the
bacterium17. Furthermore, low titers of CLas may not be detected by conventional PCR.
In uninfected citrus growing areas, the presence of psyllids is reported well before visual symptoms of HLB
appear in trees. The primary method of psyllid control thus far has been insecticide application, but increased use
of pesticides has resulted in resistance in some populations of D. citri18. Other methods to control D. citri include
releasing the parasitic wasp Tamarixia radiata19 and using RNAi approaches20–22, although the latter has not been
approved for commercial purposes. In HLB-affected areas, many growers have replaced dead or dying trees to
reduce sources of inoculum. However, psyllids are still present in Florida, and they continue to actively transmit
HLB. Early detection of CLas remains a critical goal and would be advantageous to both researchers and growers.
Many PCR-based methods have been developed for detecting CLas in psyllids23,24. However, developing an effective, accurate, and inexpensive method is required to enable earlier disease detection. In areas where Asian citrus
psyllid exists but the symptoms have not yet appeared on citrus trees, early detection of CLas-infected psyllids
would be valuable.
Laser-induced breakdown spectroscopy (LIBS) technique offers many advantages for elements analysis25–27.
It has gained a great popularity in elemental analysis because of its portability, lightning speed, low cost, nonrequirement for chemicals, minimal or no sample preparation, simultaneous determination of multiple elements,
and capability to perform express identification25–27. The technique involves short, high-intensity laser pulses
capable of ablating a small amount of material, thereby creating a momentary plasma. An optical fiber collects a
portion of the light emitted from the plasma and delivers it to a spectrometer. The captured spectra are considered
a “fingerprint” associated with a sample’s elemental composition.
In the last few years, LIBS has been used to study the effects of CLas infection on the nutritional composition
of citrus plants28,29. It was recently demonstrated that LIBS can successfully differentiate between CLas-infected
and healthy citrus plants by analyzing the major macro- and micronutrients28. LIBS analysis showed that CLas
significantly decreased the level of calcium, magnesium, and potassium in citrus plants29. Recently, it has also
been shown that combination of LIBS and Raman spectroscopy significantly improves discrimination and classification of bacterial species and strains30.
In this work, we introduce the LIBS technique for composition microanalysis of D. citri, the vector of citrus
huanglongbing. LIBS can reveal the biochemical differences between CLas-infected and non-infected Asian citrus
psyllids for immediate detection of the pathogen. To our knowledge, this is the first time that a LIBS technique has
been directly applied to differentiate between pathogen-infected and pathogen-free vectors.
Material and Methods
Asian citrus psyllid colonies. D. citri colonies were continuously reared at the Citrus Research and
Education Center, University of Florida (CREC-IFAS, UF, Lake Alfred, United States). Healthy psyllids were
maintained on CLas-free alemow trees (Citrus macrophylla) in a USDA-APHIS/CDC-approved secured growth
room (27 ± 1 °C, 65 ± 2% relative humidity, L16:D8 h photocycle). Monthly, random samples of D. citri adults and
citrus leaves were collected and tested using polymerase chain reaction (PCR) assay as previously described31 to
confirm that the plants remained CLas-free and the insects did not harbor CLas. CLas-infected psyllids colonies
were reared on HLB-symptomatic and PCR-positive/CLas-infected C. macrophylla plants and maintained in the
same conditions as described above. The CLas-infection rate was tested simultaneously (50 adult individuals per
monthly sampling). CLas-infected and uninfected D. citri colonies were maintained in separate, USDA-APHIS/
CDC-approved secured growth rooms to minimize the chance of cross-contamination. Mature adults (2–3 mm)
were collected using an aspirator for LIBS assays.
Laser-induced breakdown spectroscopy.
We developed a compositional microanalysis procedure that
allows us to obtain the elemental emission spectrum of an insect vector. For the analysis, we used a LIBS instrument, “SLIT-LIBS,” supplied by Onteko LLC (Tampa, United States), which includes a laser that emits in a “burst
mode” regime described below. In this device, the laser beam is coupled with the optical path of a slit-lamp microscope for better visualization of samples.
