Accepted Manuscript

Title: Head shape variation in response to diet in Triatoma

williami (Hemiptera, Reduviidae: Triatominae), a possible
Chagas disease vector of legal Amazônia

Authors: Rosaline Rocha Lunardi, Hugo A. Benı́tez, Thaı́s

Peres Câmara, Letı́cia Pinho Gomes, Wagner Welber
Arrais-Silva

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

To appear in:

Received date: 11-11-2016

Revised date: 3-4-2017
Accepted date: 4-4-2017

Please cite this article as: Lunardi, Rosaline Rocha, Benı́tez, Hugo A., Câmara, Thaı́s
Peres, Gomes, Letı́cia 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 Amazônia.Zoologischer Anzeiger - A Journal
of Comparative Zoology http://dx.doi.org/10.1016/j.jcz.2017.04.001

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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

1Laboratóriode 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: rosaline@terra.com.br

1
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; West-

Eberhard, 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.

2
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 above-

mentioned 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

6
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.

7
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).

8
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.

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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

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2a

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2b

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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 P(param)
Effect tr.
Centroid size
Individual 2185488.05 40472.001 54 5050.79 <.0001 n.a n.a
Error 1 440.714998 8.013 55
Shape
Effect
Individual 0.03976375 6.14E-05 648 40.82 <.0001 11.09 <0.0001
Error 1 0.00099226 1.5034E-06 660
Profile View
SS MS df F P Pillai P(param)
Effect tr.
Centroid size
Individual 1101127.227 22471.9 49 1821.82 <.0001 n.a n.a
Error 1 616.745492 12.33491 50
Shape
Effect
Individual 0.05579351 9.49E-05 588 84.51 <.0001 11.61 <0.0001
Error 1 0.00067368 1.1228E-06 600

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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 MS df F P

Sex 468461.3279 468461.3279 1 39.45 <.0001

Blood Source 3337.56206 3337.56206 1 0.28 0.5983

Individual 617544.1754 11875.84953 52 n.a n.a

Shape

Effect SS MS df F P

Sex 0.00147508 0.000245846 6 5.31 <.0001

Blood Source 0.00127951 0.000213252 6 4.61 0.0002

Individual 0.00317193 9.7899E-06 324 n.a n.a

Profile View

Centroid size

Effect SS MS df F P

Sex 271858.7489 271858.7489 1 47.44 <.0001

Blood Source 8160.938121 8160.938121 1 1.42 0.2387

Individual 269339.2275 5730.621863 47 n.a n.a

Shape

Effect SS MS df F P

Sex 0.00306804 0.00025567 12 5.94 <.0001

Blood Source 0.00098303 8.19188E-05 12 1.9 0.0316

Individual 0.02428738 4.30627E-05 564 n.a n.a

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