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Wing shape differentiation of Mepraia species (Hemiptera: Reduviidae)
Infection, Genetics and Evolution 11 (2011) 329–333
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Wing shape differentiation of Mepraia species (Hemiptera: Reduviidae) Ricardo Campos a, Carezza Botto-Mahan b, Ximena Coronado a, Nicola´s Jaramillo c, Francisco Panzera d, Aldo Solari a,* a
Programa de Biologı´a Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile Departamento de Ciencias Ecolo´gicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile c Seccio´n Gene´tica Evolutiva, Facultad de Ciencias, Universidad de La Repu´blica, Montevideo, Uruguay d Grupo de Biologı´a y Control de Enfermedades Infecciosas, Instituto de Biologı´a, Universidad de Antioquia, AA 1226, Medellı´n, Colombia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 August 2010 Received in revised form 3 November 2010 Accepted 5 November 2010 Available online 24 November 2010
Mepraia is an endemic genus found in the semiarid and arid regions of north-central Chile. Until 1998, Mepraia spinolai was the only species of the genus, distributed in coastal and interior valleys from Chile between 188 and 348S. However, on the basis of karyotype and morphological characters, coastal desert populations between 188 and 268S were ranked as a new species, Mepraia gajardoi. Recently, genetic studies using nuclear and mitochondrial markers on Mepraia populations suggest that the geographical criterion to separate the two species should be reviewed. Mepraia species show conspicuous alary polymorphism, unique in the Triatominae subfamily. Females of both species are invariably micropterous, while males of M. spinolai can be micropterous, brachypterous or macropterous, and only brachypterous in M. gajardoi. In this study, we use geometric morphometrics analyses to compare male wings of M. spinolai and M. gajardoi from natural populations, in order to examine if these two species have diverged in alary shape. As expected, we found that brachypterous wings of both species are smaller than macropterous wings of M. spinolai. Additionally, we detected clear differences in shape on wings of M. gajardoi and M. spinolai, not attributable to allometric effects. For last, a new alary phenotype, insects with vestigial wings, was described here for the first time. In conclusion, our analyses on wings of Mepraia species separate two distinct groups consistent with the two described species. However, our findings of vestigial wings in some coastal areas of the north part of Chile cannot rule out the existence of a hybrid zone. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Chagas disease Triatominae vectors Mepraia gajardoi Mepraia spinolai Alary polymorphism Brachypterous Macropterous Geometric morphometrics
1. Introduction Chagas disease is a serious human parasitic disease in the Americas caused by the flagellate protozoan Trypanosoma cruzi, and transmitted by blood-sucking insects of the subfamily Triatominae (Hemiptera: Reduviidae) (Lent and Wygodzinsky, 1979; Schofield et al., 2006). Two Triatominae genera occur in Chile: Triatoma and Mepraia. Triatoma infestans has been the main domestic vector of T. cruzi, now controlled in several countries, including Chile, through widespread interventions under the auspices of the Southern Cone Initiative (Dias and Schofield, 1999). The Mepraia genus is endemic from the semiarid and arid regions of northern and central Chile, mainly found amongst rock piles but occasionally enters and colonizes domestic and peridomestic habitats (Frı´as and Atria, 1998; Schofield et al., 1998; Cattan et al., 2002). This genus was first described with the inclusion of Triatoma spinolai Porter, 1934, as Mepraia spinolai by
* Corresponding author. Tel.: +56 2 978 6062; fax: +56 2 735 5580. E-mail address: [email protected] (A. Solari). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.11.002
Mazza et al. (1940), and revalidated by Lent et al. (1994). However, even today some authors do not agree with the existence of this genus and include the Mepraia species into Triatoma genus (Schofield and Galva˜o, 2009). Until 1998, M. spinolai was the only species of the genus, distributed in coastal and interior valleys from Chile between parallels 188 and 348S (Lent and Wygodzinsky, 1979). However, on the basis of karyotype, morphological characters, and experimental crosses, coastal desert populations between parallels 188 and 268S were ranked as a new species named Mepraia gajardoi (Frı´as et al., 1998; Jurberg et al., 2002). The remaining populations, from 268 to 348S, are distributed in the interior mountains from the Atacama to the Metropolitan Region, maintaining the name M. spinolai (Frı´as et al., 1998). Recently, genetic studies using nuclear and mitochondrial markers on Mepraia sp. populations suggest that the geographical criterion initially proposed by Frı´as et al. (1998) to separate the two Mepraia species should be reviewed (Calleros et al., 2010). According to these authors, individuals from Caleta Bandurrias (258S, Antofagasta Region) are more related to M. spinolai populations (i.e., from Atacama to Metropolitan Regions, see Table 1) based on nuclear markers (karyotype and nuclear ribosomal
R. Campos et al. / Infection, Genetics and Evolution 11 (2011) 329–333
330 Table 1 Location of collecting sites of Mepraia sp. Species
Alary phenotype
Number of adult malesa
Location name
Latitude; longitude
M. gajardoi
Br Br Br Br Ve Br/Ma Br/Ma Ma Br/Ma
9 5 8 9 10 3/7 3/5 3 3/4
El Morro, Arica, APR Playa Corazones, Arica, APR Caleta Vitor, APR Caleta Camarones, APR Medano, ANR Inca de Oro, ATR Las Chinchillas National Reserve, CR Pueblo Hundido, Pedregal, CR Til Til, MR
188280 4700 S; 188280 4700 S; 188450 4500 S; 198120 1600 S; 248360 5100 S; 268450 0800 S; 318300 2800 S; 318130 2400 S; 338060 1900 S;
Mepraia sp. M. spinolai
708190 2700 W 708190 2700 W 708200 3400 W 708160 0800 W 708330 3100 W 698540 1600 W 718060 1900 W 708570 2500 W 708550 5300 W
Br, brachypterous; Ve, vestigial wings (not included in posterior analyses); Ma, macropterous; APR, Arica and Parinacota Region; ANR, Antofagasta Region; ATR, Atacama Region; CR, Coquimbo Region; MR, Metropolitan Region. Micropterous information is not included here. a Collected as adults or nymphs reared in climatic chamber.
