Geographic distribution of sub-species of
In México, biogeography data are available for species of triatomas called Trypanosoma cruzi transmitters; for example, the phyllosoma complex is distributed in several states of the south-central southeast of the country. In contrast, Northwestern Mexico species such as Triatoma rubida are considered sylvatic and in the process of domestication. The lack of research of these northern species of the country has generated an ignorance that contrasts with a growing number of alleged new cases of Chagas disease registered in health institutions in states such as Sonora. From the six species of triatomas that are potential transmitters of the trypanosoma in the state of Sonora, Triatoma rubida is the only one that has recent studies of distribution and transmission capacity. It is important then to know the degree of domesticity of the native species with the capacity of transmission of Trypanosoma cruzi and to define areas of risk. The process of adaptation of the sylvatic triatomines to the peridomestic and the domestic habitat has been understood in terms of environmental and biological variables. In this research, the profile of cuticular hydrocarbons of a peridomestic, domestic and sylvatic population of Triatoma rubida was analyzed and compared.
- Triatoma rubida
- cuticular hydrocarbons
Chagas disease (CD) is caused in humans and animals by the parasite
In Mexico 32 species are reported; 19 belong to the gender
2. Bibliographic revision
This insect has a wide geographic distribution in the Northwest of Mexico, Nayarit, Sinaloa and Sonora, and has been found in the Southwest of the United States in the states of Arizona, California, New Mexico and Texas. It is an established species throughout its range, and there is no information available on its dispersion [18, 19]. The populations of
|Baja California Sur, Sonora|
|Isla Estanque BC., Sonora|
|Sonora, Sinaloa y Nayarit|
|Suroeste de USA, Sonora y Veracruz|
Currently in the anti-vectorial fight of CD, in endemic countries like Brazil, Argentina, Bolivia and Peru, morphometric, biochemical, molecular and genetic studies of vector species are being developed, that contribute in the decision-making for the eradication in their houses. One of these lines of work is the analysis of the cuticular hydrocarbons (CH) of the triatomines .
2.2. Cuticle of insects
The cuticle of the insect is secreted by a double layer of epidermal and hypodermic cells. The hypodermis is described as a functional syncytium and is formed as a base membrane particularly during deposition and expansion of the old cuticle. The cuticle is formed by an inner pro-cuticle composed of chitin (
2.3. Insects’ cuticular hydrocarbons
The insect’s cuticular lipids consist of aliphatic material, which forms a thin layer in its integument. These lipids or surface waxes are presented as highly stable complex mixtures with unique structural characteristics. Among its main compounds hydrocarbons (HCs), fatty alcohols and waxes of high molecular weight predominate. The main function of these lipids in the insect is to restrict water loss and avoid lethal drying. It has been shown that they also participate in the absorption of chemical substances that can affect the activity of microorganisms and intervene in various chemical communication processes [33, 34, 35].
Cuticular hydrocarbons (CH) are continuously synthesized in the insect’s intrategumenal tissue, through the enzymatic action of fatty acid synthetase (FAS), an acetyl CoA for elongation, a reductase and a decarboxylase that produces hydrocarbons and CO2. The epidermal cells responsible for its production are the oenocytes that lie beneath the hypodermis. Oenocytes transport hydrocarbons through tissues through a hemolytic lipoprotein called liporin. This lipid synthesis is considered dynamic and changes as the insect passes through its nymphal stages, stopping at the adult stage. De Renobales et al. , proposed that the hydrocarbons synthesized by the insect are stored inside their tissues until the next shedding. The insect needs a new layer of lipids as regulators of its permeability [35, 37].
Based on studies conducted in particular on nine species of triatomines of the genus
Finally, understanding how insect HCs, together with other surface lipids, are involved in the absorption of chemicals is essential for the timely and adequate vector control measures to be applied in the future [42, 43].
This research analyzes and compares the profile of cuticular hydrocarbons of a peridomestic, domestic and a sylvatic population of
3. Materials and methods
3.1. Sampling area
The city of Guaymas Sonora was chosen because it is considered, in this study, to be an endemic area of the CD. The port is situated at 110°53′34” North latitude and 27°55′30” West Greenwich, at a height of 15 m. It has a desert or hot climate, with a maximum monthly temperature of 30–35°C in the months from July to August and a minimum average monthly temperature of 18°C. Its average annual temperature is 28°C. Its vegetation is xerophytic type, where mesquite (
3.2. Sampling and capture of insects
Three districts of the City of Guaymas were monitored to collect the batch of peridomestic and domestic insects, where the epidemic was known: El Rastro, Cerro Gandareño and Yucatán. In these areas, the existence of triatomines was known, particularly
The sylvatic insects were collected from the surrounding hills, a hill in the northern part was chosen 1400 m away from the city where domestic and peridomestic insects were collected.
