Johnston´S Organ As a Mechanosensory Element for Spatial Orientation in Rhodnius Prolixus

Johnston´s organ as a mechanosensory element for spatial orientation in Rhodnius prolixus

Bibiana Ospina-Rozo; Manu Forero-Shelton, Jorge Molina

Flagellar antennae in the class Insecta generally bear two basal segments (scape and pedicel) and a segmented flagellum lacking intrinsic muscles (Schneider, 1964). In the non-muscular joint between the pedicel and the flagellum is located the Johnston’s organ (JO). This organ is a chordotonal complex consisting of sub-unities called scolopidia, each one bearing one to three specialized sensory neurons (Yack, 2004). These neurons are capable of detecting the movement of the flagellum, and transducing it into action potentials (Yack, 2004). These two features: the lack of intrinsic muscles beyond the scape and the presence of the JO are considered synapomorphic traits for the class Insecta (Kristensen, 1998; Kristensen, 1981).

The Johnston’s organ has been deeply studied in groups of Holometabolous insects, and it has been proven to have many important and diverse functions such as flight control (Sane et al., 2007), near- field hearing (Kamikouchi et al., 2009) and detection of electric fields (Greggers et al., 2013), among others. In holometabolous insects the JO can have variable number of scolopidia. Higher numbers and organization of scolopidia are considered either a strategy to enhance resolution like near-field sound detection in males of Aedes genus with 7000 scolopidia (Boo & Richards, 1975), or a way to ensure various functions as in Drosophila, where the JO consists of 200 scolopidia divided into 5 regions and capable of codifying wind direction, near-field sound and gravity direction (Kamikouchi et al., 2009).

However, the function of the JO in basal groups remains unclear, and at the same time, the basal function of the JO is unknown. Since it is a synapomorphic trait for all insects (Kristensen, 1981), the JO could have had a simpler function in basal groups, and subsequently undergone an exaptation process, getting new functions according to different selective pressures over each group of insects. A possible basal function for the JO could be gravity sensing.

That is because gravity sensing is essential for life on earth in order to ensure key processes inside the body and locomotion (Morey-Holton, 2003). Other mechanical stimuli are usually relevant only for certain groups of insects, while gravity affects all groups in the same way. And, as the JO is a graviceptor

1 in some groups of holometabolous insects (Kamikouchi et al., 2009), graviception could be a function present in basal groups and retained in some holometabolous groups.

In order to test this hypothesis it is important to evaluate if the JO can perform as a graviceptor in basal groups of insects. Therefore, we have chosen to study the Heteroptera group which is part of the most successful radiation of hemimetabolous insects (Weirauch & Schuh, 2011), and has a JO consisting of a maximum of 50 scolopidia (Rossi & Romani, 2013). Among the diversity of species of Heteroptera we decided to work with Rhodnius prolixus because this species is showing a clear negative geotactic behavior (Gaunt & Miles, 2000), which lead us to think that gravity sensing is highly important for these insects in order to orient themselves while climbing.

Our research´s aim was to determine if the Johnston’s organ (JO) of Rhodnius prolixus, being a complex of mechanosensory neurons, could act as a graviceptor bringing information about the direction of gravity, and thus helping the insect to monitor its spatial orientation.

In order to establish if the JO in R. prolixus can perform as a graviceptor, it is necessary to study the process of transduction of the gravity force. Transduction process of any stimulus has three phases as reported by Yack (2004): 1) Coupling, how certain part of the body has a structural configuration allowing it to link the stimulus with the sensory neurons. 2) Transduction, how mechanical displacement of the neuron results in variation of membrane potential. 3) Coding, production of specific patterns of electrical impulses.

Our study focusses in the coupling process of sensing gravity force with the antenna. In this phase it is necessary to know the external structure of the antenna, then the way it is affected by the stimulus action and finally the internal structure, meaning the organization and anchoring point of the JO. These three aspects have to be correlated in order to determine: 1) if the structural traits in the antenna are part of a structural design optimized to enhance the response to the gravity force action, and 2) if the anchoring point of the scolopidia is appropriate to allow their linking with the movement of the flagellum produced by gravity force. In order to explore the three aspects mentioned above and their correlations, we used basic physics and mechanical engineering approaches. Our results are divided into three parts presented here each one as an independent manuscript.

First of all, we studied the external morphology of the antenna, characterizing the length and diameter of the segments, shape and size of the non-muscular joints and cuticle thickness. Since R. prolixus is a hemimetabolous insect, and the five nymphal stages have very similar habits than the adults (Paurometabola according to McKamey (1999)), they were expected to have similar antennae. We analyzed the external morphology in the antenna of the five nymphs and the adult. These results are

2 presented in the first manuscript with emphasis in the non-muscular joints. We also evaluated thickness of the cuticle walls in the antenna of the first nymph, fifth nymph and the adults. These measurements were included as important parameters in the model developed and presented in the third manuscript.

Then, we carried out a biomechanical analysis of R. prolixus antennae by changing the insect’s spatial orientation and seen the effect of the standard earth gravity on the position of the flagellum (distal part of the antenna). Results of this process are presented in the second manuscript.

Once we had information about structure of the antenna and how it is affected by the gravity force, the next step was to observe the organization of the scolopidia in the JO inside the pedicel. Although previous studies had shown the longitudinal shape of a scolopale unit of the JO in R. prolixus (Wigglesworth & Gillet, 1934), our findings are important because they are showing the anchoring point of the scolopidia, the potential number of scolopidia in the JO, and their organization inside the pedicel. This information is available in the second manuscript too.

Our third manuscript is in a certain way a combination of the first two manuscripts. By using finite element analysis (FEA), we explored the relevance of the main structural features observed of the antenna in the process of coupling the stimulus of gravity. And, we compared how gravity force acts on the very different structural designs of the antenna in three postembryonic stages of the life cycle in R. prolixus.

