Head shape variation in response to diet in Triatoma williami (Hemiptera, Reduviidae: Triatominae), a possible Chagas disease vector of legal Amazônia

Accepted Manuscript Title: Head shape variation in response to diet in Triatoma williami (Hemiptera, Reduviidae: Triatominae), a possible Chagas disease vector of legal Amazˆonia Authors: Rosaline Rocha Lunardi, Hugo A. Ben´ıtez, Tha´ıs Peres Cˆamara, Let´ıcia Pinho Gomes, Wagner Welber Arrais-Silva PII: DOI: Reference:

S0044-5231(17)30023-2 http://dx.doi.org/doi:10.1016/j.jcz.2017.04.001 JCZ 25461

To appear in: Received date: Revised date: Accepted date:

11-11-2016 3-4-2017 4-4-2017

Please cite this article as: Lunardi, Rosaline Rocha, Ben´ıtez, Hugo A., Cˆamara, Tha´ıs Peres, Gomes, Let´ıcia Pinho, Arrais-Silva, Wagner Welber, Head shape variation in response to diet in Triatoma williami (Hemiptera, Reduviidae: Triatominae), a possible Chagas disease vector of legal Amazˆonia.Zoologischer Anzeiger - A Journal of Comparative Zoology http://dx.doi.org/10.1016/j.jcz.2017.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Head shape variation in response to diet in Triatoma williami (Hemiptera, Reduviidae: Triatominae), a possible Chagas Disease Vector of Legal Amazônia Rosaline Rocha Lunardi1 *¶, Hugo A. Benítez2¶, Thaís Peres Câmara1&, Letícia Pinho Gomes1& Wagner Welber Arrais-Silva1

1Laboratório

de Parasitos e Vetores, Campus Universitário do Araguaia, Universidade Federal de Mato Grosso, Caixa Postal 232, 78600-000, Barra do Garças, Mato Grosso, Brazil 2

Departamento de Recursos Ambientales, Facultad de Ciencias Agronómicas, Universidad de Tarapacá, Arica, Chile.

*Corresponding author E-mail: [email protected]

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Abstract Triatoma williami is naturally infected by Trypanosoma cruzi, the ethiological agent of Chagas disease, the most significant cause of morbidity and mortality in South and Central America. There is a lack of data demonstrating the bionomic aspects, the vectorial competence or the natural ecotope and the wild hosts of T. williami, although this species may be considered secondary vector because it maintains their wild condition and show synanthropic potential, colonizing the peridomiciles and frequently invading the household. The synanthropy represents a secondary adaptation by sylvatic species in response to environmental changes, and this adaptability to human dwellings depends on the plasticity of triatomine. This study describes for the first time the shape plasticity of T. williami in response to diet (blood). Two groups of triatomines was exposed to a sole blood meal source, mammalian and bird, to assess the effect of blood meal source on head morphology. The variation was analyzed using geometric morphometric tools and showed that T. williami has higher morphological variation in shape associated with blood source. This results represent an important representation of the shape adaptation of T. williami and a contribution to the knowledge of morphometrics variation of insect vector of diseases. Keywords: Morphometrics, Shape, Diet influence, Vector ecology. Introduction

Morphological adaptation has been a core element in compared biology, especially in the study of organismal diversification and evolutionary innovation (Pigliucci et al., 2006; WestEberhard, 1989, 2005). It is well known that all organisms (including the sessile animals) exhibit at least some degree of plasticity, that is, their function is affected by external conditions (Foster, 1979; Mokady et al., 1999). Despite the obvious adaptive significance of plasticity, it is clearly not without limits and apparent constraints. Organisms are not infinitely plastic but rather limited in the range of environments they can respond to.

