Morphological variability and ecological characterization of the Chagas disease vector Triatoma dimidiata (Hemiptera: Reduviidae) in El Salvador

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Morphological variability and ecological characterization of the Chagas Disease vector Triatoma dimidiata (Hemiptera: Reduviidae) in El Salvador

V´ıctor D. Carmona-Galindo Conceptualization; Methodology; Formal analysis; Resources; Data Curation; Writing Mar´ıa Fernanda Mar´ın Recinos Methodology; Formal analysis; Writing - Original Draft; Visualization , ´ Saul Hidalgo Methodology; Formal analysis; Writing - Original Draft; Visualization , ´ Alfredo Gamez Guillermo Recinos Paredes Methodology; Formal analysis; Writing - Original Draft; Visualization , Enrique Eduardo Posada Vaquerano Methodology; Formal analysis; Writing - Original Draft; Visualization , ˜ Methodology; Formal analysis; Writing - Original Draft; Visualization , Andrea Luc´ıa Romero-Magana Karla Castillo Ayala Methodology; Formal analysis; Investigation; Resources; Writing - Original Draft; Visualization; PII: DOI: Reference:

S0001-706X(19)30961-1 https://doi.org/10.1016/j.actatropica.2020.105392 ACTROP 105392

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Acta Tropica

Received date: Revised date: Accepted date:

16 July 2019 8 February 2020 8 February 2020

Please cite this article as: V´ıctor D. Carmona-Galindo Conceptualization; Methodology; Formal analysis; Resources Mar´ıa Fernanda Mar´ın Recinos Methodology; Formal analysis; Writing - Original Draft; Visualization , ´ Saul Hidalgo Methodology; Formal analysis; Writing - Original Draft; Visualization , ´ Alfredo Gamez Guillermo Recinos Paredes Methodology; Formal analysis; Writing - Original Draft; Visualization , Enrique Eduardo Posada Vaquerano Methodology; Formal analysis; Writing - Original Draft; Visualization , ˜ Methodology; Formal analysis; Writing - Original Draft; Visualization , Andrea Luc´ıa Romero-Magana Karla Castillo Ayala Methodology; Formal analysis; Investigation; Resources; Writing - Original Draft; Visualization; Morphological variability and ecological characterization of the Chagas Disease vector Triatoma dimidiata (Hemiptera: Reduviidae) in El Salvador, Acta Tropica (2020), doi: https://doi.org/10.1016/j.actatropica.2020.105392

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Research Paper

Morphological variability and ecological characterization of the Chagas Disease vector Triatoma dimidiata (Hemiptera: Reduviidae) in El Salvador

Víctor D. Carmona-Galindo1,2,*, María Fernanda Marín Recinos2, Saúl Alfredo Gámez Hidalgo2, Guillermo Recinos Paredes2, Enrique Eduardo Posada Vaquerano2, Andrea Lucía Romero-Magaña2, and Karla Castillo Ayala3

1

University of Detroit Mercy; Biology Department; Detroit, Michigan, USA

2

Universidad de El Salvador; Centro de Investigación y Desarrollo en Salud; Laboratorio de Entomología

de Vectores; San Salvador, El Salvador 3

Medical Care Development International; Zika Community Response in Guatemala and El Salvador; San

Salvador, El Salvador

_______________ *Corresponding author: [email protected]

ABSTRACT There are 8 million people with Chagas disease worldwide and in El Salvador approximately 39% of the population is at risk of contracting the disease. One of the principal challenges in mitigating Chagas is evaluating the role of the vector ecology of triatomine species in the transmission of the Trypanosoma cruzi parasite in anthropogenically modified habitats, where new patterns of transmission frequently arise. Field studies of triatomine vector ecology in El Salvador have largely focused on describing parameters that contribute to infestation patterns, which may themselves be rooted in the morphological variability that exists in triatomine populations. The objective of this study was to evaluate the morphology of the vector species Triatoma dimidiata with respect to the characteristics of the ecological landscape the vector inhabits throughout El Salvador. We used image analyses to evaluate T. dimidiata morphological variability and then used Geographic Information Systems to intersect the morphological point-data with map layers containing different environmental characteristics. Our study found that the variation in the size, shape, and coloration of T. dimidiata varied in relation to elevation, Holdridge life zone, soil type and land use. We further characterize the local morphological adaptations of T. dimidiata with respect to the local ecological, biological, and geographical conditions in El Salvador. We suggest that future studies consider a molecular exploration of local T. dimidiata species complex in El Salvador, especially since morphological studies of triatomine species complex have found that variability correlate with the genetic variability of the population.

KEYWORDS: American Trypanosomiasis, Central America, ecohealth, geographic information systems, phenotypic variation, vector ecology

INTRODUCTION Chagas disease, or American Trypanosomiasis, is a parasitic infection caused by the protozoan hemoflagellate Trypanosoma cruzi (OMS, 2016). The parasite T. cruzi is transmitted by hematophagous insect vectors in the taxonomic subfamily Triatominae, but while all triatomine species are potential vectors of T. cruzi (Klotz et al., 2010) only those species adapted to live in habitats with humans are the considered important vectors species for transmission of the disease (Bustamante et al., 2015; Nattero et al., 2016). Harbored in the gut of the Triatominae bug, the T. cruzi parasite is transmitted to humans when following a blood-meal the bug defecates and the parasite is able to enter the bloodstream at the bite site (Lent and Wygodzinsky, 1979; Jurberg and Galvao, 2006). Chagas disease is characterized by both an acute phase where the parasite enters the bloodstream and starts replicating, and a chronic phase where the parasites enter specific tissues where they keep replicating (OMS, 2016). In the chronic phase, Chagas disease is incurable and results in serious health issues as well as premature death (Rassi et al., 2010). As with most neglected tropical diseases, Chagas disproportionately affects impoverished rural areas and compounds economic burdens (Hotez et al., 2008). There are eight million people with Chagas disease worldwide (OMS, 2016), and in Latin America approximately 28 million people (one quarter of the population) are at risk of contracting the disease (OMS, 2005). In 2005, 232,000 people in El Salvador were infected with Chagas disease and it is estimated that 39% of the population is at risk for infection (Cedillos et al., 2011a; Rassi et al., 2010). Anthropogenic impacts to the environment frequently contribute to new patterns of transmission in Chagas disease (Briceño-León, 2009; Alarcón de Noya y Noya González, 2015). As such, one of the principal challenges in mitigating Chagas is evaluating the role of the vector ecology of triatomine species in the transmission of T. cruzi in anthropogenically modified habitats (Bustamante et al., 2015). Important considerations in vector ecology include habitat plasticity (de Oliveira et al., 2015), domiciliary potential (Dorn et al., 2007), and feeding preferences (Castillo Ayala et al., 2018). However,