A schematic representation for this setup is shown in Fig. 1. The pulsed (neodymium: yttrium aluminum garnet) Nd:YAG laser emits at a wavelength of 1,064 nm while working in a Q-switch regime, producing light pulses
(shots) with energy of up to 40 mJ at a repetition rate of 1 Hz. A low-power red laser was used to point where the
Nd:YAG laser would impact and ablate the sample and generate the plasma. Each laser shot consisted of a train
of three micropulses, each having a duration of 8 ns and an interval of 10–25 µs between them, resulting in an
overall shot duration of about 70–80 µs. The laser beam was focused using a 50 mm focal length lens which produced a 40 µm diameter target on the samples. The laser ablation process induced the emission of light which was
collected by an optical fiber and delivered to a cross Czerny–Turner spectrometer with a linear CCD as a detector.
The spectral resolution of the system is 0.3 nm with a spectral range of 250–800 nm.
Spectrum recording, data processing, and statistical analysis.
We used adult Asian citrus psyllids
from healthy (22 insects) and CLas-infected colonies (38 insects), the latter with an infection rate of 60% for LIBS
detection of HLB. After spectra measurements, PCR was performed on all sampled psyllids as described previously31. PCR negatives from infected colonies were excluded from the statistical analysis. Consequently, only 22
CLas-infected psyllids were included in the statistical analysis. To obtain the spectra, psyllids were fixed with a
double-sided-tape strip to a microscope slide held on a stand. Because CLas grows and multiplies in the insect’s
haemolymph, the first laser pulse was required to perforate the exoskeleton in order to gain access to the interior
of the psyllid. When the hemolymph exuded from the psyllid, a second laser pulse was delivered to ablate the
exposed hemolymph and capture it’s spectrum. In total, four shots were performed on each insect: the first one to
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Figure 1. Schematic representation of laser-induced breakdown spectroscopy system used in this study. A
stereomicroscope was modified and attached to the laser source to point laser beams on the abdomen of Asian
citrus psyllid.
Element or compound
Wavelength (nm)
Mg I
279.5
Fe II
298.9
Zn I
334.6
CN band
386.1–388.3
Cu I
406.2
Ca I
435.3
Ca I
462.1
NI
499.9
C-C
516.2
CaO band
547–55644
Fe I
566.3
CaOH
610.244
HI
656.2
Table 1. Spectra lines detected and identified in the haemolymph of D. citri using LIBS.
puncture the exoskeleton and release the haemolymph, and the others to calculate the average. The spectrometer
was connected to a computer and spectra were stored using the SpectraSuite software (Ocean Optics, Tampa,
United States). The average spectra of each sample was analyzed independently using the elemental database of
the National Institute of Standards and Technology (NIST)32 and the LIBS Army elemental database33. The data
were normalized by dividing the intensity of individual emission line by the total intensity of the total spectrum
(i.e., the sum of the thirteen intensities). Statistical analyses were performed using JMP version 9.0 (SAS Institute
Inc.). Principal component analysis (PCA) was performed using normalized data captured from the thirteen
spectral lines. In addition, t-test (p < 0.05) was used to compare the level of each spectral line (normalized intensity) in CLas-infected D. citri with that of the controls. The PCA was repeated using only five of the captured
spectral lines (Mg I, N II, CaO, Fe I, and CaOH), which were dramatically affected by CLas infection as shown
by the t-test.
Results
Thirteen peaks representing seven different elements were identified in the haemolymph of D. citri using LIBS
(Table 1).
A typical spectrum of healthy (blue) and CLas-infected (red) D. citri obtained with the LIBS after normalization is shown in Fig. 2. The wavelength of each detected peak is displayed in Fig. 2. One peak was identified for
magnesium (279.5 nm), two peaks for iron (298.9, and 566.3 nm), one peak for zinc (334.6 nm), one for copper
(406.2 nm), two for calcium (435.3, 462.1 nm), two for calcium-related compounds (CaO: 547–556 nm, CaOH:
610.2 nm), two for nitrogen (386.1–388.3 and 499.9 nm) or nitrogen-related compounds, and one for hydrogen
(656.2 nm). Only CLas-infected psyllids which were confirmed positive using PCR were included in the data analysis. In the same manner, all control psyllids were also confirmed negative by PCR. In general, the peak intensity
of most of the detected peaks was lower in CLas-infected D. citri Fig. 2. These included N II, CaO, Zn I, CaOH, Fe
II, Ca I, and Cu I. The differences between the healthy and CLas-infected spectra are also shown in yellow (Fig. 2).
LIBS successfully differentiated between CLas-infected and healthy D. citri.