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Triatomines are well known as a high plastic species, where morphological analyses have been used to understand the process of plasticity and infer about the changes of this morphometric phenomena (Dujardin et al., 2009; Hernández et al., 2011; Nattero et al., 2013). In the last few years’ studies about phenotypic plasticity using morphometric have been analysing the genetic effect of this pattern. Nattero et al. (2013) found that mostly the effect of variation of these species are related to quality of blood ingested during nymph instars and adult stages. In the last decades, evolutionary biologists have combined accurate mathematical tools that reduce error in determining the morphological changes and effects on species (Bookstein, 1991; Rohlf and Marcus, 1993; Wagner, 1984). One of the methods developed from this process is the shape analysis based on statistical multivariate techniques and novel visualisation methods. Geometric morphometrics (GMM) is a coordinate-based method, so that their primary data are 2D or 3D Cartesian coordinates of anatomically distinguishable landmarks (i.e. discrete anatomical points that are homologous among all the individuals under analysis) (Adams et al., 2004; Adams et al., 2013, Bookstein, 1998, Rohlf and Marcus, 1993, Rohlf and Slice, 1990). In the present study we will analyse the influence of the diet at the head shape variation in one of the species of the subfamily Triatominae. This subfamily includes 18 genera and 141 species (Jurberg et al., 2010). These insects are obligatory hematophagous and are potential vectors of Trypanosoma cruzi (Chagas, 1909), that is the etiologic agent of Chagas Disease or American trypanosomiasis, one of the most important parasitic diseases in Latin America (Soares et al., 2014). Although triatomines are primarily sylvatic, some species occasionally are attracted to the interior of the dwellings and a few species, such as Triatoma infestans and Rhodnius prolixus, are mainly domestic (Depickère et al., 2012; Noireau et al., 2005). The species found in sylvatic habitats generally play a limited epidemiological role compared to domestic species, but they may act as synanthropic vectors of T. cruzi and as a reinfestation source of dwellings after insecticide 3

spraying (Marti et al., 2014; Noireau et al., 2000). These species are considered secondary vectors because they maintain their wild condition, they are generally autochthonous and they show synanthropic potential, colonizing the peridomiciles and frequently invading the household where they sometimes establish colonies (Noireau et al., 2005; Rodríguez et al., 2013). The synanthropy represents a secondary adaptation by sylvatic species in response to environmental changes, and this adaptability depends on the plasticity of triatomines (Forattini and Service, 1989). In general, all triatomines are considered plastic insects that develop rapid morphological changes in response to environmental variability. The organisms that have this flexibility in the expression of a character have a potential advantage for dealing with environmental heterogeneity (Ernande and Dieckmann, 2004). This ability named phenotypic plasticity, is defined as the capacity of a single genotype to exhibit variable phenotypes in response to variation in the environment (Fordyce, 2006; Whitman and Agrawal, 2009; Whitman and Ananthakrishnan, 2009), and is considered essential for understanding the development and maintenance of morphological variation (Nattero et al., 2013a; Pigliucci, 2005; Pigliucci et al., 2006). A better understanding of different aspects in adaptation and evolution of Triatominae populations may contribute to the selection of appropriate vector control strategies in endemic areas. In this way, the quantitative study of phenotypic traits and nutritional variables could be informative about the population structure and the relative mobility of vectors (Hernández et al., 2011). In this context, morphological plasticity in response to blood source may have important ecological consequence and may predict the synanthopic success of species. Blood source would influence on different phenotypic dimensions in ontogenetic trajectory, and represent environments that are different enough to cause significant variations in head shape and size of triatomines (Nattero et al., 2013a). 4

Therefore, the principal aims of this study was investigate and quantify the phenotypic shape variation of T. williami triggered by two different blood sources, in mammals and birds.

Materials and Methods Data collection Adult specimens of T. williami were spontaneously collected between May and August, 2012 in the outside of a military building located in a natural reserve, in the municipality of Barra do Garças, Mato Grosso. The nymphs of the first generation of wild parental were randomly selected, forming two groups with 100 specimens each. The groups were fed at a determined source from the first nymphal stage and therefore there was no selection according to sex (macroscopically visible only in adults). The accumulated mortality was 57% in the group fed in mammals and 84% in the group fed on birds, until the N5 stage. The insects were identified through their external morphological characters, according to Lent and Wygodzinsky (1979). They were maintained in a controlled environment at a temperature of 27 ± 1oC and relative humidity of 70% ± 10, fed weekly on quail (Coturnix coturnix). The bird group (AV) was fed exclusively on quail (average feeding time 32.9 min) and the mammal group (MAM) exclusively on mice (average feeding time 50.7 min) (Mus musculus).

