Introduction
Chagas disease, or American trypanosomiasis, affects millions of people, particularly in the Americas (de Fuentes-Vicente et al., Reference de Fuentes-Vicente, Gutiérrez-Cabrera, Flores-Villegas, Lowenberger, Benelli, Salazar-Schettino and Córdoba-Aguilar2018; Dye-Braumuller et al., Reference Dye-Braumuller, Gorchakov, Gunter, Nielsen, Roachell, Wheless, Debboun, Murray and Nolan2019). This disease is caused by the parasite Trypanosoma cruzi, with hematophagous triatomines, commonly known as kissing bugs, serving as its vectors (Cruz-López et al., Reference Cruz-López, Malo, Rojas and Morgan2001). These insects seek refuge in the cracks and fissures of human dwellings or animal shelters such as chicken coops or corrals. Most triatomines are nocturnally active, feeding on their hosts and spending daylight hours in these refuges (Taneja and Guerin, Reference Taneja and Guerin1997). Three triatomine species recognized as key vectors of T. cruzi are Triatoma pallidipennis, T. infestans, and Rhodnius prolixus (Ramsey et al., Reference Ramsey, Townsend Peterson, Carmona-Castro, Moo-Llanes, Nakazawa, Butrick, Tun-Ku, de la Cruz-Félix and Ibarra-Cerdeña2015; de Fuentes-Vicente et al., Reference de Fuentes-Vicente, Gutiérrez-Cabrera, Flores-Villegas, Lowenberger, Benelli, Salazar-Schettino and Córdoba-Aguilar2018).
Limiting physical interaction between triatomines and humans represents a key strategy for Chagas disease control, and one specific mechanism involves the use of lures to attract the insects into traps (Rojas de Arias et al., Reference Rojas de Arias, Abad-Franch, Acosta, López, González, Zerba, Tarelli and Masuh2012; Cardozo et al., Reference Cardozo, Fiad, Crocco and Gorla2020). This concept is not novel: chemically-baited traps are extensively employed against hematophagous insects (Ryelandt et al., Reference Ryelandt, Noireau and Lazzari2011; Mota et al., Reference Mota, Vitta, Lorenzo-Figueiras, Barezani, Zani, Lazzari, Diotaiuti, Jeffares, Bohman and Lorenzo2014), and lures may encompass semiochemicals (Bohman et al., Reference Bohman, Weinstein, Unelius and Lorenzo2018), synthetic compounds (Guidobaldi and Guerenstein, Reference Guidobaldi and Guerenstein2016; Reference Guidobaldi and Guerenstein2013), or volatiles from various sources (Taneja and Guerin, Reference Taneja and Guerin1997). In fact, volatiles from fermented sources have been tested to attract mosquitoes (Aldridge et al., Reference Aldridge, Britch, Allan, Tsikolia, Calix, Bernier and Linthicum2016), bed bugs (Singh et al., Reference Singh, Wang and Cooper2015), and triatomines (Guerenstein et al., Reference Guerenstein, Lorenzo, Núñez and Lazzari1995; Pires et al., Reference Pires, Lazzari, Diotaiuti and Lorenzo2000; Cardozo et al., Reference Cardozo, Fiad, Crocco and Gorla2020). In this context, fermented products could serve as effective lures for triatomines, being both cost-effective and easy to prepare. Furthermore, only alcohol fermentation products have been tested for triatomines, leaving room to explore the impact of volatiles from other fermentation sources, such as lactic acid fermentation, acetic acid fermentation, and butyric acid fermentation, as the fermentation type significantly influences the chemical composition and volatile profiles (Januszek and Satora, Reference Januszek and Satora2021). Additionally, protein-based products could act as effective attractants due to their nitrogen-derived compounds (Taneja and Guerin, Reference Taneja and Guerin1997).
In this study, we present the results obtained from testing the biological effects of volatiles derived from: (a) various fermentation types; (b) homemade fermented fruits; and (c) commercial insect attractants. Two-choice tests were conducted using a group of fifth-instar nymphs from Triatoma pallidipennis, T. infestans, and Rhodnius prolixus.