The principal
component analysis (PCA) generated using the normalized intensity of the thirteen detected peaks is shown in
Fig. 3A,B. PC1 and PC2 accounted about 80% of the variation (Fig. 3A). As shown in the score plot (Fig. 3A), the
CLas-infected D. citri were separated from the healthy D. citri, indicating that their elemental profile was different
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Figure 2. Typical spectra of healthy and CLas-infected Asian citrus psyllid (PCR-positive). Yellow spectrum is a
subtraction between healthy and CLas-infected psyllids spectra.
from that of healthy psyllids. The CLas-infected D. citri clustered in the left side of the score plot, whereas the
healthy psyllids clustered to the right of the plot. The loading plot (Fig. 3B) showed that most of the detected
peaks were higher in the control psyllids (first and fourth quadrants). The loading plot also showed that H I
(656.2 nm), and Mg I (279.5 nm), were not important in the model because they lay in between the two groups
(Fig. 3B).
The Student’s t-test CLas-infected D. citri adults were deficient in most detected elements. When the intensities of the detected peaks were compared between the CLas-infected and healthy D.
citri we found many differences. The C-C I (279.5 nm) peak was not significantly different (P > 0.9430) between
the CLas-infected and healthy psyllids (Fig. 3C). The intensities of Fe II (at 298.9 nm), Zn I, C-N, and H I peak in
CLas-infected psyllids shows small differences between healthy and CLas-infected psyllids (Fig. 3C). However, the
intensity of Fe I (566.3 nm), Cu I, Ca I (435.3 nm), Ca I (462.1 nm), N II, CaO band (547–556), and CaOH (610.2)
peaks in CLas-infected psyllids were dramatically lower (P < 0.0000) than those of healthy psyllids (Fig. 3C).
These results indicated that Fe I (566.3 nm), Ca I (435.3 nm), Ca I (462.1 nm), N II (499.9), C-C (516.2), CaO
band (547–556), and CaOH (610.2) peaks were the best markers for differentiation between the CLas-infected
and healthy psyllids. Furthermore, these results suggested that CLas-infected D. citri were deficient in zinc, iron,
copper, magnesium, calcium, and nitrogen.
Filtering the data showed that only five peaks were necessary to discriminate between healthy
and CLas-infected psyllids. Using the results from Fig. 3C, we refined the PCA model by using only five
peaks (N II, Mg I, CaO, Fe I, and CaOH) (Fig. 3D,E). The scatter plot generated using these five peaks showed
better separation between CLas-infected and healthy psyllids (Fig. 3D), indicating that the eliminated peaks
were not significant for the model. The CLas-infected psyllids clustered together in the left of the plot and were
totally separated from the controls, which clustered together in the right side of the score plot (Fig. 3D). In
this analysis, PC1 and PC2 accounted for about 96% of the variation (Fig. 3E). In agreement with the Student’s
t-test, all five selected peaks (N II, C-C, CaO, Fe I, and CaOH) were significantly lower in CLas-infected psyllids
(Fig. 3E). These selected peaks correlated with the control psyllids and appeared on the right side of the loading
plot (Fig. 3E).
Discussion
Evaluation of the classification model.
Thirteen peaks from seven different elements were detected in
healthy and CLas-infected D. citri. We decided to implement PCA because it can efficiently identify outliers and
has been successfully used for classification of LIBS data34. The PCA generated using all of the detected peaks
showed the existence of two main clusters (healthy and CLas-infected D. citri). This result indicated that LIBS can
be used to differentiate between CLas-infected and healthy psyllids. Previous studies on D. citri showed that CLas
infection can produce a large number of nutritional changes in its host insect35–38.
It has also been shown that LIBS can be successfully used to differentiate between HLB-symptomatic and
healthy citrus leaves29, but no clear separation was observed. Unfortunately, no PCR was performed in the previous study to confirm the presence of the CLas titer in the leaves. Twenty-nine peaks from nine different elements
were identified in citrus leaves, however only thirteen peaks were found to useful for the multivariate analysis29.
Because some elements such as hydrogen, oxygen, and nitrogen exist at high background levels in the ambient
atmosphere, these elements were hard to measure in citrus as the LIBS was conducted at ambient conditions29. In
a similar study, LIBS was also used to differentiate between CLas-infected and healthy citrus plants by analyzing
the major macro- and micronutrients28. Analysis of the LIBS using soft independent modeling of class analogy
(SIMCA) data was able to detect CLas-infected plants from the first month28.