Morphometric Analysis Eight anatomical landmarks (based on their anatomical traits) were digitized in the head of T. williami, in dorsal and profile view in order to have the complete head variation in a 2D view (Fig. 1), using the software program tpsDIG V2.17 (Rohlf, 2013), for which coordinates were generated from all the landmarks and the head shape information was extracted using a full 5

Procrustes fit. Procrustes superimposition is a procedure that removes the information of size, position and orientation in order to obtain shape variables (Rohlf and Slice, 1990). For the dorsal view, the symmetry was taken into account, and the symmetric component of shape was calculated from the averages of original and reflected copies (Klingenberg et al., 2002). In order to avoid any inaccuracy in the sampling and landmarking process it is critically important to perform a procedure to test the measurement error (ME), which was conducted comparing the original dataset with a control of repeated measures comparing the values of the MS of the individual values with the error by a Procustes ANOVA (e.g. Klingenberg and McIntyre, 1998). In order to quantify the shape variation related with the shape dimensions, a principal component analysis (PCA) was carried out, and to evaluate the differences between sexual dimorphism and their differences in diets a canonical variate analysis (CVA) was performed Both analysed traits were evaluated by a two way ANOVA in order to find a significant influence in shape. The results were reported as Procrustes distances and the respective P values for these distances, after permutation tests (10000 runs), were reported. All the abovementioned analyses were performed using MorphoJ v1.05d (Klingenberg, 2011).

Size and Centroid size comparison A proxy of size was used in order to get the maximum of information from the morphological plasticity effect of the blood meal differences. The proxy of size was the centroid size (Bookstein, 1986, 1989, Mosimann, 1970). The centroid size (CS) defined as the square root of the sum of squared distances of a set of landmarks from their centroid (Dryden and Mardia, 1998). The level of statistic differentiation between shape and centroid size was assessed by

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computing a two way ANOVA using two factors, sex and food source. The results were reported as sums of squares (SS) and mean squares (MS), which are dimensionless (Arnqvist and Martensson, 1998; Cardini and Elton, 2007). Results The Procrustes ANOVA for assessing the measurement error of both views (dorsal and profile) showed that the MS for individual variation exceeded the measurement error (Table 1). The PCA shows that the first few PC’s accumulated most shape variation in very few dimensions for both views. In the head dorsal view, the first three PC’s account for 80.3% (PC1: 50.3%, PC2: 21.1%, PC3: 8.9%) and for the head profile view the first two PC’s account for 62.3% (PC1: 27.4%, PC2: 21.4%, PC3: 13.4%) providing a reasonable approximation of the total amount of variation. The average head shape shows that blood feed affects the morphology and they are also clearly differentiated by sex. In the individuals (male and female average morphology) that were feeding with the mammal blood, the variation at the dorsal view was principally narrowing in the landmarks 1 and 8 (Apex of gena) and a symmetric left broadening of the central landmaks of the head, making the morphology a bit narrow between the tip of the head and the intersection of the eye (Fig 2A). For the profile view, the morphology shows a broadening of the landmarks 4 and 5 with Ocelo anterior and the intersection of the head base and neck, and narrowing in the landmark 1 and 8 (Fig 2B). For the individuals feeding the bird blood, the dorsal shape show a vector movement of the landmarks 1 and 8 showing a broadening pattern and global expansion of to the central landmarks giving a thinner morphology. For the profile shape the landmarks displacement were principally from the central landmarks. Where a narrowed variation was noticeable of the landmark 5 on the intersection of head base and neck.

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The grouped CVA showed a strong differentiation between diet and sex that were quantified comparing the Procustes distances. Graphically this variation can also be observed using the first two CVs are aligned with the major axes of variation among groups. Therefore, they account for the maximum amount of among-group variance relative to within-group variance (Fig 3 a and b). In order to confirm the significance of the diet and sex variation the ANOVA of the two investigated positions, showed highly significant differences for shape between sex and diet, nevertheless, the centroid size only shows differences between the sex and not clear differences on the diet (Table 2).

Discussion This study has used geometric morphometrics to analyse the phenotypic plasticity of the T. williami as a consequence of the blood source diet in mammals and birds. This tool was found to be useful for exploring and evaluating the levels of morphological variation and sexual shape dimorphism, confirming a significant variation due to the blood sources in mammals and birds. Our results demonstrate that the diets on two food sources, with complete different nutritional background (mammal and birds) induced phenotypic plasticity. Findings confirm previous studies in T. infestans suggesting that morphological expression on the phenotype is a consequence of developmental allocation to tissue growth that maintains growth and development of head (Nattero et al., 2013a; Nattero et al., 2013b). The individuals that are feeding on the mammals blood shown a wider and shorter morphology effect of the narrowed landmarks 1 and 8 in contrast to the individuals feeding on bird blood, where in accordance with previous studies of Nattero et al. (2013a), the effect of the bird diet on the morphology generates a thinner head shape (Fig. 2).