given the great morphological variation of triatomine vector species (Nattero et al., 2016; Panzera et al., -Palacio et al., 2015) has reinforced ecological and geographic isolating mechanisms in triatome populations (Bargues et al., 2017) resulting in cryptic speciation (Dorn et al., 2018; Lima-Cordon et al., 2019; Pavan et al., 2015). The vector Triatoma dimidiata may represent a species complex of five subspecies (Bargues et al., 2017) each with unique morphological, ecological, behavioral, and molecular considerations (Bustamante et al., 2004; Pavan et al., 2015; Panzera et al., 2006). An important first step in evaluating the local vector ecology of triatomines species is the delimitation of clear morphological diagnostic characters (Bargues et al., 2017; Oliveira et al., 2017). Field studies of triatomine ecology in El Salvador have largely focused on describing parameters that contribute to infestation patterns (Bustamante Zamora, 2015; Cedillos et al., 2012; Cedillos, 1975). Infestation patterns of triatomine vector species, however, may be rooted in the morphological variability that exists in population (Bargues et al., 2017) as well as the characteristics of their landscape ecology (Fernández et al., 2014). The objective of this study was to evaluate the morphology of the vector species Triatoma dimidiata with respect to the ecological characteristics of the landscape the vector inhabits in El Salvador. We hypothesized that the variation in the size, shape, and coloration of T. dimidiata would be related to changes in elevation, Holdridge life zone, soil type and land use of field sites where the vector persists in El Salvador.

MATERIALS AND METHODS Image Analysis The specimens of T. dimidiata used in this study were collected by the Ministry of Health from domiciliary habitats between Jan-Sep 2013 using national protocols for Chagas disease (MINSAL, 2011) and included the location (latitude and longitude) where each specimen was collected. A total of 52

individuals of T. dimidiata (23 males and 29 females) preserved for the same duration in ethanolglycerol solution (19:1) were selected for this study, representing five Departments from across El Salvador: Santa Ana (n=14 samples), Morazán (n=10 samples), San Vicente (n=4 samples), La Unión (n=12 samples), and San Miguel (n=12 samples). The dorsal and ventral views of the specimens were digitally photographed using a Canon Powershot A2200 camera under laboratory conditions. The digital images were used to collect a series of color and morphological measurements using Sigma Scan Pro (v5) from several dorsal and ventral structures. The dorsal structures considered were: Wing (Figure 1A), spots on dorsal connexivial plates (Figure 1B), body (Figure 1C), and light region on dorsal connexivial plates (Figure 1D). The ventral structures considered were: light region on ventral connexivial plate (Figure 1E) and ventral dark region (Figure 1F). The color measurements included: total pixel Intensity, average pixel intensity, average red pixel intensity, average blue pixel intensity, and average green pixel intensity. The morphological measurements included: area, shape, and proportion of the area of a given structure (dorsal or ventral) to body area. Shape was calculated using formula (1):

s W

“P”

P

(1)

2 A

“A”

f

u u

l

A total of 50 variables consisting of the longitude, latitude, color measurements, and morphological measurements for T. dimidiata were tabulated as a geographic information system (GIS) shapefile format (*.shp).

Geographic Information Systems The map layers for Elevation (Curvas a Nivel), Holdridge Life Zone (Zonas de Vida), Soil Type (Pedologico), and Land Use (Corine) for the country of El Salvador were made available by the GIS Laboratory in the Physics Department at the College of Natural Sciences and Mathematics in the

l.

University of El Salvador. The Elevation map contained information on elevational changes, the Holdridge Life Zone map contained the distribution of two forest types (tropical moist humid subtropical forest [bh-S] and humid tropical forest [bh-T]), the Soil Type map contained the distribution of three different soils (Grumosols, Red Clayish Latosols, and Acidic Clayish Latosols), and the Land Use map contained information on the distribution of six land management types (Urban, Agroforestry, Scrubland, Mixed Agriculture and Pastures, sugarcane, and Coffee). The GIS point map containing T. dimidiata distribution data (location as well as both color and morphology measurements) was intersected with each of the GIS polygon map layers containing ecological data (Elevation, Holdridge Life Zone, Soil Type, and Land USE) using the software QGIS (v 2.18) and the resulting GIS shapefile (*.shp) containing the combined spatial information was exported as a spreadsheet file (*.csv). The number of T. dimidata specimens representing each of the independent categories in the combined spatial information file were as follows: elevation: 50 total samples, Holdridge Life Zone: 44 total samples (bh-S: 28 samples, and bh-T: 16 samples), soil type: 46 total samples (grumosols: 10 samples, red clayish latosols: 25 samples, acidic clayish latosols: 11 samples), and land use: 48 total samples (urban: 13 samples, Agroforestry: 7 samples, Scrubland: 9 samples, Mixed Agriculture and Pastures: 5 samples, sugarcane: 5 samples, coffee: 9 samples).

Statistical Analysis The distribution of all morphological and color variables measured from the T. dimidiata image analysis was each evaluated using a Shapiro-Wilk test for normality. The relationship of all morphological and color variables with elevation was evaluated using a Pearson correlation (parametric data) and Spearman rank-correlation (non-parametric data). The differences in mean for all morphological and color variables with respect to Holdridge Life Zone were evaluated using a Student Ttest (parametric data) and Mann-Whitney U (non-parametric data). The differences in mean for all

morphological and color variables with respect to both Land Use as well as Soil Type were evaluated using an ANOVA (parametric data) and Kruskal-Wallis (non-parametric data). Statistical analyses were conducted using the software package STATISTICA (v6).

RESULTS The underlying distributions for morphological and color characteristics measured during the image analysis of T. dimidiata specimens are listed in Table 1.

Elevation With higher elevations (Table 2), T. dimidiata had a smaller and less circular light region on dorsal connexivial plate, a larger wing size, and a body with darker coloration (in terms of total pixel intensity) (Figures 2A-2C). With lower elevations (Table 2), T. dimidiata had a larger and more circular light region on dorsal connexivial plate, a smaller wing size, and a lighter body coloration (in terms of lower total pixel intensity) (Figures 2D). There were no significant correlations between elevation and the remaining morphological and color variables measured from T. dimidiata (p>0.05).

Holdridge Life Zone In humid subtropical forest (bh-S) habitats (Table 3), T. dimidiata had the light region on the dorsal connexivial plates with a lighter hue (in terms of total pixel intensity), a less circular body shape, and less circular spots on the dorsal connexivial plates (Figures 3a-c). In humid tropical forest (bh-T) habitats (Table 3), T. dimidiata had the light region on the dorsal connexivial plates with a darker hue (in terms of total pixel intensity), a more circular body shape, and more circular spots on the dorsal connexivial plates (Figures 3a-c). There were no significant differences between Holdridge Life Zone

categories in terms of the remaining morphological and color variables measured from T. dimidiata (p>0.05).