DNA, including the two internal transcribed spacers ITS-1 and ITS-2, and the 5.8S rRNA gene). However, the results with mitochondrial marker (cytochrome oxidase I gene) indicate a closer relationship of these individuals with M. gajardoi populations (i.e., Arica Region, see Table 1). Calleros et al. (2010) suggest that this disagreement between nuclear and mitochondrial markers in the individuals from Caleta Bandurrias may be the result of introgression due to past hybridization events or the retention of ancestral polymorphisms. Mepraia species show conspicuous alary polymorphism, unique in the Triatominae subfamily (Lent and Wygodzinsky, 1979; Schofield et al., 1998). Alary polymorphism occurs quite commonly in other reduviid subfamilies and frequently, although not exclusively, in deserticolous species. Females of both species are invariably micropterous (wingless), while males of M. spinolai are micropterous, brachypterous (wings shorter or equal than the length of the abdomen, respectively) or macropterous (wings larger than the length of the abdomen). Only 5% of M. spinolai males present brachypterous or macropterous wings (Botto-Mahan, 2004) and males of M. gajardoi are invariably brachypterous (Frı´as et al., 1998). Because wings are bi-dimensional rigid structures, they are well suited for geometric morphometric work (Dujardin, 2008). Geometric morphometrics is a modern technique that analyzes the variation of morphological shape, which captures as much biological information as possible from an anatomical structure (Rohlf and Marcus, 1993). This technique has been applied to the Triatominae subfamily for taxonomic purposes (Matias et al., 2001; Villegas et al., 2002), to distinguish between laboratory reared and field specimens (Jaramillo et al., 2002), to correlate with chromatic variation of T. infestans across several countries (Gumiel et al., 2003), to define the spatial structuring of T. infestans populations at a finer geographic scale (Schachter-Broide et al., 2004), and to retrieve relevant information about origins of a population, even from a single individual (Dujardin, 2008). Due to inexpensive cost of this approach, it could be commonly applied in entomological surveillance programs (Dujardin, 2008). In this study, we used geometric morphometrics analyses to compare wings of M. spinolai and M. gajardoi males collected from natural populations, in order to determine whether or not the two species have developed wing shape differences.
from instars III, IV and V were regularly fed with laboratory mice and reared under optimal growing conditions in a climatic chamber at 26 8C, 70% relative humidity, and 14:10-h light:dark photoperiod, until they reached sexual maturity (Botto-Mahan, 2009). 2.2. Handling and data processing of wings Adult males were sacrificed and wings carefully removed. Fresh wings were dry mounted between two slides and then photographed with a digital camera (PowerShot SD600 digital ELPH, 6.0 megapixels, Canon). Then, we selected eight landmarks (Fig. 1) on the right wing of each insect using the software COOWin (Dujardin, 2004) as previously described (Dujardin, 2008). The selected anatomical points became two-dimensional coordinate arrays that were submitted to Generalize Procrustes Analysis algorithm (GPA) (Rohlf and Slice, 1990; Bookstein, 1991; Rohlf and Marcus, 1993) using the software MOG (Dujardin, 2003). At the end of the process a matrix of continuous variables was built, which contains the wing shape information, and was free of variation in digitizing location, orientation, and scale present in the raw coordinates. Those shape variables are computed as non-uniform (partial warps) and uniform components, which describe regional and global deformations of the wing architecture, respectively (Bookstein, 1991). Previous to GPA an isometric estimator of size variation, the centroid-size, was calculated as the square root of the sum of the squared distances between the center of the configuration of landmarks and each individual landmark (Bookstein, 1991). 2.3. Size variation
[(Fig._1)TD$IG]
Wing samples were divided into three groups represented by M. gajardoi brachypterous, M. spinolai brachypterous, and M. spinolai macropterous. The centroid-sizes of the different species were compared by non-parametric analyses based on permutations (1000 runs), allowing the comparison of both means and ˜ o et al., 2009). Bonferroni correction variances of size (Caro-Rian was applied for the significance level (0.05).
2. Materials and methods 2.1. Insects Several field trips were carried out to collect nymphs and adults of Mepraia sp. in the coast and interior valleys of Chile (Table 1). Insects were manually collected by trained people. The collected specimens were brought to the laboratory, and the adults were classified in the two species according the morphological criteria suggested by Frı´as et al. (1998). Nymphs
Fig. 1. The eight landmark positions on the right wing of Mepraia species used in this study. Numbers refer to the order of collection.
[(Fig._2)TD$IG]
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Fig. 2. Photographs showing the different types of wings and connexivum colour in males of Mepraia sp.: (A) M. gajardoi brachypterous, (B) Mepraia sp. with vestigial wings (Antofagasta Region; not included in the analyses), (C) M. spinolai micropterous (not included in the analyses), (D) M. spinolai brachypterous, and (E) M. spinolai macropterous.