Once collected, the two pairs of main and secondary wings were extracted from the adult insects which were wrapped in foil and transported to the laboratory of parasitology at the University of Sonora, North Caborca Unit. For the identification of the morphological characteristics that define
3.3. Analysis and extraction of hydrocarbons
Cuticular hydrocarbons (CHs) were extracted following the methods of Juárez and Blomquist , and Juárez et al. . Each pair of specimen wings were given a washing treatment with 2 mL of distilled water twice to remove any contaminants such as feces or soil particles. They were then transferred to a 4 mL glass vial with a screw cap, Teflon septum and properly labeled. A 1 mL of high-performance liquid chromatography (HPLC) grade hexane was added with 99% purity (Sigma-Aldrich, México). With this solvent they were kept for 1 day, at a temperature of 28°C for the extraction of the cuticular lipids.
3.4. Isolation of hydrocarbons
The next step consisted of separating the hydrocarbons from the other cuticular components (lipids, waxes, etc.) present in the extract. For this purpose, the lipid mixture was reconstituted from each vial with hot hexane and then applied to a mini glass column (10 × 5 mm ID) with 1.74 g of silica gel, 60% pore and 70-230 (Sigma-Aldrich, México; cat. 288,624). Previously equilibrated with hexane, the elution was carried out with 4 mL of hexane for each sample (4 mL/mg). The silica from the column was renewed every three samples (Figure 2).
3.5. Obtaining the chromatographic profiles of each population
Once the methodology was adjusted, 3 μl of each of the hydrocarbon samples extracted from
3.6. Identification of HC by GC-MS
For the identification of the linear hydrocarbons, an HP 6890 chromatograph coupled to an Agilent 5975C VL mass spectrometer was used. GC conditions were HP-5MS nonpolar column of 30 × 0.25 mm ID × 0.25 μm film; helium carrier gas at 1.5 mL/min constant flow; oven temperature programmed 50°C (1 min) to 200–50°C /min, then to 320–3°C/min (25 min) and the injector was operated in split-less mode at 320°C. The conditions of the MS detector were ionization energy of 70 eV; transfer line at 320°C; the ionization chamber at 230°C and the quadrupole at 150°C. For the analysis of the collected data, an MSD ChemStation Agilent Technologies Inc. was used.
4. Statistical analysis
We estimated central tendency measures and compared the relative means of abundance of hydrocarbons between genera; the significance was tested by a nonpaired t (Excel 2006 package), after normalization of the data with arcosene. Relative means (Tukey’s post hoc test) were compared between the three populations through a one-way analysis of variance (ANOVA). The data were presented in tables and graphs. All tests were estimated at one tail and values of p < 0.05 were considered as statistically significant. For these analyses the statistical package BioStat 2007 was used.
5.1. Collection of insects
A total of 120 peridomestic, 50 domestic and 50 sylvatic specimens were collected. Of the 220 insects, there were nymphs of second stage (NII; 1.4%), nymphs of the third stage (NIII; 11.4%), nymphs of the fourth instar (NIV; 17.3%), 53.6% were nymphs of the fifth instar (NV; 53.6%), 6.8% were adult females (AF) and 9.5% were adult males (AM). No specimens of the first nymphal period were found in all three populations.
5.2. Analysis of the hydrocarbons: Retention times obtained
The gas chromatographic standardization process allowed to obtain the retention times of 14 commercial hydrocarbons standards, AccuStandard Brand, Inc. USA (purity 99%), which were used to estimate Kovats indexes for each sample analyzed (Table 2).
|Standard||Name||Retention Time (Minutes)|
5.3. Identification of the cuticular hydrocarbons profile of
The 35 components of
The chromatographic profile was similar for the three populations, and the hydrocarbons corresponded to n-alkanes with a continuous series of C25, C27, C29, C31, C33 and C35. In addition, the Kovats indices identified C35.52, C36.00, C37.74, C37.75, C38.00, C39.41, C39.60 and C39.83, which are likely representations of branched isomers of alkanes. The location of the methyl branches of these hydrocarbons, by the proposal of Katritzky et al. , was estimated. The Kovats indexes: IK3552, IK3600, IK3774, IK3775, IK3800, IK3941, IK3960 and IK3983 therefore correspond to the hydrocarbons described in Table 3.