Soil Type In grumosol soils (Table 4), T. dimidiata had the light region on ventral connexivial plate with a lighter hue (in terms of total pixel intensity)(Figure 4E), the ventral dark region with a darker hue in terms of (blue, green and average pixel intensity) (Figures 4A-B and 4D) as well as a lighter hue (in terms of total pixel intensity) (Figure 4C), the light region on the dorsal connexivial plates with a lighter hue (in terms of red, green, average and total pixel intensity) (Figure 5A-D), spots on the dorsal connexivial plates (Figure 5E), wings (Figure 6A), and body (Figure 6B) with a lighter hue (in terms of total pixel intensity), a larger light region on ventral connexivial plate (Figure 7C), a less circular light region on ventral connexivial plate (Figure 7A) as well as ventral dark region (Figure 7B), a smaller light region on the dorsal connexivial plates (Figure 8A) as well as spots on the dorsal connexivial plates (Figure 8B), less circular spots on the dorsal connexivial plates (Figure 8C), a smaller body (Figure 9B), as well as proportionally smaller light region on the dorsal connexivial plates (Figure 8E), spots on the dorsal connexivial plates (Figure 8D), and wings (Figure 9A). In red clayish latosol soils (Table 4), T. dimidiata had the light region on ventral connexivial plate with a darker hue (in terms of total pixel intensity) (Figure 4E), and the ventral dark region with a lighter hue (in terms of blue, green and average pixel intensity) (Figures 4A, 4B, 4D) as well as a darker hue (in terms of total pixel intensity) (Figure 4C), the light region on dorsal connexivial plates with a darker hue (in terms of red, green and average pixel intensity) (Figures 5A, 5C, 5D) as well as an intermediate hue (in terms of total pixel intensity) (Figure 5B), spots on dorsal connexivial plates with an intermediate hue (in terms of total pixel intensity) (Figure 5E), wings as well as a the entire body with a darker hue (in terms of total pixel intensity) (Figures 6A-B), intermediate size in the light region on ventral connexivial

plate (Figure 7C), a more circular light region on ventral connexivial plate (Figure 7A), a larger light region on dorsal connexivial plates (Figure 8A) as well as spots on dorsal connexivial plates (Figure 8B), more circular spots on the dorsal connexivial plates shapes (Figure 8C), larger body (Figure 9B), as well as proportionally larger light regions on dorsal connexivial plates (Figure 7D), spots on the dorsal connexivial plates (Figure 8D), as well as wings (Figure 9A). In acidic clayish latosol soils (Table 4), T. dimidiata had the light region on ventral connexivial plate with a darker hue (in terms of total pixel intensity) (Figure 4E), and the ventral dark region with a darker hue (in terms of blue, green and total pixel intensity) (Figure 4B-D) as well as an intermediate hue (in terms of average pixel intensity) (Figure 4A), the light region on the dorsal connexivial plates with a darker hue (in terms of red, green, average and total pixel intensity) (Figure 5A-D), spots on the dorsal connexivial plates with a darker hue (in terms of total pixel intensity) (Figure 5E), both wings and entire body with a darker hue (in terms of total pixel intensity) (Figure 6A-B), a smaller light region on ventral connexivial plate (Figure 7C), a less circular Light region on ventral connexivial plate (Figure 7A) as well as ventral dark region (Figure 7B), a smaller light region on the dorsal connexivial plates (Figure 8A) as well as spots on the dorsal connexivial plates (Figure 8B), less circular spots on the dorsal connexivial plates shape (Figure 8C), a smaller body (Figure 9B), and a proportionally larger light region on the dorsal connexivial plates (Figure 7D), spots on dorsal connexivial plates (Figure 8D), as well as wings (Figure 9A). There were no significant differences among soil type categories in terms of the remaining morphological and color variables measured from T. dimidiata (p>0.05).

Land Use In urban habitats (Table 5), T. dimidiata had the ventral dark region with a darker hue (in terms of total pixel intensity (Figure 10A) as well as a lighter hue (in terms of red, green and average pixel

intensity) (Figures 10B-D), light region on ventral connexivial plate with a darker hue (in terms of total pixel intensity) (Figure 10E) as well as a lighter hue (in terms of blue and average pixel intensity) (Figures 10F-G), spots on the dorsal connexivial plates with a lighter hue (in terms of red, green, blue and average pixel intensity) (Figures 10A-C, 10G) as well as with a darker hue (in terms of total pixel intensity) (Figure 11E), light region on the dorsal connexivial plates with a lighter hue (in terms of blue pixel intensity) (Figure 11D) as well as a darker hue (in terms of total pixel intensity) (Figure 11F), a body with a lighter hue (in terms of red pixel intensity) (Figure 12A) as well as a darker hue (in terms of total pixel intensity) (Figure 12B), wings with a darker hue (in terms of total pixel intensity) (Figure 12C), a smaller light region on dorsal connexivial plates (Figure 14A), a less circular light region on the dorsal connexivial plates (Figure 14C), more circular spots on the dorsal connexivial plates (Figure 14D), a smaller light region on ventral connexivial plate (Figure 13A), a larger ventral dark region (Figure 13B), a larger body (Figure 15C), and proportionally larger wings (Figure 15B), spots on the dorsal connexivial plates (Figure 14B), light region on the dorsal connexivial plates (Figure 14A) as well as ventral dark region (Figure 13B). In agroforestry habitats (Table 5), T. dimidiata had the ventral dark region with a darker hue (in terms of total pixel intensity) (Figure 10A) as well as a lighter hue (in terms of red, green and average pixel intensity) (Figures 10B-D), a light region on ventral connexivial plate with a darker hue (in terms of total pixel intensity) (Figure 10E) as well as an intermediate hue (in terms of blue and average pixel intensity) (Figures 10F-G), spots on dorsal connexivial plates with a lighter hue (in terms of red, green, blue and average pixel intensity) (Figure 11A-C, 11G) as well as a darker hue (in terms of total pixel intensity) (Figure 11E), the light region on dorsal connexivial plates with a lighter hue (in terms of blue and total pixel intensity) (Figure 11D-F), a body with a lighter hue (in terms of red pixel intensity) (Figure 12A) as well as a darker hue (in terms of total pixel intensity) (Figure 12B), wings with a darker hue (in terms of total pixel intensity) (Figure 12C), a larger light region on the dorsal connexivial plates (Figure