2.4. Shape variation
3.2. Size variation
Groups were compared by the relative warps analysis, a principal component analysis on the shape variables matrix. The software MOG (Dujardin, 2003) was used to compute the relative warps. This analysis allows to visualize the distribution of individuals on the first two relative warps, which accounts of most of the shape variation, and thus to detect the formation of clusters. Euclidean distances were calculated between consensus shape for each group; then statistical significance was estimated among distances using a null model of 1000 permutations corrected by the Bonferroni method using the software COV (Dujardin, 2006).
Mepraia spinolai macropterous males showed the larger wing size and wings of M. spinolai brachypterous males were included in the M. gajardoi wing size range (Fig. 3). Wing size analyses show statistically significant differences between M. spinolai macropterous males and brachypterous males of both Mepraia species (P < 0.0001 for both comparisons). No statistically significant differences were detected between the brachypterous males of Mepraia species (P = 0.715). The range in size was larger for M. gajardoi brachypterous males.
2.5. Allometry
The GPA algorithm showed two clearly distinguishable shapes between M. gajardoi and M. spinolai groups, with almost identical shape between brachypterous and macropterous M. spinolai males (Fig. 4). Lack of perfect superposition means shape differences, which can then be located on the wing (Fig. 4). Inspection of specimens of each group projected onto the two first principal
We also examined the shape changes resulting from changes in size, i.e., allometry. This kind of analysis is necessary to evaluate the possible shape differences explained by size variation or growth. Allometric effects were examined using the software COV (Dujardin, 2006) through a multivariate regression analysis between size and shape to test for equal slopes and, if proceed, a multivariate covariance analysis (MANCOVA) to test for significant differences among groups. The shape variables operated as dependent variables, while the groups and the centroid-size were used as independent variables (Dujardin, 2006).
3.3. Shape variation
[(Fig._3)TD$IG]
3. Results
Fig. 3. Centroid-size for wings of Mepraia species. Centroid-size variation presented as quantile plots. Vertical lines under the quantiles are individuals. Each box shows the median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. Units are pixels converted to millimeters.
3.1. Studied insects A total of 31 males of M. gajardoi (all brachypterous) and 28 males of M. spinolai (9 brachypterous and 19 macropterous) were used in this study (Table 1). Morphological examination of the studied specimens indicates that wing phenotypes and connexivum characteristics differed as previously described (Frı´as et al., 1998; Jurberg et al., 2002). Specifically, brachypterous males of M. gajardoi presented reddish little spots on the border of the connexivum, and micropterous, brachypterous and macropterous males of M. spinolai always showed a continuous reddish/orange border in the connexivum (Fig. 2). For the first time, we observed a new alary phenotype, all adult males of Mepraia sp. (N = 10) obtained from the coastal area of Antofagasta Region showed vestigial small wings (i.e., reaching up to the first abdominal segment) and reddish medium spots in the border of the connexivum (Fig. 2B). The adult condition of these individuals was tested by the observation of spermatozoons and meiotic cells in their gonads (data not shown). Vestigial wings of these adult males were not included in our morphometric analyses.
[(Fig._4)TD$IG]
Fig. 4. Shape comparison among M. gajardoi, M. spinolai brachypterous and M. spinolai macropterous using the software MOG. Each configuration represents the average configuration of each group. Each circle represents a landmark following the same numbers as shown in Fig. 1. Lack of perfect superposition means shape difference, which can then be located on the wing.
[(Fig._5)TD$IG]
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Fig. 5. Inspection of specimens of each group projected onto the two first principal components (PC or relative warps) of shape variables (partial warps plus uniform components) of Mepraia species wings. PC1 and PC2 explain 85% of the shape variation showing a clear-cut differentiation along the horizontal axis (PC1) between the two species. Ranges: PC1 from 0.0157 to 0.102, and PC2 from 0.045 to 0.046.
components (PC1 and PC2, hereafter) obtained from shape variables, show two significantly different clusters in wing shape (M. gajardoi versus M. spinolai brachypteruos P < 0.0001, M. gajardoi versus M. spinolai macropterous P < 0.0001, M. spinolai brachypterous versus macropterous P = 0.508; Fig. 5). PC1 and PC2 explained 85% of the total shape variation. 3.4. Allometry Allometric effects between M. gajardoi and M. spinolai showed a contribution of size in wing shaping (P < 0.0001). Results from comparison among the slopes of growth model for the three groups (i.e., M. gajardoi, M. spinolai brachypterous, and M. spinolai macropterous) did not show statistically significant differences (Wilks lambda = 0.490, F = 1.502, P = 0.09), therefore, the hypothesis of a common allometric model could not be rejected. According to this, we removed the size effect in our shape analyses. Differences between shapes remained significant between M. gajardoi and both brachypterous and macropterous M. spinolai (P < 0.0001 and P = 0.002, respectively) and, unlike our previous result, marginally significant for the comparison between the two wing phenotypes of M. spinolai (P = 0.085). 4. Discussion Differentiation of triatomine species within a similar complex or subspecies is a main question to be explained as well as their origin, distribution, dispersion, or epidemiological importance (Bustamante et al., 2004; Costa et al., 2009). Two different species have been described in the Mepraia genus, presenting remarkable geographical variation in abdomen colouration, ecological distribution, antennal phenotype and male wing polymorphism (Frı´as et al., 1998; Moreno et al., 2006). According to Frı´as et al. (1998), Mepraia species inhabit places with different ecological characteristics, e.g., M. gajardoi inhabits northern coastal areas, while M. spinolai dwells within the valleys and some coastal areas. However, these coastal areas exhibit more temperate climate and greater ecological diversity than those inhabited by M. gajardoi. In our study, as expected, we found that brachypterous wings of both species are smaller than macropterous wings of M. spinolai (Fig. 3). However, an overlap is observed in the wing size, appearing some brachypterous wings larger than macropterous. This overlap is due to the procedure of wing morph classification in relation to abdomen length. Then, when wings are shorter or equal than the length of the abdomen insects are considered brachypterous and when wings are larger than the length of the abdomen, macropterous. However, in this analysis of size there is no