The t-test showed that most of the differences between CLas-infected and healthy psyllids were observed
in the following peaks: N II, CaO, Mg1, CaOH, and Fe I. Consequently, we eliminated the rest of the peaks
and refined the PCA analysis using these five peaks which significantly improved the separation between the
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Figure 3. Differentiation between healthy and CLas-infected Asian citrus psyllid using elements identified
by LIBS. (A) Principal component analysis of all identified elements (n = 22). (B) PCA-loading-plot for all
identified elements. (C) Signal intensity of all elements identified by LIBS in healthy and CLas-infected Asian
citrus psyllid. (D) Principal component analysis of five significant identified elements (n = 22). (E) PCAloading-plot for five significant identified elements identified elements. Bars represent standard errors. P-values
were calculated using the Student’s t-test.
CLas-infected and healthy psyllids. This result showed that the reduced PCA model (5 peaks from four elements)
can be successfully used to differentiate between CLas-infected and healthy psyllids. The reduction in the number
of wavelengths required for the efficient classification of CLas-infected and healthy insects makes it possible to
create a portable and low-cost instrument which does not require a spectrometer. This portable detector could be
built using just a few selective filters.
Nutritional Changes in CLas-infected D. citri.
The LIBS compositional analysis showed that
CLas-infected adult psyllids were low in iron, zinc, copper, magnesium, nitrogen, and calcium, indicating that
CLas-infected psyllids were under nutritional stress. Previous studies on citrus plants showed that CLas infection
can produce a large number of nutritional changes. Zinc, magnesium, iron, nitrogen, and phosphorus were lower
in CLas-infected plants compared to healthy plants39. LIBS also showed that CLas-infected citrus plants were also
deficient in magnesium, potassium, calcium, copper, silicon, sodium, and titanium29. In addition, aluminum,
silicon, titanium, manganese, nickel, copper, zinc, rubidium, strontium, and zirconium were also present at low
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levels in CLas-infected ‘pineapple’ sweet orange juice40. It was also reported that CLas-infected citrus trees were
deficient in phosphorus, indicating that phosphorus could be required for the growth of CLas41. The previous
results together indicated that CLas could acquires these elements from its host. In fact, many researchers believe
that CLas pathogenicity is due to nutrient depletion and energy parasitism42. Many elements such as iron, zinc,
copper, and manganese could be essential for the growth of CLas because they act as cofactors for various essential enzymes. The presence of the znuABC genes, which are responsible for the import of zinc, indicated that zinc
was an essential element for CLas43. They suggested that the uptake of zinc by CLas from its host plant results in
zinc deficiency. The reduction in micro and macronutrients in CLas-infected citrus may also result from root
damage. It is believed that plugging of the phloem by CLas could stop the circulation of the phloem sap from the
leaves (source) to the roots (sink), which may compromise root function, decrease root mass, lower the ability to
absorb water and minerals, and ultimately lead to tree death29.
This study showed that LIBS can successfully measure various elements in the haemolymph of small insects
like D. citri. This method is fast (less than 5 min/insect) and does not require sample preparation as in the case of
inductively coupled plasma optical emission spectroscopy (ICP-OES). In addition, our results showed that LIBS
can be performed on small samples (~0.01–1 µL) and enables simultaneous multi-elemental analysis.
Conclusion
Herein we demonstrated that LIBS enables a fast, objective, and reliable diagnosis of the CLas pathogen in D. citri
psyllids. The PCA analysis showed that LIBS could be successfully used to differentiate between CLas-infected
and healthy D. citri. In addition, the t-test applied on the LIBS spectra provided insights about the elemental
changes in CLas-infected psyllids. Considering the great separation between the CLas-infected and healthy psyllids, our results suggested that LIBS could be used for rapid screening of CLas in D. citri. Finally, LIBS could also
be extended to study other plant insect-borne diseases such as citrus tristeza virus and potato zebra chip disease
as well as human insect-borne diseases such as zika and malaria.
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Acknowledgements
We thank our labs’ members for the helpful discussion and technical assistance. We acknowledge Shelley E. Jones,
Lorraine Jones, and Floyd Butz for maintaining insect colonies. Teresa Flores thanks CONACYT for the financial
support during her sabbatical year.
Author Contributions
N.K., E.E. and L.P.C. conceived the experiments. N.K., A.P.F., T.F.R. and L.P.C. conducted the experiments. N.K.,
A.P.F., P.G.B., T.F.R. and L.P.C. analyzed the results. N.K. and L.P.C. wrote the manuscript with input from all
other authors.
Additional Information
Competing Interests: The authors declare no competing interests.
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