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Therefore, the results suggest that differences observed in the head shape of T. williami on two food sources could be a plastic response to interaction between insect and host. Triatomines obtain their blood feed directly from arterioles or venules of their vertebrate hosts, causing the feeding performance differed greatly among species mainly due to factors related to the host physiology and the insect feeding apparatus (Paim et al., 2011; Sant'Anna et al., 2001). The feeding efficiency of triatomines is directly influenced by the anti-haemo static activity of the saliva; in response to haemostasis of a particular host, it would facilitate the maintenance of a steady flow of blood during feeding (Araujo et al., 2011; Nattero et al., 2013a). The saliva profile of T. williami remains unknown, and it cannot support any explanation about differences in blood ingestion or feeding process. In contrast, it is widely recognized that birds and mammals exhibit differences in the haemostatic features, although basic mechanisms of haemostasis are conserved in both (Guarneri et al., 2000; Lewis, 1996; Nattero et al., 2013b). Birds have thrombocytes that perform a similar function to mammalian platelets, but are less effective, since they do not respond with the same intensity to platelet aggregation inducers (Araujo et al., 2009; Araujo et al., 2011; Martinez-Abadias et al., 2011). Birds also appear to lack some coagulation factors, particularly in the intrinsic coagulation system (Lewis, 1996). As a consequence of differences in the haemostatic mechanisms and blood viscosity, higher in mammals compared to birds (Baskurt, 2007), the sucking process of triatomines may require more mechanical effort in mammals that was principally reflected on a wider head, the morphology adapted to more “complicated type of food” (Fig. 2). The differences in blood features presented by birds and mammals may affect the ingestion of blood by triatomines. This blood intake capacity of the insect is promoted by the cibarial pump, the musculature of which occupies practically the full length of the head (Bennet-Clark, 1963). This powerful pump creates negative pressure between the opening of the food canal and the lumen of the alimentary compartment; therefore, blood viscosity, size of red blood cells and 9

their capacity to deform can be directly associated with the muscular effort of the cibarial pump (Kingsolver and Daniel, 1995). Considering that cibarial pump muscles nearly fill the head capsule, that viscosity of the diet interferes with the blood sucking and that the haemostasis mechanism is different between birds and mammals. The phenotypic plasticity observed in the head shape of T. williami could be due to an adaptation to its hosts, with different development of the cibarial pump muscles. This possibility was shown in T. infestans, that had greater development of the pump muscles, when fed on mammal compared to birds, resulting in higher head shapes changes (Nattero et al., 2013a; Nattero et al., 2013b). In addition to the factors stated above, several other factors may also affect insect feeding, especially on live hosts, the blood diet varies on the same host depending on the blood flow of the cannulated vessel (Araujo et al., 2009). The sexual dimorphism presented in head shape and size of T. williami (Fig. 3), with females greater than males, occur frequently among triatomine species (Gaspe et al., 2012; Nattero et al., 2013a; Vargas et al., 2006). In many species, shape and size variationare related with nutritional quality of blood (Feliciangeli et al., 2007; Guarneri et al., 2000). It is worth mentioning that T. williami did not show variations in the head centroid size related to the blood source, unlike the observations made in T. infestans feeding on birds or mammals, whose head was greater in feeding on mammals, and the body was greater in those feeding on birds (Nattero et al., 2013a). The lack of association between these sizes would indicate more variation in the insect feeding behavior or in the host blood nutritional value, whereas size is often considered a more labile character than shape (Dujardin et al., 2014). Size variation is more influenced by environmental factors, whereas shape variation has a stronger genetic component (Klingenberg, 2002; 2010; Klingenberg et al., 2010; Klingenberg and Leamy, 2001). In contrast, T. williami head centroid size was more stable than shape, when exposed to different blood diet sources. Despite the 10