14A), more circular light region on dorsal connexivial plates (Figure 14C), more circular spots on dorsal connexivial plates (Figure 14D), a smaller light region on ventral connexivial plate (Figure 13A), a smaller ventral dark region (Figure 13B), a larger body (Figure 15C), and proportionally smaller wings (Figure 15B), spots on the dorsal connexivial plates (Figure 14F), light region on the dorsal connexivial plates (Figure 14E), as well as ventral dark region (Figure 13C). In scrubland habitats (Table 5), T. dimidiata had the ventral dark region with a darker hue (in terms of total pixel intensity) (Figure 10A) as well as a lighter hue (in terms of red, green and average pixel intensity) (Figures 10B-D), a light region on ventral connexivial plate with a darker hue (in terms of total pixel intensity) (Figure 10E) as well as a lighter hue (in terms of blue and average pixel intensity) (Figures 10F-G), spots on the dorsal connexivial plates with a lighter hue (in terms of red, green, blue and average pixel intensity) (Figures 10A-C, 10G) as well as a darker hue (in terms of total pixel intensity) (Figure 10E), light region on dorsal connexivial plates with a lighter hue (in terms of blue pixel intensity) (Figure 11D) as well as a darker hue (in terms of total pixel intensity) (Figure 11F), a body with a lighter hue (in terms of red pixel intensity) (Figure 12A) as well as a darker hue (in terms of total pixel intensity) (Figure 12B), wings with a darker hue (in terms of total pixel intensity) (Figure 12C), smaller light region on the dorsal connexivial plates (Figure 14A), less circular light region on dorsal connexivial plates (Figure 14C), more circular spots on dorsal connexivial plates (Figure 14D), smaller light region on ventral connexivial plate (Figure 13A), a larger ventral dark region (Figure 13B), a larger body (Figure 15C), as well as proportionally larger wings (Figure 15B), spots on dorsal connexivial plates (Figure 14F), light region on dorsal connexivial plates (Figure 14E) and ventral dark region (Figure 13C). In mixed agriculture and pastures habitats (Table 5), T. dimidiata had the ventral dark region with a lighter hue (in terms of total pixel intensity) (Figure 10A) as well as a lighter hue (in terms of red, green and average pixel intensity) (Figure 10B-D), a light region on ventral connexivial plate with a lighter hue (in terms of total pixel intensity) (Figures 10E) as well as a darker hue (in terms of blue and

average pixel intensity) (Figures 10F-G), spots on dorsal connexivial plates with a lighter hue (in terms of red, green, blue, average and total pixel intensity) (Figures 11A-C, 11E, 11G), a light region on dorsal connexivial plates with a lighter hue (in terms of blue and total pixel intensity) (Figures 11D-F), a body with a lighter hue (in terms of red pixel intensity) (Figure 12A) as well as a darker hue (in terms of total pixel intensity) (Figure 12B), wings with a darker hue (in terms of total pixel intensity) (Figure 12C), smaller light region on the dorsal connexivial plates (Figure 14A), a less circular light region on dorsal connexivial plates (Figure 14C), less circular spots on the dorsal connexivial plates (Figure 14D), a smaller light region on ventral connexivial plate (Figure 13A), a larger ventral dark region (Figure 13B), a larger body (Figure 15C), as well as proportionally smaller wings (Figure 15B), spots on dorsal connexivial plates (Figure 14F), light region on the dorsal connexivial plates (Figure 14E), and proportionally larger ventral dark region (Figure 13C). In sugarcane habitats (Table 5), T. dimidiata had the ventral dark region with an intermediate hue (in terms of total pixel intensity) (Figure 10A) as well as a darker hue (in terms of red, green and average pixel intensity) (Figures 10B-D), a light region on ventral connexivial plate with a lighter hue (in terms of total pixel intensity) (Figure 10E) as well as an intermediate and darker hue (in terms of blue and average pixel intensity respectively) (Figures 10F-G), spots on the dorsal connexivial plates with a lighter hue (in terms of red, green, blue and average pixel intensity) (Figures 11A-C, 11G) as well as an intermediate hue (in terms of total pixel intensity) (Figure 11E), the light region on dorsal connexivial plates with a darker hue (in terms of blue pixel intensity) (Figure 11D) as well as an intermediate hue (in terms of total pixel intensity) (Figure 11F), a body with a darker hue (in terms of red pixel intensity) (Figure 12A) as well as a lighter hue (in terms of total pixel intensity) (Figure 12B), wings with a lighter hue (in terms of total pixel intensity) (Figure 12C), smaller light region on dorsal connexivial plates (Figure 14A), a less circular light region on dorsal connexivial plates (Figure 14C), more circular spots on dorsal connexivial plates (Figure 14D), a smaller light region on ventral connexivial plate (Figure 13A), a

larger ventral dark region (Figure 13B), a larger body (Figure 15C), as well as proportionally smaller wings (Figure 15B), spots on dorsal connexivial plates (Figure 14F), light region on the dorsal connexivial plates (Figure 14E), and proportionally larger ventral dark region (Figure 13C). In coffee habitats (Table 5), T. dimidiata had the ventral dark region with a lighter hue (in terms of green, red, average and total pixel intensity) (Figures 10A-D), a light region on ventral connexivial plate with a lighter hue (in terms of total pixel intensity) (Figure 10E) as well as an intermediate hue (in terms of blue and average pixel intensity) (Figures 10F-G), spots on dorsal connexivial plates with a lighter hue (in terms of red, green, blue and average pixel intensity) (Figures 11A-C, 11G) as well as an intermediate hue (in terms of total pixel intensity) (Figure 11E), the light region on the dorsal connexivial plates with a lighter hue (in terms of blue pixel intensity) (Figure 11D) as well as an intermediate hue (in terms of total pixel intensity) (Figure 11F), a body with a lighter hue (in terms of red pixel intensity) (Figure 12A) as well as a darker hue (in terms of total pixel intensity) (Figure 12B), wings with a darker hue (in terms of total pixel intensity) (Figure 12C), a smaller light region on dorsal connexivial plates (Figure 14A), a less circular light region on the dorsal connexivial plates (Figure 14C), more circular spots on dorsal connexivial plates (Figure 14D), a smaller light region on ventral connexivial plate (Figure 13A), a larger ventral dark region (Figure 13B), a larger body (Figure 15C), as well as proportionally smaller wings (Figure 15B), spots on the dorsal connexivial plates (Figure 14F), light region on the dorsal connexivial plates (Figure 14E), and a proportionally larger ventral dark region (Figure 13C). There were no significant differences among land use categories in terms of the remaining morphological and color variables measured from T. dimidiata (p>0.05).

DISCUSSION The variation of T. dimidiata in terms of the size, shape and coloration changed predictably in relation to elevation, Holdridge Life Zone, soil type, and land use across El Salvador. Morphological

plasticity allows insect populations to respond to variability in the physical environment resulting from changes associated with ecological gradients, both direct and indirect community interactions, as well as climate change (Hodkinson, 2005). Elevational gradients can affect insect morphology in terms of color polymorphism, wing-size variability, and insect-size variability as a result of changes in thermal tolerance and needs, partial pressures of atmospheric gases, fecundity, and genetic factors (Hodkinson, 2005). Large-scale habitat changes have been associated with both genetic (Dorn et al., 2007) and morphological (Fergnani et al., 2013) variability in Triatomine species assemblages, but they have also been associated with changes in general insect behavior like locomotion, feeding behavior, and sensory ability (Yates et al., 2014). Interacting species can also impact insect morphology via changes in population density, parasitism, and predation rates (Hodkinson, 2005). Climate change can contribute to insect range expansion and contraction, as well as altitudinal and latitudinal shifts in distribution (Hodkinson, 2005) by further compounding effects from direct and indirect community interactions (Rasmann et al., 2014). Field studies characterizing the micro- and macro-climatic preferences of Triatomine species have focused on habitat selectivity, which is bound in terms of temperature, relative humidity, and light environment (Dorn et al., 2007; Dorn et al., 2018; Lima-Cordon et al., 2019; Fergnani et al., 2013; Lorenzo et al., 2000; Minoli, 2004). However, expanding on the regional phenotypic variability described by Bustamante et al. (2014), this study was able to characterize the local morphological variation of T. dimidiata with respect to the local ecological, biological, and geographical conditions in El Salvador. While both the time-scale and factors driving T. dimidiata variability in El Salvador remain unexplored, it is important to note that Triatomine species can undergo rapid morphological changes in order to adapt to new habitats (Dujardin et al., 1999), especially when the vector species exhibit a greater range of ecotopes (Noireau et al., 2005). Fergnani et al. (2013) suggests that at the distributional limits, Triatomine species assemblages may be shaped by environmental filtering. As such, it is possible that