Materials and methods
Insects
Insects were reared in the Instituto Nacional de Salud Pública. The colonies of different triatomines were established approximately five years ago, constantly adding the first generation of adults from wild insects. We used fifth-stage nymphs of T. pallidipennis, T. infestans, and R. prolixus maintained under controlled temperature, humidity, and photoperiod (28 ± 2°C, 60 ± 5 Rh, and 12 : 12 h). Insects were used after a 15-day period of starvation and were taken from approximately six cohorts of the brood. Triatomines were provided throughout the duration of the experiments.
Fermented products
Fermentation types
Lactic fermentation . We used a similar procedure previously reported with slight modifications (Panesar et al., Reference Panesar, Kennedy, Knill and Kosseva2010). The Lactiplantibacillus plantarum TEJ 7 strain was reactivated in De Man, Rogosa and Sharp (MRS) agar (Sigma Aldrich, Toluca) at 37°C for 48 h, with a pH 6.5 ± 0.2. MRS agar typically contains 1.0% universal peptone, 1.0% meat extract, 0.4% yeast extract, 2.0% D(+)-glucose, 0.5% sodium acetate trihydrate, 0.2% dipotassium hydrogen phosphate, 0.2% triammonium citrate, 0.02% magnesium sulphate heptahydrate, 0.005% manganese sulphate tetrahydrate, and 1.0% agar. To prepare the preinoculum, a colony of approximately 1 mm in diameter of L. plantarum TEJ 7 was transferred with a bacteriological loop to 10 mL of MRS broth, incubated for 48 h at 37°C and 150 rpm. Subsequently, the preinoculum was transferred to 50 ml of MRS broth under the same conditions. Subsequently, the whey was supplemented with 5 g l−1 of yeast extract, and the pH was adjusted to 6.2 ± 0.2 with 1 N HCl. Then, it was sterilized at 121°C for 15 min. In 250 ml capacity flasks, 50 ml of supplemented serum were placed and inoculated at 4% v/v with MRS broth at a concentration of 4 × 106 colony-forming units ml−1 of L. plantarum TEJ 7. Fermentation conditions were 37°C for 36 h at 150 rpm.
Alcoholic fermentation . The Saccharomyces cerevisiae CDBB-L-328 strain was reactivated in Yeast extract Peptone Dextrose (YDP) agar (20 g l−1 casein peptone, 20 g l−1 dextrose, 10 g l−1 yeast extract and 15 g l−1 bacteriological agar) at 30°C for 24 h, at pH 6.5 ± 0.2 (Nurhayati et al., Reference Nurhayati, Mayzuhroh, Arindhani and Caroenchai2016). For the preparation of the preinoculum, a yeast colony (approximately 1 mm in diameter) was transferred to 10 ml of YPD broth at pH 6.5 ± 0.2, incubated for 24 h at 30°C and 180 rpm. Subsequently, the preinoculum was transferred to 50 ml of broth under the same conditions. Raw sugar cane cubes were dissolved in water until a concentration of 24 °Brix was obtained, and it was supplemented with 2.0 g l−1 ammonium sulphate, the pH was adjusted to 4.5 ± 0.2 with 1 N HCl. Subsequently, it was sterilized at 121°C for 15 min. Fifty ml of the supplemented raw sugar were inoculated at 5% v/v with YPD broth at a concentration of 1.45 × 107 yeast cells ml−1. The fermentation conditions were at 30°C for 72 h at 180 rpm.