correction for body size (i.e., total length from the clypeus to the last segment of the abdomen). This means that there is great variation in body size of macropterous males, therefore, small macropterous males present wings shorter than some brachypterous ones. A second finding of our study is the clear difference detected on wing shape between M. gajardoi and M. spinolai (Fig. 5). Even though, allometry analyses showed that size explained part of the variation in wing shape, by removing the effect of size the differences in wing shape remained significant. Interestingly, when allometry effect was removed in the comparison between both wing phenotypes of M. spinolai, we detected marginally significant differences in wing shapes. It is necessary to increase the number of M. spinolai brachypterous specimens in order to withdraw more robust conclusions in within species comparison. According to Frı´as et al. (1998), insects of Antofagasta Region show brachypterous wing morph. However, we found only insects with vestigial wings (Fig. 2B), suggesting that the geographical criterion to separate the two Mepraia species it is not clear. In fact, Calleros et al. (2010) suggest that the genetic divergence of these populations is in the range of those reported in the triatomine species closely related and should take into account the possibility of hybridization as an important source of genetic variation within the genus. Species with consistent morphological differences would arise through divergent ecological adaptation (Dujardin et al., 1999). This agrees with several studies suggesting that ecological factors constitute the main guiding force for speciation in triatomines, and morphological differentiation could be faster than the installation of genetic or reproductive barriers (Dujardin et al., 1999; MasComa and Bargues, 2009). Due to the possible existence of gene flow among populations of M. gajardoi and M. spinolai, it could not be said, under the biological criterion, that they are distinct species. Thus, the morphological species could be ecological species, a vision that fits the concept of ‘‘evolutionary unit’’ in which two populations become in different species if they follow different evolutionary destinations (Dujardin et al., 1999). This would suggest that M. gajardoi and M. spinolai populations are in a recent stage of speciation, founding differences in morphology and cytogenetics, but not necessarily strong molecular differences, especially in the contact region between two species. The existence of successful crosses, even at low frequencies (6–10%), as reported by Frı´as et al. (1998), would enable the existence of hybrids. Therefore, we cannot discard the possibility of an hybrid zone between M. spinolai and M. gajardoi, localized in the coastal areas of Antofagasta Region and the northern part of Atacama Region, similar to those reported for T. infestans and Triatoma platensis (Panzera et al., 1995; Pereira et al., 1996; Pe´rez et al., 2005) or as described in the Triatoma brasiliensis species complex (Costa et al., 2009). Genetic analyses by different markers of the individuals with vestigial wings could clarify its origin. In conclusion, the geometric morphometrics analyses on wings of Mepraia species allow us to separate two distinct groups consistent with the two described species (Frı´as et al., 1998; Jurberg et al., 2002). However, according to our findings of individuals with vestigial wings and molecular evidence reported by Calleros et al. (2010), we cannot rule out the existence of a hybrid zone in some coastal areas of the north part of Chile. Acknowledgements We thank I. Co´rdova for helping to collect insect at the field, N. Villarroel for laboratory assistance, and E. Ma´rquez for helping RC to use geometric morphometrics softwares. We especially thank B. Arriaza for showing us insect collecting sites, and A, Bacigalupo for sharing insect wings. CONAF – Coquimbo Region allowed part of
R. Campos et al. / Infection, Genetics and Evolution 11 (2011) 329–333
this research at the Reserva Nacional Las Chinchillas. This work was financed by FONDECYT 1085154 (AS), and partially by PBCT/ PSD-66 and FONDECYT 11090086 (CBM). R Campos was supported by a CONICYT – PhD fellowship. References Bookstein, F.L., 1991. Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, Cambridge, p. 435. Botto-Mahan, C., 2004. Modificacio´n fenotı´pica inducida por para´sitos: la interaccio´n Mepraia spinolai–Trypanosoma cruzi. PhD Thesis, Universidad de Chile, Santiago, 102 pp. Botto-Mahan, C., 2009. Trypanosoma cruzi induces life-history trait changes in the wild kissing bug Mepraia spinolai: implications for parasite transmission. Vector Borne Zoonotic Dis. 9, 505–510. Bustamante, D.M., Monroy, C., Menes, M., Rodas, A., Salazar-Schettino, PM., Rojas, G., Pinto, N., Guhl, F., Dujardin, J.P., 2004. Metric variation among geographic populations of the Chagas vector Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) and related species. J. Med. Entomol. 