Triatomine cryptic species may still share many genetic similarities while at the same time undergoing rapid morphological adaptations to new environmental conditions (Dujardin et al., 1999; Noireau et al., 1997). The variability of T. dimidiata in El Salvador also underscores the role of studying the ecology of vector populations to mitigate Chagas disease (Peterson, 2015), not just to characterize the richness of local vector-

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l., 2018) but to also characterize how anthropogenic

effects may contribute to domiciliation (Alarcón de Noya y Noya González, 2015; Buitrago et al., 2016). We suggest that future studies consider a molecular exploration of local T. dimidiata species complex in El Salvador, especially since morphological studies of triatomine species complex have found that variability correlates with the genetic variability of the population (Costa et al., 2016; Nattero et al., 2016).

ACKNOWLEDGEMENTS We would like to thank the Center for Global Health Research (CENSALUD) at the University of El Salvador for logistical support as well as the Department of Vector Studies at Salvadoran Ministry of Health for shared access to the T. dimidiata specimen collection. We also thank the Leishmaniasis Transmission Lab at the Instituto Oswaldo Cruz for excellent suggestions on improving the manuscript. Funding support for Dr. Víctor D. Carmona-Galindo was awarded by the Core Fulbright U.S. Scholar Program.

Conflict of Interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

Credit Author Statement Víctor Daniel Carmona-Galindo: Conceptualization, Methodology, Formal analysis, Resources, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Project administration, Funding acquisition María Fernanda Marín Recinos: Methodology, Formal analysis, Writing - Original Draft, Visualization Saúl Alfredo Gámez Hidalgo: Methodology, Formal analysis, Writing - Original Draft, Visualization Guillermo Recinos Paredes: Methodology, Formal analysis, Writing - Original Draft, Visualization Enrique Eduardo Posada Vaquerano: Methodology, Formal analysis, Writing - Original Draft, Visualization Andrea Lucía Romero-Magaña: Methodology, Formal analysis, Writing - Original Draft, Visualization Karla Castillo Ayala: Methodology, Formal analysis, Investigation, Resources, Writing - Original Draft, Visualization, Supervision, Project administration

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dimidiata with different epidemiological importance as Chagas disease vectors. Trop Med Int Health. 2006; 11: 1092103. Pavan M.G., Rivas G.B.S., Dias F.B.S., Gurgel-Gonçalves R. Looks Can be Deceiving: Cryptic Species and Phenotypic Variation in Rhodnius spp., Chagas Disease Vectors. In: Pontarotti P. (eds) Evolutionary Biology: Biodiversification from Genotype to Phenotype. Springer, Cham. 2015. Peterson JK, Bartsch SM, Lee BY, y Dobson AP. Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control. Parasit Vectors. 2015; 8: 537. R

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interactions along elevation gradients. Functional Ecology. 2014; 28(1): 46-54 Rassi A, Rassi A, Marin-Neto JA. Chagas disease. Lancet. 2010; 375 (9723): 1388–1402. Yates ML, Andrew NR, Binns M, and Gibb H. Morphological traits: predictable responses to macrohabitats across a 300 km scale. PeerJ 2014; 2: e271.

Table 1. Underlying distribution of continuous-scale morphological, color, and ecological characteristics associated with the image analysis of T. dimidiata in El Salvador. Characteristics Ecological

Parametric Distribution (p>0.05)

Non-Parametric Distribution (p<0.05)  Elevation (W=0.9525)

Wing

 Average blue pixel intensity

 Average green pixel intensity

(W=0.9646)  Average red pixel intensity (W=0.9793)  Average pixel intensity (W=0.9647)

(W=0.9499)  Total pixel intensity (W=0.4935)  Proportion to body area (W=0.8991)  Shape (W=0.7167)

 Area (W=0.9791) Spots on Dorsal Connexivial Plates

 Average blue pixel intensity (W=0.9730)  Average green pixel intensity (W=0.9616)

 Average red pixel intensity (W=0.9513)  Total pixel intensity (W=0.8231)  Proportion to body area (W=0.7146)  Shape (W=0.9209)

 Average pixel intensity (W=0.9562)  Area (W=0.9796) Body

 Average blue pixel intensity

 Total pixel intensity (W=0.4695)

(W=0.9832)  Average green pixel intensity (W=0.9605)  Average red pixel intensity (W=0.9798)  Average pixel intensity (W=0.9761)  Area (W=0.9766)  Shape (W=0.9809) Light Region on Dorsal Connexivial Plates

 Average pixel intensity (W=0.9837)

 Average blue pixel intensity (W=0.9456)  Average green pixel intensity (W=0.9539)

 Average red pixel intensity (W=0.9446)  Total pixel intensity (W=0.9522)  Proportion to body area (W=0.7882)  Area (W=0.8402)  Shape (W=0.6467) Light Region on Ventral Connexivial Plate

 Average blue pixel intensity (W=0.9750)  Average red pixel intensity (W=0.9597)

 Average green pixel intensity (W=0.9229)  Average pixel intensity (W=0.9286)  Total pixel intensity (W=0.6209)  Proportion to body area (W=0.7259)  Area (W=0.8373)  Shape (W=0.7604)

Ventral Dark Region

 Shape (W=0.9843)

 Average blue pixel intensity (W=0.9406)  Average green pixel intensity (W=0.8852)  Average red pixel intensity (W=0.8901)  Average pixel intensity (W=0.8937)  Total pixel intensity (W=0.5832)  Proportion to body area (W=0.7449)  Area (W=0.9357)

Table 2. Correlations between elevation and both morphological and color characteristics of T. dimidiata in El Salvador. T. dimidiata Body Region

Variability with Elevation

Wing

 Proportion to body area was positively correlated (r=0.3075, p=0.0282, Figure 2d)

Light Region on Dorsal

 Total pixel intensity was negatively correlated (r=-0.3777, p=0.0063, Figure 2a)

Connexivial Plates

 Area was negatively correlated (r=-0.2819, p=0.0451, Figure 2b)  Shape was negatively correlated (r=-0.2912, p=0.0382, Figure 2c)

Table 3. Changes in the morphology and color of T. dimidiata with respect to Holdridge Life Zone in El Salvador. T. dimidiata Body Region

Variability with Holdridge Life Zones

Light Region on Dorsal

 Total pixel intensity was greater in humid subtropical forest (bh-S) than in humid tropical forest

Connexivial Plates

(bh-T) (U=180, p=0.0323, Figure 3a).  Shape was greater in humid subtropical forest (bh-S) than in humid tropical forest (bh-T) (U=161, p=0.0118, Figure 3b).