Acetic acid fermentation. We followed a similar procedure already reported elsewhere (Roda et al., Reference Roda, Lucini, Torchio, Dordoni, De Faveri and Lambri2017). This fermentation was carried out in two consecutive steps, an alcoholic fermentation using S. cerevisiae CDBB-L-328 strain (see above), and acetic fermentation using Acetobacter aceti CDBB-B-1237 strain (Baena-Ruano et al., Reference Baena-Ruano, Santos-Dueñas, Mauricio and García-García2010; Min Kyung and Young-Suk, Reference Min Kyung and Young-Suk2019). The strain A. aceti CDBB-B-1237 was reactive in mannitol agar (D-mannitol 25 g l−1, yeast extract 5 g l−1, casein peptone 3 g l−1 and bacteriological agar 15 g l−1) at 30°C for 72 h, at pH = 7 ± 0.2. For the preparation of the preinoculum, a colony of approximately 1 mm in diameter of A. aceti CDBB-B-1237 was transferred to 10 ml of mannitol agar, it was incubated for 72 h at 30°C and 180 rpm. The pineapples were washed, and the shell was removed, 500 g of pineapple shell was crushed to produce a homogeneous mixture. Subsequently, to obtain the pineapple must, 250 g of previously obtained shell were used, and distilled water was added to reach a ratio of 1 : 2 shell: water and the mixture was placed in hermetic containers. Samples were then subjected to a physical pretreatment by high pressure autoclaving (15 lb in2, 121°C for 15 min). Once the treatment was finished, it was filtered to remove residues. Subsequently, the total soluble solids content (°Brix) was measured with a refractometer (HANNA® HI96801, Rhode USA). The must previously obtained was supplemented with 2.0 g l−1 of ammonium sulphate, the pH was adjusted to 4.5 with 1 N HCl, and sterilized for 15 min at 121°C. 50 ml of the supplemented must was placed in flasks of 250 mL capacity, and inoculated at 5% v/v with YPD broth with a concentration of 1.45 × 107 cells ml−1 of S. cerevisiae strain. The fermentation conditions were set as 30°C for 72 h at 180 rpm. After this time, 500 ml of the previously fermented must were placed and inoculated at 5% v/v with mannitol broth with a concentration of 1.45 × 107 cells ml−1 of A. aceti CDBB-B-1237 strain. Fermentation conditions were set at 30°C for 30 days.
Home-made fermented products
We fermented fruits: blackberry, blueberry, raspberry, strawberry, and a mixture of these four fruits. For this, we put 1 kg of fruit (250 g of each in the mixture), 60 g of sugar, and 4 l of water into a 6 l glass container, and let the mixture for one week at 25°C, and 1 atm, 80% RH. We filtered the juice, which was then stored at 0°C for 2 days to stop the fermentation, which was then used as a lure.
Commercial lures and chemicals
Three commercially available protein and food-based attractive baits were chosen: Ceratrap® and SWD Lure® (obtained from Bioiberica, Barcelona, Spain), and Susbin (from Mendoza, Argentine) which were used with no further modifications. Torula yeast was obtained from Somos (Mexico City, Mexico). Ten pellets of 5 g each were suspended in 1 l of water, and the dissolution was used in the experiments. Standard compounds used for the identification (> 95%): Ethanol, isopentyl alcohol, 2-ethyl-1-hexanol, ethyl acetate, acetic acid, isopentyl acetate, methyl butanoate, ethyl hexanoate, limonene, linalool, α-copaene, α-pinene, nonanal, heptane, octane, p-cresol were obtained from Sigma Aldrich (Toluca, Mexico).
Bioassays
The insects' response to the volatile compounds emitted by the ferments or the commercial products was evaluated in a double-choice bioassay. The olfactometer design was modified from elsewhere (Weeks et al., Reference Weeks, Logan, Birkett, Pickett and Cameron2013). A rectangular acrylic cage (90 × 20 × 12 cm) was adapted by producing a hole on each end of the cage. The resulting two holes were hermetically connected to two pots of 250 ml (10 cm i.d.) each (fig. 1). While we placed 10 ml of the treatment to be evaluated in one pot (either the fermented product or commercial insect attractant), 10 ml of water was placed as a control in the opposite pot. The pots had a mesh to prevent insects from falling into the liquid. The plastic pots were too slippery for the insects to climb and return to the arena. Five fifth-instar nymphs (15 to 30 days after ecdysis) were used, due to availability. Insects were placed in a container for 5 min to acclimatize at the centre of the arena, and then were released. After 3 h, the number of insects that fell into the pots (treatment or control) was recorded. The bioassays were carried out between 18 : 00 and 23 : 00 h (due to the insect's nocturnal activities) at a temperature of 30 ± 2°C and 95% RH. Twenty-five replicates were performed for each treatment. After each replicate, the olfactometers were rinsed with water and soap, dried, and then cleaned with hexane and ethanol to eliminate chemical interference. Treatments were rotated to avoid positional bias. We used five olfactometers simultaneously; experiments were carried out from January 2021 to August 2022. We used insects from the same colony
Illustration of the olfactometers used in this work. (A) Plan view, internal diameter of the hole 10 cm, distance between holes 70 cm. (B) Side view, a cage of 90 × 20 × 12 cm. Pots were plastic pots and measured 25 × 10 cm (height × diameter). The treatment pot contained fermented products or commercial attractants. The control pot contained water.