41, 296–301. Calleros, L., Panzera, F., Bargues, M.D., Monteiro, F.A., Klisiowicz, D.R., Zuriaga, M.A., Mas-Coma, S., Pe´rez, R., 2010. Systematics of Mepraia (Hemiptera–Reduviidae): cytogenetic and molecular variation. Infect. Genet. Evol. 10, 221–228. ˜ o, H., Jaramillo, N., Dujardin, J.P., 2009. Growth changes in Rhodnius Caro-Rian pallescens under simulated domestic and sylvatic conditions. Infect. Genet. Evol. 9, 162–168. ˜ a, M., Canals, M., 2002. Abundance Cattan, P.E., Pinochet, A., Botto-Mahan, C., Acun of Mepraia spinolai in a Periurban zone of Chile. Mem. Inst. Oswaldo Cruz 97, 285–287. Costa, J., Peterson, A.T., Dujardin, J.P., 2009. Morphological evidence suggests homoploid hybridization as a possible mode of speciation in the Triatominae (Hemiptera, Heteroptera, Reduviidae). Infect. Genet. Evol. 9, 263–270. Dias, J.C.P., Schofield, C.J., 1999. The evolution of Chagas disease (American Trypanosomiasis) control after 90 years since Carlos Chagas discovery. Mem. Inst. Oswaldo Cruz 94, 103–121. Dujardin, J.P., 2003. Geometric morphometrics (Procrustes superimposition, thinplate spline analyses) (MOG). http://www.mpl.ird.fr/morphometrics. Dujardin, J.P., 2004. Anatomical landmarks collection (COO). http://www.mpl.ird.fr/ morphometrics. Dujardin, J.P., 2006. Multivariate analysis of covariance (COV). http://www. mpl.ird.fr/morphometrics. Dujardin, J.P., 2008. Morphometrics applied to medical entomology. Infect. Genet. Evol. 8, 875–890. Dujardin, J.P., Panzera, P., Schofield, J.C., 1999. Triatominae as a model of morphological plasticity under ecological pressure. Mem. Inst. Oswaldo Cruz 94, 223– 228. Frı´as, D., Atria, J., 1998. Chromosomal variation, macroevolution and possible parapatric speciation in Mepraia spinolai (Porter) (Hemiptera: Reduviidae). Genet. Mol. Biol. 21, 179–184. Frı´as, D.A., Henry, A.A., Gonza´lez, C.R., 1998. Mepraia gajardoi: a newspecies of Triatominae (Hemiptera: Reduviidae) from Chile and its comparison with Mepraia spinolai. Rev. Chil. Hist. Nat. 71, 177–188.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Wing shape differentiation of Mepraia species (Hemiptera: Reduviidae) Ricardo Campos a, Carezza Botto-Mahan b, Ximena Coronado a, Nicola´s Jaramillo c, Francisco Panzera d, Aldo Solari a,* a
Programa de Biologı´a Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago 7, Chile Departamento de Ciencias Ecolo´gicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile c Seccio´n Gene´tica Evolutiva, Facultad de Ciencias, Universidad de La Repu´blica, Montevideo, Uruguay d Grupo de Biologı´a y Control de Enfermedades Infecciosas, Instituto de Biologı´a, Universidad de Antioquia, AA 1226, Medellı´n, Colombia b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 August 2010 Received in revised form 3 November 2010 Accepted 5 November 2010 Available online 24 November 2010
Mepraia is an endemic genus found in the semiarid and arid regions of north-central Chile. Until 1998, Mepraia spinolai was the only species of the genus, distributed in coastal and interior valleys from Chile between 188 and 348S. However, on the basis of karyotype and morphological characters, coastal desert populations between 188 and 268S were ranked as a new species, Mepraia gajardoi. Recently, genetic studies using nuclear and mitochondrial markers on Mepraia populations suggest that the geographical criterion to separate the two species should be reviewed. Mepraia species show conspicuous alary polymorphism, unique in the Triatominae subfamily. Females of both species are invariably micropterous, while males of M. spinolai can be micropterous, brachypterous or macropterous, and only brachypterous in M. gajardoi. In this study, we use geometric morphometrics analyses to compare male wings of M. spinolai and M. gajardoi from natural populations, in order to examine if these two species have diverged in alary shape. As expected, we found that brachypterous wings of both species are smaller than macropterous wings of M. spinolai. Additionally, we detected clear differences in shape on wings of M. gajardoi and M. spinolai, not attributable to allometric effects. For last, a new alary phenotype, insects with vestigial wings, was described here for the first time. In conclusion, our analyses on wings of Mepraia species separate two distinct groups consistent with the two described species. However, our findings of vestigial wings in some coastal areas of the north part of Chile cannot rule out the existence of a hybrid zone. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Chagas disease Triatominae vectors Mepraia gajardoi Mepraia spinolai Alary polymorphism Brachypterous Macropterous Geometric morphometrics
1. Introduction Chagas disease is a serious human parasitic disease in the Americas caused by the flagellate protozoan Trypanosoma cruzi, and transmitted by blood-sucking insects of the subfamily Triatominae (Hemiptera: Reduviidae) (Lent and Wygodzinsky, 1979; Schofield et al., 2006). Two Triatominae genera occur in Chile: Triatoma and Mepraia. Triatoma infestans has been the main domestic vector of T. cruzi, now controlled in several countries, including Chile, through widespread interventions under the auspices of the Southern Cone Initiative (Dias and Schofield, 1999). The Mepraia genus is endemic from the semiarid and arid regions of northern and central Chile, mainly found amongst rock piles but occasionally enters and colonizes domestic and peridomestic habitats (Frı´as and Atria, 1998; Schofield et al., 1998; Cattan et al., 2002). This genus was first described with the inclusion of Triatoma spinolai Porter, 1934, as Mepraia spinolai by
* Corresponding author. Tel.: +56 2 978 6062; fax: +56 2 735 5580. E-mail address: [email protected] (A. Solari). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.11.002
Mazza et al. (1940), and revalidated by Lent et al. (1994). However, even today some authors do not agree with the existence of this genus and include the Mepraia species into Triatoma genus (Schofield and Galva˜o, 2009). Until 1998, M. spinolai was the only species of the genus, distributed in coastal and interior valleys from Chile between parallels 188 and 348S (Lent and Wygodzinsky, 1979). However, on the basis of karyotype, morphological characters, and experimental crosses, coastal desert populations between parallels 188 and 268S were ranked as a new species named Mepraia gajardoi (Frı´as et al., 1998; Jurberg et al., 2002). The remaining populations, from 268 to 348S, are distributed in the interior mountains from the Atacama to the Metropolitan Region, maintaining the name M. spinolai (Frı´as et al., 1998). Recently, genetic studies using nuclear and mitochondrial markers on Mepraia sp. populations suggest that the geographical criterion initially proposed by Frı´as et al. (1998) to separate the two Mepraia species should be reviewed (Calleros et al., 2010). According to these authors, individuals from Caleta Bandurrias (258S, Antofagasta Region) are more related to M. spinolai populations (i.e., from Atacama to Metropolitan Regions, see Table 1) based on nuclear markers (karyotype and nuclear ribosomal