Spots on Dorsal Connexivial Plates

 Shape was greater in humid subtropical forest (bh-S) than in humid tropical forest (bh-T) (U=179, p=0.0307, Figure 3c).

Table 4. Changes in the morphology and color of T. dimidiata with respect to soil type in El Salvador. T. dimidiata Body Region

Variability with Soil Types

Wing



Total pixel intensity (H2,46=14.719, p=0.0006) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 6A).



Proportion to body area (H2,46=6.126, p=0.0468) was greatest in habitats with red clayish latosols and acidic clayish latosols, and lowest in habitats with grumosols (Figure 9A).

Spots on Dorsal



Connexivial Plates

Total pixel intensity (H2,46=24.372, p=0.0000) was greatest in habitats with grumosols, intermediate in habitats with red clayish latosols, and lowest in habitats with acidic clayish latosols (Figure 5E).



Area (F2,43=8.124, p=0.0010) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Fisher's LSD, p<0.05, Figure 8B).



Shape (H2,46=14.012, p=0.0009) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Figure 8C).



Proportion to body area (H2,46=7.994, p=0.0184) was greatest in habitats with red clayish latosols and acidic clayish latosols, and lowest in habitats with grumosols (Figure 8D).

Body



Total pixel intensity (H2,46=18.083, p=0.0001) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 6B).



Area (F2,43=5.331, p=0.0085) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Fisher's LSD, p<0.05, Figure 9B).

Light Region on Dorsal



Connexivial Plates

Average green pixel intensity (H2,46=8.784, p=0.0124) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 5A).



Total pixel intensity (F2,43=11.060, p=0.0001) was greatest in habitats with grumosols, intermediate in habitats with red clayish latosols, and lowest in habitats with acidic clayish latosols (Fisher's LSD, p<0.05, Figure 5B).



Average red pixel intensity (H2,46=8.470, p=0.0145) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 5C).



Average pixel intensity (F2,43=4.670, p=0.0146) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Fisher's LSD,

p<0.05, Figure 5D). 

Area (H2,46=13.620, p=0.0011) was greatest in habitats with red clayish latosols, intermediate in habitats with acidic clayish latosols and lowest in habitats with grumosols (Figure 8A).



Proportion to body area (H2,46=13.489, p=0.0012) was greatest in habitats with red clayish latosols and acidic clayish latosols, and lowest in habitats with grumosols (Figure 8E).

Light Region on Ventral



Connexivial Plate

Total pixel intensity (H2,46=19.054, p=0.0001) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 4E).



Shape (H2,46=8.827, p=0.0121) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Figure 7A).



Area (H2,46=10.753, p=0.0046) was greatest in habitats with grumosols, intermediate in habitats with red clayish latosols, and lowest in habitats with acidic clayish latosols (Figure 7C).

Ventral Dark Region



Average pixel intensity (F2,43=6.213, p=0.0043) was greatest in habitats with red clayish latosols, intermediate in habitats with acidic clayish latosols, and lowest in habitats with grumosols (Fisher's LSD, p<0.05, Figure 4A).



Average blue pixel intensity (F2,43=10.523, p=0.0002) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Fisher's LSD, p<0.05, Figure 4B).



Total pixel intensity (H2,46=12.507, p=0.0019) was greatest in habitats with grumosols, and lowest in habitats with both red clayish latosols and acidic clayish latosols (Figure 4C).



Average green pixel intensity (F2,43=5.086, p=0.0104) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Fisher's LSD, p<0.05, Figure 4D).



Shape (F2,43=3.865, p=0.0286) was greatest in habitats with red clayish latosols, and lowest in habitats with both grumosols and acidic clayish latosols (Fisher's LSD, p<0.05, Figure 7B).



Proportion to body area (H2,46=6.236, p=0.0443) was greatest in habitats with grumosols, intermediate in habitats with acidic clayish latosols and lowest in habitats with red clayish latosols (Figure 7D).

Table 5. Changes in the morphology and color of T. dimidiata with respect to land use in El Salvador. T. dimidiata Body Region

Variability with Land Use

Wing

 Total pixel intensity (H5,45=17.760, p=0.0033) was greatest in areas with sugarcane, and lowest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee (Figure 12C).  Area (F5,39=2.461, p=0.0496) was greatest in agroforestry and scrubland, intermediate in urban infrastructure as well as mixed agriculture and pastures, and lowest in sugarcane and coffee (Fis

’ LSD, < .

,F u

A.

 Proportion to body area (H5,45=30.286, p<0.0001) was greatest in urban infrastructure and scrubland, and lowest in agroforestry, mixed agriculture and pastures, sugarcane, as well as coffee (Figure 15B). Spots on Dorsal Connexivial Plates

 Average red pixel intensity (H5,45=17.079, p=0.0044) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, and lowest in areas with sugarcane (Figure 11A).  Average green pixel intensity (F5,39=5.034, p=0.0012) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, l

u

F

’ LSD, < .

,F u

B.

 Average pixel intensity (H5,45=15.465, p=0.0085) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, and lowest in areas with sugarcane (Figure 11C).  Total pixel intensity (H5,45=17.121, p=0.0043) was greatest in areas with mixed agriculture and pastures, intermediate in areas with sugarcane and coffee, and lowest in areas with urban infrastructure, agroforestry, scrubland (Figure 11E).  Average blue pixel intensity (F5,39=3.871, p=0.0061) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, l

u

F

’ LSD, P< .

,F u

.

 Area (F5,39=2.881, p=0.0262) was greatest in agroforestry habitats, intermediate in urban infrastructure, scrubland, mixed agriculture and pastures, as well as sugarcane, and lowest

ff

l

F

’ LSD, P< .

,F u

4B .

 Shape (H5,45=14.582, p=0.0123) was greatest in urban infrastructure, agroforestry, scrubland, sugarcane, as well as coffee, and was lowest in mixed agriculture and pastures (Figure 14D).  Proportion to body area (H5,45=25.549, p=0.0001) was greatest in urban infrastructure and scrubland, and lowest in agroforestry, mixed agriculture and pastures, sugarcane, as well as coffee (Figure 14F). Body

 Average red pixel intensity (F5,39=3.612, p=0.0088) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, and lowest in areas w

u

F

’ LSD, < .

,F u

A.

 Total pixel intensity (H5,45=21.180, p=0.0007) was greatest in areas with sugarcane, and lowest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee (Figure 12B).  Area (F5,39=5.274, p=0.0009) was greatest in urban infrastructure, agroforestry, scrubland, ll

x

ul u

u

,

l

u

ff

F

’

LSD, p<0.05, Figure 15C). Light Region on Dorsal Connexivial Plates

 Average blue pixel intensity (F5,39=5.104, p=0.0011) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, l

u

F

’ LSD, < .