Headspace volatiles collection (SPME)
Volatiles were collected by solid phase micro extraction (SPME) using a poly-dimethylsiloxane fibre (SUPELCO, Deisenhofen, Germany). Ten millilitres of fermented juice or commercial attractant were placed in a 20 ml flask. The flask was covered with aluminium foil to prevent volatile compounds from escaping. After 10 min of volatile equilibration, an SPME fibre was then inserted into the vial through the foil and exposed to the volatiles. The exposure time was one minute. Conversely, an identical empty flask was used at the same time to determine the chemical background (emitted by the vial or in the surrounding air). We used the same SPME fibre for each replicate of the treatment during the study and followed the recommendation of the SPME guidelines (Romeo, Reference Romeo2009). Four replicates for each treatment were done.
Chemical identification
SPME samples were analysed on a GC–MS Shimadzu GC-2010 plus, Triple-Quadrupole TQ8040 (Texas). A CB-5MS capillary column (30 m × 0.25 mm ID, Agilent Tech, Santa Clara, CA) was temperature programmed from 50°C (held for 2 min) to 280°C at 15°C min−1, then held at 280°C for 10 min. The temperature of the injector was held at 250°C. Ionization was carried out by electron impact at 70 eV, and 250°C. Compounds were identified comparing the normal alkane retention indices (Kovats and Arithmetic) and mass spectra with those reported in the NIST library. Some compounds were confirmed by comparing the retention indices and mass spectra with those of pure standards (Sigma-Aldrich, Toluca, Mexico). The relative amount (percentage) of a given component was calculated relative to the sum of all areas under the peaks of a chromatogram. Percentages are provided due to the limitations implied by the SPME analysis used for quantification (Romeo, Reference Romeo2009; Alborn et al., Reference Alborn, Bruton and Beck2021). However, we used this technique because it possesses various advantages, including higher sensitivity, ease of handling, shorter adsorption time, and no solvent peak in GC, thus, molecules with lower molecular weight can be measured (Jalili et al., Reference Jalili, Barkhordari and Ghiasvand2020; Cagliero et al., Reference Cagliero, Mastellone, Marengo, Bicchi, Sgorbini and Rubiolo2021).
Statistical analysis
Data were analysed using R (R Development Core Team, 2023). Before formal analysis, we determined if the data met the assumptions of normality and homogeneity of variances by using the Shapiro-Wilk and the Levene tests, respectively. The data from the double-choice bioassays in the rectangular arena were Box-Cox transformed as needed (Box and Cox, Reference Box and Cox1964) and analysed using a two-tailed Williams-corrected t-test with a 95% confidence interval.
Results
Fermentation types
T. infestans was attracted to volatiles from alcoholic fermentation (t = −2.28, df = 47, P < 0.05), yet R. prolixus preferred the control over volatiles (t = 2.6, df = 47, P < 0.05). T. pallidipennis was attracted to volatiles from lactic fermentation (t = −3.37, df = 47, P < 0.01), however, T. infestans was attracted to the control (t = 2.14, df = 47, P < 0.05). T. pallidipennis (t = 2.54, df = 47, P < 0.05) and T. infestans (t = 4.31, df = 47, P < 0.001) preferred the control over volatiles from lactic acid fermentation (fig. 2).
Responses of T. pallidipennis, T. infestans, and R. prolixus to volatiles from different types of fermentations. Twenty-five replicates were performed for each treatment. AF; alcoholic fermentation, LF; lactic fermentation, AAF; acetic acid fermentation.
The most abundant volatiles found in alcoholic fermentation were ethanol (32.46%), acetoin (24.77%), isopentyl formate (20.93%), and isopentyl acetate (10.46%). Meanwhile, the most abundant compounds detected in the headspace of lactic fermentation were acetoin (44.60%), acetic acid (42.49%), and ethanol (5.31%). Finally, most abundant volatiles found in acetic acid fermentation were acetoin (57.65%), ethyl acetate (23.82%), and isopentyl acetate (7.46%) (table 1).
Volatiles emitted from the different types of fermentation (percentages ± SE)
KRI, Kovats Retention Index; LRI, Library Retention Index (NIST database: https://webbook.nist.gov/chemistry/).
Alcoholic fermentation (AF), lactic fermentation (LF), and acetic acid fermentation (AAF).
a Identification based on the comparison of chromatographic and mass-spectra data with NIST library.
b Identification based on comparison with standard.