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330 Table 1 Location of collecting sites of Mepraia sp. Species
Alary phenotype
Number of adult malesa
Location name
Latitude; longitude
M. gajardoi
Br Br Br Br Ve Br/Ma Br/Ma Ma Br/Ma
9 5 8 9 10 3/7 3/5 3 3/4
El Morro, Arica, APR Playa Corazones, Arica, APR Caleta Vitor, APR Caleta Camarones, APR Medano, ANR Inca de Oro, ATR Las Chinchillas National Reserve, CR Pueblo Hundido, Pedregal, CR Til Til, MR
188280 4700 S; 188280 4700 S; 188450 4500 S; 198120 1600 S; 248360 5100 S; 268450 0800 S; 318300 2800 S; 318130 2400 S; 338060 1900 S;
Mepraia sp. M. spinolai
708190 2700 W 708190 2700 W 708200 3400 W 708160 0800 W 708330 3100 W 698540 1600 W 718060 1900 W 708570 2500 W 708550 5300 W
Br, brachypterous; Ve, vestigial wings (not included in posterior analyses); Ma, macropterous; APR, Arica and Parinacota Region; ANR, Antofagasta Region; ATR, Atacama Region; CR, Coquimbo Region; MR, Metropolitan Region. Micropterous information is not included here. a Collected as adults or nymphs reared in climatic chamber.
DNA, including the two internal transcribed spacers ITS-1 and ITS-2, and the 5.8S rRNA gene). However, the results with mitochondrial marker (cytochrome oxidase I gene) indicate a closer relationship of these individuals with M. gajardoi populations (i.e., Arica Region, see Table 1). Calleros et al. (2010) suggest that this disagreement between nuclear and mitochondrial markers in the individuals from Caleta Bandurrias may be the result of introgression due to past hybridization events or the retention of ancestral polymorphisms. Mepraia species show conspicuous alary polymorphism, unique in the Triatominae subfamily (Lent and Wygodzinsky, 1979; Schofield et al., 1998). Alary polymorphism occurs quite commonly in other reduviid subfamilies and frequently, although not exclusively, in deserticolous species. Females of both species are invariably micropterous (wingless), while males of M. spinolai are micropterous, brachypterous (wings shorter or equal than the length of the abdomen, respectively) or macropterous (wings larger than the length of the abdomen). Only 5% of M. spinolai males present brachypterous or macropterous wings (Botto-Mahan, 2004) and males of M. gajardoi are invariably brachypterous (Frı´as et al., 1998). Because wings are bi-dimensional rigid structures, they are well suited for geometric morphometric work (Dujardin, 2008). Geometric morphometrics is a modern technique that analyzes the variation of morphological shape, which captures as much biological information as possible from an anatomical structure (Rohlf and Marcus, 1993). This technique has been applied to the Triatominae subfamily for taxonomic purposes (Matias et al., 2001; Villegas et al., 2002), to distinguish between laboratory reared and field specimens (Jaramillo et al., 2002), to correlate with chromatic variation of T. infestans across several countries (Gumiel et al., 2003), to define the spatial structuring of T. infestans populations at a finer geographic scale (Schachter-Broide et al., 2004), and to retrieve relevant information about origins of a population, even from a single individual (Dujardin, 2008). Due to inexpensive cost of this approach, it could be commonly applied in entomological surveillance programs (Dujardin, 2008). In this study, we used geometric morphometrics analyses to compare wings of M. spinolai and M. gajardoi males collected from natural populations, in order to determine whether or not the two species have developed wing shape differences.
from instars III, IV and V were regularly fed with laboratory mice and reared under optimal growing conditions in a climatic chamber at 26 8C, 70% relative humidity, and 14:10-h light:dark photoperiod, until they reached sexual maturity (Botto-Mahan, 2009). 2.2. Handling and data processing of wings Adult males were sacrificed and wings carefully removed. Fresh wings were dry mounted between two slides and then photographed with a digital camera (PowerShot SD600 digital ELPH, 6.0 megapixels, Canon). Then, we selected eight landmarks (Fig. 1) on the right wing of each insect using the software COOWin (Dujardin, 2004) as previously described (Dujardin, 2008). The selected anatomical points became two-dimensional coordinate arrays that were submitted to Generalize Procrustes Analysis algorithm (GPA) (Rohlf and Slice, 1990; Bookstein, 1991; Rohlf and Marcus, 1993) using the software MOG (Dujardin, 2003). At the end of the process a matrix of continuous variables was built, which contains the wing shape information, and was free of variation in digitizing location, orientation, and scale present in the raw coordinates. Those shape variables are computed as non-uniform (partial warps) and uniform components, which describe regional and global deformations of the wing architecture, respectively (Bookstein, 1991). Previous to GPA an isometric estimator of size variation, the centroid-size, was calculated as the square root of the sum of the squared distances between the center of the configuration of landmarks and each individual landmark (Bookstein, 1991). 2.3. Size variation
[(Fig._1)TD$IG]
Wing samples were divided into three groups represented by M. gajardoi brachypterous, M. spinolai brachypterous, and M. spinolai macropterous. The centroid-sizes of the different species were compared by non-parametric analyses based on permutations (1000 runs), allowing the comparison of both means and ˜ o et al., 2009). Bonferroni correction variances of size (Caro-Rian was applied for the significance level (0.05).