, Figure 11D).

 Total pixel intensity (F5,39=10.385, p=0.0000) was greatest in areas with agroforestry and mixed agriculture and pastures, intermediate in areas with coffee, and lowest in areas with ub

f

u u ,

ubl

,

u

F

’ LSD, p<0.05, Figure 11F).

 Area (H5,45=21.054, p=0.0008) was greatest in agroforestry habitats and lowest in urban infrastructure, scrubland, mixed agriculture and pastures, sugarcane, as well as coffee (Figure 14A).  Shape (H5,45=19.047, p=0.0019) was greatest in agroforestry habitats and lowest in urban infrastructure, scrubland, mixed agriculture and pastures, sugarcane, as well as coffee (Figure 14C).  Proportion to body area (H5,45=3.669, p=0.0000) was greatest in urban infrastructure and

scrubland, and lowest in agroforestry, mixed agriculture and pastures, sugarcane, as well as coffee (Figure 14E). Light Region on Ventral Connexivial Plate

 Total pixel intensity (H5,45=17.698, p=0.0033) was greatest in areas with mixed agriculture and pastures, sugarcane, as well as coffee, and lowest in areas with urban infrastructure, agroforestry, scrubland (Figure 10E).  Average pixel intensity (H5,45=11.340, p=0.0450) was greatest in areas with urban infrastructure and scrubland, intermediate in areas with sugarcane and coffee, and lowest in areas with agroforestry and mixed agriculture and pastures (Figure 10F).  Average blue pixel intensity (F5,39=4.553, p=0.0023) was greatest in areas with urban infrastructure, and intermediate-high in areas with scrubland, intermediate-low in areas with coffee, and lowest in areas with agroforestry, mixed agriculture and pastures, and u

, F

’ LSD, < .

,F u

.

 Area (H5,45=15.119, p=0.0099) was greatest in scrubland and lowest in urban infrastructure, agroforestry, mixed agriculture and pastures, sugarcane, and coffee (Figure 13A). Ventral Dark Region

 Total pixel intensity (H5,45=16.285, p=0.0061) was greatest in areas with both mixed agriculture and pastures as well as coffee, intermediate in areas with sugarcane, and lowest in areas with urban infrastructure, agroforestry and scrubland (Figure 10A).  Average pixel intensity (H5,45=13.278, p=0.0209) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, and lowest in areas with sugarcane (Figure 10B).  Average red pixel intensity (H5,45=16.835, p=0.0048) was greatest in areas with urban infrastructure, agroforestry, scrubland, intermediate in areas with mixed agriculture and pastures as well as coffee, and lowest in areas with sugarcane (Figure 10C).  Average green pixel intensity (H5,45=11.134, p=0.0488) was greatest in areas with urban infrastructure, agroforestry, scrubland, mixed agriculture and pastures, as well as coffee, and lowest in areas with sugarcane (Figure 10D).  Area (H5,45=12.205, p=0.0321) was greatest in urban infrastructure, scrubland, mixed agriculture and pastures, sugarcane, as well as coffee, and lowest in agroforestry (Figure 13B).

 Proportion to body area (H5,45=14.575, p=0.0123) was greatest in urban infrastructure, scrubland, mixed agriculture and pastures, sugarcane, as well as coffee, and lowest in agroforestry (Figure 13C).

Figure 1. Regions of T. dimidiata considered for morphology and color variation: (A) Wing , (B) Spots on Dorsal Connexivial Plate, (C) Body, (D) Light Region on Dorsal Connexivial Plate, (E) Light Region on Ventral Connexivial Plate, (F) Ventral Dark Region.

Figure 2. Correlation of elevation with morphology and color of T. dimidiata: (A) Total Pixel Intensity of Body, (B) Area of Light Region on Dorsal Connexivial Plate, (C) Shape of Light Region on Dorsal Connexivial Plate, and (D) Wing Size.

Figure 3. Changes in morphology and color of T. dimidiata with respect to Holdridge Life Zones: (A) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (B) Shape of Light Region on Dorsal Connexivial Plates, (C) Shape of Spots on Dorsal Connexivial Plates.

Figure 4. Changes in coloration of T. dimidiata ventral regions in relation to soil type: (A) Average Pixel Intensity of Ventral Dark Region, (B) Average Blue Pixel Intensity of Ventral Dark Region, (C) Total Pixel Intensity of Ventral Dark Region, (D) Average Green Pixel Intensity of Ventral Dark Region, (E) Total Pixel Intensity of Light Region on Ventral Connexivial Plate. Bars denote 95% confidence intervals, and

parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 5. Changes in coloration of T. dimidiata dorsal regions in relation to soil type: (A) Average Green Pixel Intensity of Light Region on Dorsal Connexivial Plates, (B) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (C) Average Red Pixel Intensity of Light Region on Dorsal Connexivial Plates, (D) Average Pixel Intensity of Light Region on Dorsal Connexivial Plates, (E) Total Pixel Intensity of Spots

on Dorsal Connexivial Plates. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 6. Changes in coloration of T. dimidiata anatomical structures in relation to soil type: (A) Total Pixel Intensity of Wing, (B) Total Pixel Intensity of Body. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 7. Changes in morphology of T. dimidiata ventral regions in relation to soil type: (A) Shape of Light Region on Ventral Connexivial Plate, (B) Shape of Ventral Dark Region, (C) Area of Light Region on Ventral Connexivial Plate, (D) Proportion of Ventral Dark Region to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 8. Changes in morphology of T. dimidiata dorsal regions in relation to soil type: (A) Light Region on Dorsal Connexivial Plates Area, (B) Area of Spots on Dorsal Connexivial Plates, (C) Shape of Spots on Dorsal Connexivial Plates, (D) Proportion of Spots on Dorsal Connexivial Plates to Body Area, (E) Proportion of LIght Region on Dorsal Connexicivial Plates to Body Area. Bars denote 95% confidence

intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 9. Changes in morphology of T. dimidiata anatomical structures in relation to soil type: (A) Proportion of Wing to Body Area, (B) Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05). Structural variation by soil type