shape fits, the idea of a genetic trait, however, also varies according to the environmental conditions, as observed here in T. williami and also T. dimidiate (e.g. Dujardin et al., 2009). We showed for the first time that T. williami has higher morphological plasticity in shape associated with blood source, in spite of the presence of stasis in size. However, there still remains the challenge to test whether plasticity confers a fitness advantage to culminate in domiciliation. Acknowledgements This work was supported by Fundação de Amparo à Pesquisa do Estado de Mato Grosso, Conselho Nacional do Desenvolvimento Científico and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. References Adams, D.C., Rohlf, F.J., Slice, D.E., 2004. Geometric morphometrics: ten years of progress following the 'revolution'. Italian Journal of Zoology 71, 5-16. Adams, D.C., Rohlf, F.J., Slice, D.E., 2013. A field comes of age: geometric morphometrics in the 21st century. Hystrix-Italian Journal of Mammalogy 24, 7-14. Araujo, R.N., Pereira, M.H., Soares, A.C., Pereira, I.D., Diotaiuti, L., Gontijo, N.F., Lehane, M.J., Guarneri, A.A., 2009. Effect of intestinal erythrocyte agglutination on the feeding performance of Triatoma brasiliensis (Hemiptera: Reduviidae). Journal of insect physiology 55, 862-868. Araujo, R.N., Soares, A.C., Gontijo, A.F., Guarneri, A.A., Pereira, M.H., Gontijo, N.F., 2011. Electromyogram of the cibarial pump and the feeding process in hematophagous Hemiptera. INTECH Open Access Publisher.

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Figure Legends

Figure 1: Representation of the 8 morphological landmarks identified in two head views of Triatoma williami. A: Dorsal View, B: Profile View.

Figure 2: Wireframe representation of the phenotypic plasticity found between the two blood food sources AV: Coturnix coturnix and MAM: Mus musculus A: Dorsal View, B: Profile View.

Figure 3: Canonical variate analysis of the head shape comparing the sexual shape dimorphism and blood food sources shape for the different views A: Dorsal View, B: Profile View. The wireframe represents the average head shape by sex and blood food source.

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Fig.1

19

2a

20

2b

21

Fig.3a

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Fig.3b

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Table 1. Measurement error procrustes analysis of variance for both centroid size and head shape of Triatoma williami.Sums of squares (SS) and mean squares (MS) are in units of Procrustes distances (dimensionless) Dorsal View

SS

MS

df

F

P

Pillai tr.

P(param)

5050.79 <.0001

n.a

n.a

Effect Individual 2185488.05 Error 1 440.714998

Centroid size 40472.001 54 8.013 55 Shape

Effect Individual 0.03976375 Error 1 0.00099226 Profile View

SS

6.14E-05 1.5034E-06

648 660

40.82

<.0001

11.09

<0.0001

MS

df

F

P

Pillai tr.

P(param)

1821.82 <.0001

n.a

n.a

84.51

11.61

<0.0001

Effect Individual 1101127.227 Error 1 616.745492

Centroid size 22471.9 49 12.33491 50 Shape

Effect Individual 0.05579351 Error 1 0.00067368

9.49E-05 1.1228E-06

588 600

<.0001

24

Table 2: Procrustes ANOVA for both centroid size and shape of Triatomawilliami. Sums of squares (SS) and mean squares (MS)are in units of Procrustes distances (dimensionless). Dorsal View Centroid size Effect

SS

Sex

468461.3279 468461.3279 1

Blood Source 3337.56206 Individual

MS

3337.56206

df

1

617544.1754 11875.84953 52

F

P

39.45 <.0001 0.28

0.5983

n.a

n.a

F

P

Shape Effect

SS

MS

Sex

0.00147508

0.000245846 6

5.31

<.0001

Blood Source 0.00127951

0.000213252 6

4.61

0.0002

Individual

9.7899E-06

324 n.a

n.a

MS

df

P

0.00317193

df

Profile View Centroid size Effect

SS

Sex

271858.7489 271858.7489 1

Blood Source 8160.938121 8160.938121 1 Individual

F

47.44 <.0001 1.42

269339.2275 5730.621863 47 n.a

0.2387 n.a

Shape Effect

SS

MS

df

F

Sex

0.00306804

0.00025567

12 5.94

P <.0001

Blood Source 0.00098303

8.19188E-05 12 1.9

0.0316

Individual

4.30627E-05 564 n.a

n.a

0.02428738

25