|Retention rate||Type of hydrocarbon|
|3574||03 Methyl Pentacontane|
|3600||3x Dimethyl Pentacontane|
|3752||13, 23 Dimethyl Heptatriacontane|
|3775||15,19,23 Dimethyl Heptatriacontane|
|3800||3x Dimethyl Octariacontane|
|3941||x Trimethyl Nonatriacontane|
|3960||xx Dimethyl Nonatriacontane|
|3983||15.19, 23 Trimethyl Nonatriacontane|
5.4. Quantification in percent of area of hydrocarbons analyzed
The relative amounts in the percent of area of each linear and branched hydrocarbon analyzed for each population were obtained. In Table 4, the data for the peridomestic, domestic and sylvatic
|1||n-25||2500||6.59 ± 2.3||5.50 ± 2.3||4.50a ± 0.09||7.56b ± 0.9||5.54 ± 1.3||6.18 ± 1.0|
|2||n-27||2700||16.50a ± 1.3||20.90b ± 2.0||19.00 a ± 3.2||27.88b ± 0.8||23.00 ± 1.5||26.64 ± 2.6|
|3||n-29||2900||9.33 ± 1.2||10.80 ± 2.0||12.82 ± 1.6||13.83 ± 0.8||15.29 ± 0.9||16.10 ± 1.9|
|4||n-31||3100||11.86 ± 1.8||10.00 ± 1.4||15.69a ± 1.2||9.41b ± 0.9||14.08 ± 0.7||12.17 ± 2.3|
|5||n-33||3300||14.06 ± 2.7||12.00 ± 1.8||22.29a ± 1.7||9.61b ± 0.6||15.72a ± 1.3||12.29b ± 1.7|
|6||n-35||3500||5.19 ± 1.7||5.20 ± 0.4||2.98 ± 0.3||3.82 ± 0.5||2.59 ± 1.3||3.03 ± 0.5|
|7||03 Methyl Pentacontane||3574||2.74 ± 0.3||2.90 ± 0.2||1.55 ± 0.3||1.81 ± 0.3||1.84 ± 1.0||2.00 ± 0.3|
|8||3x Dimethyl Pentacontane||3600||2.76 ± 0.6||2.00 ± 0.4||1.53 ± 0.1||1.47 ± 0.3||1.70 ± 0.5||1.40 ± 0.3|
|9||13, 23 Dimethyl Heptatriacontane||3572||6.55 ± 0.2||7.40 ± 1.0||5.61 ± 0.9||7.73 ± 1.2||5.77 ± 0.3||6.00 ± 0.3|
|10||15,19,23 Dimethyl Heptatriacontane||3775||7.07 ± 0.8||6.80 ± 1.3||4.84 ± 2.7||4.73 ± 1.2||4.13 ± 1.0||4.04 ± 1.4|
|11||3x Dimethyl Octariacontane||3800||5.63 ± 1.1||4.90 ± 0.5||3.02 ± 1.3||4.00 ± 0.2||3.38 ± 0.6||3.00 ± 1.6|
|12||x Trimethyl Nonatriacontane||3941||2.14 ± 0.3||1.90 ± 0.3||1.96 ± 0.3||2.22 ± 0.6||1.90 ± 0.2||1.70 ± 1.2|
|13||xx Dimethyl Nonatriacontane||3960||4.79 ± 0.8||4.50 ± 0.9||3.16 ± 0.9||3.57 ± 0.8||3.18 ± 0.6||3.00 ± 1.1|
|14||15.19, 23 Trimethyl Nonatriacontane||3983||4.79 ± 1.9||5.20 ± 0.9||2.56 ± 2.2||3.50 ± 0.9||3.07 ± 0.7||3.00 ± 2.2|
In Table 5, the total amounts for linear and branched hydrocarbons are presented for each of the three populations of
5.5. Comparison of the three populations of
To analyze the differences between
In the comparison of groups of males, significant differences were also found between domestic males and peridomestic males in HC27 (p = 0.01) and HC29 (p = 0.03), whereas when comparing domestic with sylvatic males, there were significant differences in HC33 (p = 0.002). On the other hand, when comparing the populations of females with males, significant differences were observed in the population of domestic females compared to that of peridomestic and sylvatic males in the HC 29 (p = 0.01), 31 (p = 0.03) and 33 (p = 0.001). Meanwhile, the population of peridomestic females, when compared to domestic males and sylvatic males, had significant differences in HC27 (p = 0.007), HC29 (p = 0.01) and HC33 (p = 0.0009). Finally, in the comparison of populations of sylvatic versus domestic males and peridomestic males, significant differences were observed for HC27 (p = 0.0001) and HC29 (p = 0.002).
6.1. Domestic population
Based on the graphical representation of the chromatographic profiles, the identification of five of their linear hydrocarbons by mass spectrometry and the statistical analysis when comparing the relative means between genders, we suggest how the typical profile of domestic Triatoma rubida is described in Figure 7.
When comparing this profile of hydrocarbons obtained with studies of other triatomines, a clear differentiation of species can be seen. For example, the literature reports that
6.2. Peridomestic population
The GC–MS analysis showed that the chromatographic profiles of the HC in
For this population, odd strands prevailed predominantly, C27, C29, C31 and C33, which together represented 76.41% in females and 76.22% in males. The total relative amount of branched hydrocarbons in males and females was 23.59% and 23.78%, respectively. The relative amount of the pentacosane hydrocarbon was 49.64%. Based on the graphical representation of the peridomestic rubida species chromatographic profiles, in their identification by mass spectrophotometry and in the statistical analysis when comparing the relative means between genders. We can suggest as typical profile of Triatoma rubida peridomestic described in Figure 8.
6.3. Sylvatic population
The analysis of
Based on the graphical representation of the chromatograms of sylvatic
Juárez et al. , suggested that quantitative rather than qualitative differences support the idea that cuticular hydrocarbons represent primitive characteristics for
Therefore, species of triatomines of dry regions present their cuticular profiles as more abundant and complex than their congeners of humid regions. Among Triatoma,
This research provides basic knowledge on the cuticular lipids of Triatoma rubida. A unique and very different profile of cuticula hydrocarbons was obtained from