Figure 10. Changes in color of T. dimidiata ventral regions in relation to land use: (A) Total Pixel Intensity of Ventral Dark Region, (B) Average Pixel Intensity of Ventral Dark Region, (C) Average Red Pixel Intensity of Ventral Dark Region, (D) Average Green Pixel Intensity of Ventral Dark Region, (E) Total Pixel Intensity of Light Region on Ventral Connexivial Plate, (F) Average Pixel Intensity of Light Region on Ventral Connexivial Plate, (G) Average Blue Pixel Intensity of Ventral Abdominal Light Region. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 11. Changes in color of T. dimidiata dorsal regions in relation to land use: (A) Average Red Pixel Intensity of Spots on Dorsal Connexivial Plates, (B) Average Green Pixel Intensity of Spots on Dorsal Connexivial Plates, (C) Average Pixel Intensity of Spots on Dorsal Connexivial Plates, (D) Average Blue Pixel Intensity of Light Region on Dorsal Connexivial Plates, (E) Total Pixel Intensity of Spots on Dorsal Connexivial Plates, (F) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (G) Average Blue Pixel Intensity of Spots on Dorsal Connexivial Plates. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 12. Changes in color of T. dimidiata anatomical structures in relation to land use: (A) Average Red Pixel Intensity of Body, (B) Total Pixel Intensity of Body, (C) Total Pixel Intensity of Wing. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 13. Changes in morphology of T. dimidiata ventral regions in relation to land use: (A) Area of Light Region on Ventral Connexivial Plate, (B) Area of Ventral Dark Region, (C) Proportion of Ventral Dark Region to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 14. Changes in morphology of T. dimidiata dorsal regions in relation to land use: (A) Area of Light Region on Dorsal Connexivial Plates, (B) Area of Spots on Dorsal Connexivial Plates, (C) Shape of Light Region on Dorsal Connexivial Plates, (D) Shape of Spots on Dorsal Connexivial Plates, (E) Proportion of Light Region on Dorsal Connexivial Plates to Body Area, (F) Proportion of Total Spot Area on Dorsal

Connexivial Plates to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 15. Changes in morphology of T. dimidiata anatomical structures in relation to land use: (A) Wing Area, (B) Proportion of Wing Area to Body Area, (C) Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

TABLE AND FIGURE CAPTIONS Table 1. Underlying distribution of continuous-scale morphological, color, and ecological characteristics associated with the image analysis of T. dimidiata in El Salvador.

Table 2. Correlations between elevation and both morphological and color characteristics of T. dimidiata in El Salvador.

Table 3. Changes in the morphology and color of T. dimidiata with respect to Holdridge Life Zone in El Salvador.

Table 4. Changes in the morphology and color of T. dimidiata with respect to soil type in El Salvador.

Table 5. Changes in the morphology and color of T. dimidiata with respect to land use in El Salvador.

Figure 1. Regions of T. dimidiata considered for morphology and color variation: (A) Wing , (B) Spots on Dorsal Connexivial Plate, (C) Body, (D) Light Region on Dorsal Connexivial Plate, (E) Light Region on Ventral Connexivial Plate, (F) Ventral Dark Region.

Figure 2. Correlation of elevation with morphology and color of T. dimidiata: (A) Total Pixel Intensity of Body, (B) Area of Light Region on Dorsal Connexivial Plate, (C) Shape of Light Region on Dorsal Connexivial Plate, and (D) Wing Size.

Figure 3. Changes in morphology and color of T. dimidiata with respect to Holdridge Life Zones: (A) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (B) Shape of Light Region on Dorsal Connexivial Plates, (C) Shape of Spots on Dorsal Connexivial Plates.

Figure 4. Changes in coloration of T. dimidiata ventral regions in relation to soil type: (A) Average Pixel Intensity of Ventral Dark Region, (B) Average Blue Pixel Intensity of Ventral Dark Region, (C) Total Pixel Intensity of Ventral Dark Region, (D) Average Green Pixel Intensity of Ventral Dark Region, (E) Total Pixel Intensity of Light Region on Ventral Connexivial Plate. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 5. Changes in coloration of T. dimidiata dorsal regions in relation to soil type: (A) Average Green Pixel Intensity of Light Region on Dorsal Connexivial Plates, (B) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (C) Average Red Pixel Intensity of Light Region on Dorsal Connexivial Plates, (D) Average Pixel Intensity of Light Region on Dorsal Connexivial Plates, (E) Total Pixel Intensity of Spots on Dorsal Connexivial Plates. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 6. Changes in coloration of T. dimidiata anatomical structures in relation to soil type: (A) Total Pixel Intensity of Wing, (B) Total Pixel Intensity of Body. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 7. Changes in morphology of T. dimidiata ventral regions in relation to soil type: (A) Shape of Light Region on Ventral Connexivial Plate, (B) Shape of Ventral Dark Region, (C) Area of Light Region on Ventral Connexivial Plate, (D) Proportion of Ventral Dark Region to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 8. Changes in morphology of T. dimidiata dorsal regions in relation to soil type: (A) Light Region on Dorsal Connexivial Plates Area, (B) Area of Spots on Dorsal Connexivial Plates, (C) Shape of Spots on Dorsal Connexivial Plates, (D) Proportion of Spots on Dorsal Connexivial Plates to Body Area, (E) Proportion of LIght Region on Dorsal Connexicivial Plates to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 9. Changes in morphology of T. dimidiata anatomical structures in relation to soil type: (A) Proportion of Wing to Body Area, (B) Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05). Structural variation by soil type

Figure 10. Changes in color of T. dimidiata ventral regions in relation to land use: (A) Total Pixel Intensity of Ventral Dark Region, (B) Average Pixel Intensity of Ventral Dark Region, (C) Average Red Pixel Intensity of Ventral Dark Region, (D) Average Green Pixel Intensity of Ventral Dark Region, (E) Total Pixel Intensity of Light Region on Ventral Connexivial Plate, (F) Average Pixel Intensity of Light Region on Ventral Connexivial Plate, (G) Average Blue Pixel Intensity of Ventral Abdominal Light Region. Bars denote 95%

confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 11. Changes in color of T. dimidiata dorsal regions in relation to land use: (A) Average Red Pixel Intensity of Spots on Dorsal Connexivial Plates, (B) Average Green Pixel Intensity of Spots on Dorsal Connexivial Plates, (C) Average Pixel Intensity of Spots on Dorsal Connexivial Plates, (D) Average Blue Pixel Intensity of Light Region on Dorsal Connexivial Plates, (E) Total Pixel Intensity of Spots on Dorsal Connexivial Plates, (F) Total Pixel Intensity of Light Region on Dorsal Connexivial Plates, (G) Average Blue Pixel Intensity of Spots on Dorsal Connexivial Plates. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 12. Changes in color of T. dimidiata anatomical structures in relation to land use: (A) Average Red Pixel Intensity of Body, (B) Total Pixel Intensity of Body, (C) Total Pixel Intensity of Wing. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 13. Changes in morphology of T. dimidiata ventral regions in relation to land use: (A) Area of Light Region on Ventral Connexivial Plate, (B) Area of Ventral Dark Region, (C) Proportion of Ventral Dark Region to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 14. Changes in morphology of T. dimidiata dorsal regions in relation to land use: (A) Area of Light Region on Dorsal Connexivial Plates, (B) Area of Spots on Dorsal Connexivial Plates, (C) Shape of Light

Region on Dorsal Connexivial Plates, (D) Shape of Spots on Dorsal Connexivial Plates, (E) Proportion of Light Region on Dorsal Connexivial Plates to Body Area, (F) Proportion of Total Spot Area on Dorsal Connexivial Plates to Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).

Figure 15. Changes in morphology of T. dimidiata anatomical structures in relation to land use: (A) Wing Area, (B) Proportion of Wing Area to Body Area, (C) Body Area. Bars denote 95% confidence intervals, and parametric comparisons use letters to denote significant differences between means (Fisher LSD, P<0.05).