Home-made fermented fruits
Volatiles from the fermentation of blackberry attracted T. pallidipennis (t = −3.98, df = 47, P < 0.001), but these volatiles were not preferred by T. infestans (t = 8.11, df = 47, P < 0.001). Volatiles from the control were preferred over volatiles from blueberry fermentation by T. pallidipennis (t = 3.92, df = 47, P < 0.001), and T. infestans (t = 8.19, df = 47, P < 0.001). Also, T. pallidipennis preferred the control over volatiles when tested with raspberry fermentation (t = 4.24, df = 47, P < 0.001). Volatiles from other home-made fermented fruits evaluated did not present a behavioural effect over the insects (fig. 3).
Responses of T. pallidipennis, T. infestans, and R. prolixus to volatiles from fermented fruits. Twenty-five replicates were performed for each treatment.
Most abundant compounds found in fermented blackberry head space were ethyl acetate (65.97%), isopentyl acetate (21.86%), and ethanol (10.48%). Major fermented blueberry volatiles were ethyl acetate (69.63%), isopentyl acetate (14.76%), linalool (4.79%), and ethanol (4.69%). In the fermented raspberry volatiles, we found isopentyl acetate (50.75%), ethyl acetate (34.44%) as the most abundant compounds. In volatiles from fermented strawberries, the major compound was ethyl acetate (75.83%), with other minor compounds. Meanwhile, in the headspace of the fermented four berries, we found ethyl acetate (63.04%) and ethanol (21.23%) as most abundant compounds (table 2)
Volatiles emitted from fermented fruits (percentages ± SE)
KRI, Kovats Retention Index; LRI, Library Retention Index (NIST database: https://webbook.nist.gov/chemistry/).
a Identification based on the comparison of chromatographic and mass-spectra data with NIST library.
b Identification based on comparison with standard.
Commercial insect attractants
Volatiles from Ceratrap were attractive to T. infestans (t = −4.32, df = 47, P < 0.001) and R. prolixus (t = − 5.33, df = 47, P < 0.001). T. pallidipennis (t = 4.49, df = 47, P < 0.001) and T. infestans (t = 2.22, df = 47, P < 0.01) preferred the control over volatiles from the SWD lure. The same election for the control was observed with volatiles from Torula yeast volatiles with T. pallidipennis (t = 5.32, df = 47, P < 0.001) and T. infestans (t = 3.27, df = 47, P < 0.01). Volatiles from other commercial attractants used in this study did not present a behavioural effect on the triatomines (fig. 4).
Responses of T. pallidipennis, T. infestans, and R. prolixus to volatiles from commercial insects' lures. Twenty-five replicates were performed for each treatment.
Ceratrap emitted acetic acid as the most abundant volatile (66.95%), and other minor compounds as indole, p-cresol and a pyrazine. The most abundant volatiles found in SWD lure were acetic acid (67.39%), isopentyl acetate (17.51%), and acetoin (7.22%). Head space volatiles from Torula yeast found in most abundant amounts were hexanoic acid (20.5%), limonene (17.9%) and 2-heptanone (16.9%) (table 3).
Volatiles (percentages ± SE) emitted from commercial insects' attractants
KRI, Kovats Retention Index; LRI, Library Retention Index (NIST database: https://webbook.nist.gov/chemistry/).
a Identification based on the comparison of chromatographic and mass-spectra data with NIST library.
b Identification based on comparison with standard.
Discussion
While nymphs showed attraction responses to some of the ferments (from both, different fermentation process and home-made fruit fermentations) as well as commercial attractants, they also showed no response to other ferments. Moreover, volatiles found in different fermentation types are according to those reported elsewhere (Min Kyung and Young-Suk, Reference Min Kyung and Young-Suk2019; Januszek and Satora, Reference Januszek and Satora2021). For example, while T. pallidipennis was attracted to lactic fermentation volatiles, they preferred the volatiles from the control over the volatiles produced from acetic acid fermentation. Meanwhile insects of this species did not present a preference between control and alcoholic fermentation volatiles. Fermentation increases the molecular diversity of bioactive metabolites that can be obtained from the substrates (Omarini et al., Reference Omarini, Achimón, Brito and Zygadlo2020). However, in the fermentation of the fruits (also alcoholic fermentations), we detected a behavioural response of T. pallidipennis. This highlights the importance of the substrate in the fermentation process on the behavioural response of the insects (Piñero et al., Reference Piñero, Godoy-Hernandez, Giri and Wen2022; Batallas and Evenden, Reference Batallas and Evenden2023). Besides, volatile profiles differ qualitatively, so that while some compounds were common in different treatments, they may be present in different relative amounts (with the limitations of the SPME (Romeo, Reference Romeo2009; Alborn et al., Reference Alborn, Bruton and Beck2021)). Note that attracting response can be dose-dependent. Actually, in the case of triatomines, acetic acid and isobutanoic acid act as repellents at high doses but act as attractants at low doses (Palottini and Manrique, Reference Palottini and Manrique2016).