2. Materials and methods 2.1. Insects Several field trips were carried out to collect nymphs and adults of Mepraia sp. in the coast and interior valleys of Chile (Table 1). Insects were manually collected by trained people. The collected specimens were brought to the laboratory, and the adults were classified in the two species according the morphological criteria suggested by Frı´as et al. (1998). Nymphs
Fig. 1. The eight landmark positions on the right wing of Mepraia species used in this study. Numbers refer to the order of collection.
[(Fig._2)TD$IG]
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331
Fig. 2. Photographs showing the different types of wings and connexivum colour in males of Mepraia sp.: (A) M. gajardoi brachypterous, (B) Mepraia sp. with vestigial wings (Antofagasta Region; not included in the analyses), (C) M. spinolai micropterous (not included in the analyses), (D) M. spinolai brachypterous, and (E) M. spinolai macropterous.
2.4. Shape variation
3.2. Size variation
Groups were compared by the relative warps analysis, a principal component analysis on the shape variables matrix. The software MOG (Dujardin, 2003) was used to compute the relative warps. This analysis allows to visualize the distribution of individuals on the first two relative warps, which accounts of most of the shape variation, and thus to detect the formation of clusters. Euclidean distances were calculated between consensus shape for each group; then statistical significance was estimated among distances using a null model of 1000 permutations corrected by the Bonferroni method using the software COV (Dujardin, 2006).
Mepraia spinolai macropterous males showed the larger wing size and wings of M. spinolai brachypterous males were included in the M. gajardoi wing size range (Fig. 3). Wing size analyses show statistically significant differences between M. spinolai macropterous males and brachypterous males of both Mepraia species (P < 0.0001 for both comparisons). No statistically significant differences were detected between the brachypterous males of Mepraia species (P = 0.715). The range in size was larger for M. gajardoi brachypterous males.
2.5. Allometry
The GPA algorithm showed two clearly distinguishable shapes between M. gajardoi and M. spinolai groups, with almost identical shape between brachypterous and macropterous M. spinolai males (Fig. 4). Lack of perfect superposition means shape differences, which can then be located on the wing (Fig. 4). Inspection of specimens of each group projected onto the two first principal
We also examined the shape changes resulting from changes in size, i.e., allometry. This kind of analysis is necessary to evaluate the possible shape differences explained by size variation or growth. Allometric effects were examined using the software COV (Dujardin, 2006) through a multivariate regression analysis between size and shape to test for equal slopes and, if proceed, a multivariate covariance analysis (MANCOVA) to test for significant differences among groups. The shape variables operated as dependent variables, while the groups and the centroid-size were used as independent variables (Dujardin, 2006).
3.3. Shape variation
[(Fig._3)TD$IG]
3. Results
Fig. 3. Centroid-size for wings of Mepraia species. Centroid-size variation presented as quantile plots. Vertical lines under the quantiles are individuals. Each box shows the median as a line across the middle and the quartiles (25th and 75th percentiles) as its ends. Units are pixels converted to millimeters.
3.1. Studied insects A total of 31 males of M. gajardoi (all brachypterous) and 28 males of M. spinolai (9 brachypterous and 19 macropterous) were used in this study (Table 1). Morphological examination of the studied specimens indicates that wing phenotypes and connexivum characteristics differed as previously described (Frı´as et al., 1998; Jurberg et al., 2002). Specifically, brachypterous males of M. gajardoi presented reddish little spots on the border of the connexivum, and micropterous, brachypterous and macropterous males of M. spinolai always showed a continuous reddish/orange border in the connexivum (Fig. 2). For the first time, we observed a new alary phenotype, all adult males of Mepraia sp. (N = 10) obtained from the coastal area of Antofagasta Region showed vestigial small wings (i.e., reaching up to the first abdominal segment) and reddish medium spots in the border of the connexivum (Fig. 2B). The adult condition of these individuals was tested by the observation of spermatozoons and meiotic cells in their gonads (data not shown). Vestigial wings of these adult males were not included in our morphometric analyses.
[(Fig._4)TD$IG]
Fig. 4. Shape comparison among M. gajardoi, M. spinolai brachypterous and M. spinolai macropterous using the software MOG. Each configuration represents the average configuration of each group. Each circle represents a landmark following the same numbers as shown in Fig. 1. Lack of perfect superposition means shape difference, which can then be located on the wing.