Volatiles in high relative amounts from different fermentation processes were mostly the same, except for the lactic fermentation, which had acetic acid in high amounts. The same phenomenon was observed with homemade fermented fruits. The volatile compounds produced during fermentation are similar to those found in human emanations (Bernier et al., Reference Bernier, Kline, Barnard, Schreck and Yost2000; Zhang et al., Reference Zhang, Cai, Ruan and Li2005). Thus, volatile compounds could be responsible for the behavioural response of insects towards ferments. Future studies should be carried out to study the attraction or repellence evoked by the individual compounds or mixtures in triatomines. Additionally, the possible effect of other compounds over the most abundant compound should be analysed. For example, it was observed that the acetic acid fermentation where acetic acid was the most abundant volatile, was not attractive for T. pallidipennis. However, the same compound in a higher dose showed attraction in experiments with Ceratrap ®. It is possible that minor compounds could act as synergists, antagonists, or inhibitors (Chang et al., Reference Chang, Liu, Ai, Jiang, Dong and Wang2017). Thus, commercial lures could be used to modulate the behaviour of triatomines with the aim of controlling these insects. Therefore, volatiles from Ceratrap could be used as lures in traps, while SWD lure and Torula yeast could function as dislodging or repellent agents (Minoli et al., Reference Minoli, Palottini, Crespo and Manrique2013). Volatiles found in commercial products are in accordance with those previously reported (Biasazin et al., Reference Biasazin, Chernet, Herrera, Bengtsson, Karlsson, Lemmen-Lechelt and Dekker2018; Feng et al., Reference Feng, Bruton, Park and Zhang2018; Gómez-Escobar et al., Reference Gómez-Escobar, Alavez-Rosas, Castellanos, Quintero-Fong, Liedo and Malo2022).
Fermentation can be used for the production of environmentally friendly bioactive products from easily obtained and low-cost substrates. In this regard, we found that T. pallidipennis was attracted to volatiles from lactic fermentation and fermented blackberry; T. infestans was attracted to volatiles from alcoholic fermentation and Ceratrap®; and R. prolixus was attracted to volatiles from Ceratrap®. While several products could be good candidates as repellents or dislodging agents, T. pallidipennis could be repelled with volatiles from acetic acid fermentation, fermented blueberry, raspberry and SWD lure. Also, T. infestans could be dislodged using volatiles from lactic fermentation, fermented blueberry, SWD lure, and Torula yeast. Finally, R. prolixus could be possibly repelled or dislodged with volatiles from alcoholic fermentation. However, studies focusing on dislodging or repellent activity are necessary. Furthermore, additional studies about the aging of fermented products and commercial attractants are needed. Increasing the availability and diversity of natural bioactive compounds will allow for more effective control of triatomines using natural bioactive compounds in combination with commercial products to achieve synergistic effects and reduce toxicity (Omarini et al., Reference Omarini, Achimón, Brito and Zygadlo2020). In summary, volatiles derived from lactic fermentation, certain fermented fruits, and commercial lures attracted triatomines, while other products exhibited possible repellent or dislodging properties. Therefore, our list of products can be used as promising tools for triatomine control and, thus, Chagas disease.
Acknowledgments
This work was supported by CONAHCyT (Ciencia de Frontera 2019, clave 376136, número 292), CONTex grants (Unifying Texan and Mexican efforts towards controlling Chagas disease by deducing parasite-vector dynamics) and DGAPA-PAPIIT (IN218824).
Competing interests
The authors have no competing interests to declare that are relevant to the content of this article.
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