[(Fig._5)TD$IG]
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Fig. 5. Inspection of specimens of each group projected onto the two first principal components (PC or relative warps) of shape variables (partial warps plus uniform components) of Mepraia species wings. PC1 and PC2 explain 85% of the shape variation showing a clear-cut differentiation along the horizontal axis (PC1) between the two species. Ranges: PC1 from 0.0157 to 0.102, and PC2 from 0.045 to 0.046.
components (PC1 and PC2, hereafter) obtained from shape variables, show two significantly different clusters in wing shape (M. gajardoi versus M. spinolai brachypteruos P < 0.0001, M. gajardoi versus M. spinolai macropterous P < 0.0001, M. spinolai brachypterous versus macropterous P = 0.508; Fig. 5). PC1 and PC2 explained 85% of the total shape variation. 3.4. Allometry Allometric effects between M. gajardoi and M. spinolai showed a contribution of size in wing shaping (P < 0.0001). Results from comparison among the slopes of growth model for the three groups (i.e., M. gajardoi, M. spinolai brachypterous, and M. spinolai macropterous) did not show statistically significant differences (Wilks lambda = 0.490, F = 1.502, P = 0.09), therefore, the hypothesis of a common allometric model could not be rejected. According to this, we removed the size effect in our shape analyses. Differences between shapes remained significant between M. gajardoi and both brachypterous and macropterous M. spinolai (P < 0.0001 and P = 0.002, respectively) and, unlike our previous result, marginally significant for the comparison between the two wing phenotypes of M. spinolai (P = 0.085). 4. Discussion Differentiation of triatomine species within a similar complex or subspecies is a main question to be explained as well as their origin, distribution, dispersion, or epidemiological importance (Bustamante et al., 2004; Costa et al., 2009). Two different species have been described in the Mepraia genus, presenting remarkable geographical variation in abdomen colouration, ecological distribution, antennal phenotype and male wing polymorphism (Frı´as et al., 1998; Moreno et al., 2006). According to Frı´as et al. (1998), Mepraia species inhabit places with different ecological characteristics, e.g., M. gajardoi inhabits northern coastal areas, while M. spinolai dwells within the valleys and some coastal areas. However, these coastal areas exhibit more temperate climate and greater ecological diversity than those inhabited by M. gajardoi. In our study, as expected, we found that brachypterous wings of both species are smaller than macropterous wings of M. spinolai (Fig. 3). However, an overlap is observed in the wing size, appearing some brachypterous wings larger than macropterous. This overlap is due to the procedure of wing morph classification in relation to abdomen length. Then, when wings are shorter or equal than the length of the abdomen insects are considered brachypterous and when wings are larger than the length of the abdomen, macropterous. However, in this analysis of size there is no
correction for body size (i.e., total length from the clypeus to the last segment of the abdomen). This means that there is great variation in body size of macropterous males, therefore, small macropterous males present wings shorter than some brachypterous ones. A second finding of our study is the clear difference detected on wing shape between M. gajardoi and M. spinolai (Fig. 5). Even though, allometry analyses showed that size explained part of the variation in wing shape, by removing the effect of size the differences in wing shape remained significant. Interestingly, when allometry effect was removed in the comparison between both wing phenotypes of M. spinolai, we detected marginally significant differences in wing shapes. It is necessary to increase the number of M. spinolai brachypterous specimens in order to withdraw more robust conclusions in within species comparison. According to Frı´as et al. (1998), insects of Antofagasta Region show brachypterous wing morph. However, we found only insects with vestigial wings (Fig. 2B), suggesting that the geographical criterion to separate the two Mepraia species it is not clear. In fact, Calleros et al. (2010) suggest that the genetic divergence of these populations is in the range of those reported in the triatomine species closely related and should take into account the possibility of hybridization as an important source of genetic variation within the genus. Species with consistent morphological differences would arise through divergent ecological adaptation (Dujardin et al., 1999). This agrees with several studies suggesting that ecological factors constitute the main guiding force for speciation in triatomines, and morphological differentiation could be faster than the installation of genetic or reproductive barriers (Dujardin et al., 1999; MasComa and Bargues, 2009). Due to the possible existence of gene flow among populations of M. gajardoi and M. spinolai, it could not be said, under the biological criterion, that they are distinct species. Thus, the morphological species could be ecological species, a vision that fits the concept of ‘‘evolutionary unit’’ in which two populations become in different species if they follow different evolutionary destinations (Dujardin et al., 1999). This would suggest that M. gajardoi and M. spinolai populations are in a recent stage of speciation, founding differences in morphology and cytogenetics, but not necessarily strong molecular differences, especially in the contact region between two species. The existence of successful crosses, even at low frequencies (6–10%), as reported by Frı´as et al. (1998), would enable the existence of hybrids. Therefore, we cannot discard the possibility of an hybrid zone between M. spinolai and M. gajardoi, localized in the coastal areas of Antofagasta Region and the northern part of Atacama Region, similar to those reported for T. infestans and Triatoma platensis (Panzera et al., 1995; Pereira et al., 1996; Pe´rez et al., 2005) or as described in the Triatoma brasiliensis species complex (Costa et al., 2009). Genetic analyses by different markers of the individuals with vestigial wings could clarify its origin. In conclusion, the geometric morphometrics analyses on wings of Mepraia species allow us to separate two distinct groups consistent with the two described species (Frı´as et al., 1998; Jurberg et al., 2002). However, according to our findings of individuals with vestigial wings and molecular evidence reported by Calleros et al. (2010), we cannot rule out the existence of a hybrid zone in some coastal areas of the north part of Chile. Acknowledgements We thank I. Co´rdova for helping to collect insect at the field, N. Villarroel for laboratory assistance, and E. Ma´rquez for helping RC to use geometric morphometrics softwares. We especially thank B. Arriaza for showing us insect collecting sites, and A, Bacigalupo for sharing insect wings. CONAF – Coquimbo Region allowed part of
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