Our findings support the hypothesis that the JO in R. prolixus could act as a graviceptor. The design of the antenna seems to be the key element to make it flexible enough to perform as a mechanical sensor. Also the antenna is bended by gravity only in specific areas located very close to the anchoring point of the scolopidia in the JO, which was confirmed by our biomechanical analysis and computerized modeling. In conclusion, the flagellar antenna of Rhodnius prolixus could be acting as a coupling organ for mechanical information possibly codified by the Johnston’s Organ. Also, gravity is a mechanical stimulus capable of affecting the flagellum position in accordance to changes in insect’s position.

In order to study the second and third phases of the process suggested by Yack (2004), electrophysiological studies are still needed. This kind of studies could lead to understand which part of the information of the movement caused by gravity over the flagellum, is being codified and sent to mechanosensory processing centers in the brain, via the antennal nerve.

The methodology described here is appropriate to study any kind of graviceptor under the real magnitude and direction of the gravity force stimulus. Also, our results are useful to understand the characteristics of the gravity stimulus. This information has to be used to determine the better way to administrate the

3 gravity stimulus to the antenna, and to interpret the obtained results in different stages of postembryonic development in insects.

References

 Boo, K.S., & Richards, A.G. (1975) Fine structure of scolopidia in Johnston’s organ of male Aedes aegypti (L.) (Diptera: Culicidae). International Journal of Insect Morphology and Embryology, 4(6), 549–566.  Gaunt, M., & Miles, M. (2000). The ecotopes and evolution of triatomine bugs (Triatominae) and their associated trypanosomes. Memórias Do Instituto Oswaldo Cruz, 95(4), 557–65.  Greggers, U., Koch, G., Schmidt, V., Dürr, A., Floriou-servou, A., Piepenbrock, D., Göpfert, M., & Menzel, R. (2013). Reception and learning of electric fields in bees. Proceedings of the Royal Society, 280(1759), 20130528.  Kamikouchi, A., Inagaki, H. K., Effertz, T., Hendrich, O., Fiala, A., Göpfert, M. C., & Ito, K. (2009). The neural basis of Drosophila gravity-sensing and hearing. Nature, 458(7235), 165– 171.  Kristensen, N.P. (1981). Phylogeny of Insect Orders. Annual Review of Entomology, 26(1), 135-157.  Kristensen, N. P. (1998). The groundplan and basal diversification of the hexapods. Arthropod Relationships, 55, 281-293.  McKamey, S.H. (1999). Biodiversity of tropical Homoptera, with the first data from Africa. American Entomologist-Lanham-, 45(4), 213-222.  Morey-Holton, E. (2003). The impact of gravity on life. In: Evolution on planet earth: The impact of the physical environment, New York, Academic Press, pp. 143 – 160.  Rossi, M.V., & Romani, R. (2013). The Johnston’s organ of three homopteran species: A comparative ultrastructural study. Arthropod Structure & Development, 42(3), 219–228.  Sane, S. P., Dieudonné, A., Willis, M. A., & Daniel, T. L. (2007). Antennal mechanosensors mediate flight control in moths. Science, 315(5813), 863–866.  Schneider, B.D. (1964) Insect Antennae. Annual Review of Entomology, 9(1), 103–122.  Weirauch, C., & Schuh, R.T. (2011). Systematics and Evolution of Heteroptera: 25 Years of Progress. Annual Review of Entomology, 56, 487–510.  Wigglesworth, V. B., & Gillet, J.D. (1934). The function of the antennae in Rhodnius prolixus (Hemiptera) and the mechanism of orientation to the host. Journal of Experimental Biology,11, 120-139.  Yack, J. E. (2004). The structure and function of auditory chordotonal organs in insects. Microscopy Research and Technique, 63(6), 315–337. 4

Structure and postembryonic development of intersegmental nodules in the non-muscular joints of Rhodnius prolixus antennae

Bibiana Ospina-Rozo1; Manu Forero-Shelton2, Jorge Molina3

1. M. Sc. CIMPAT - Departamento de Ciencias Biológicas – Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected] 2. Dr. Sc. Grupo de Biofísica - Departamento de Física - Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected] 3. Dr. rer. nat. CIMPAT - Departamento de Ciencias Biológicas – Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected]

Abstract

The flagellar antennae of Insecta consist of two basal segments and the distal segmented flagellum lacking intrinsic muscles. The flexibility and structure of the antennae depend on the properties of their non-muscular joints. The Heteropteran antenna is divided into four long segments and has two intersegmental nodules in the non-muscular joints. Little is known about the structure, properties or function of these nodules. The aim of this study was to characterize the structure and postembryonic development of different regions in the non-muscular joints of the antenna of Rhodnius prolixus, and measure their rigidity. Using SEM imaging, we tracked the changes in shape and size of both intersegmental nodules over the course of the hemimetabolous insect life cycle. The nodule between the two flagellar segments (intraflagelloid) is a sclerite already present from the first nymphal stage, while the nodule between pedicel and flagellum (former preflagelloid, now prebasiflagellite) originates by gradual separation of the basis of the basiflagellum during postembryonic development. Using AFM, we established a qualitative correlation between the topography of the surface and the rigidity of the joint between pedicel and flagellum. Antennal pedicel, basiflagellum and prebasiflagellite have similar rigidity, while those regions connecting the nodule with each segment are more flexible, signaling the presence of two sub-articulations.

Keywords: Rhodnius prolixus, antennal joints, atomic force microscopy, scanning electron microscopy, flagellar segments.

Introduction

All members in the Insecta class s. str. have a pair of antennae (Schneider, 1964). These are very important structures with different functions such as codifying various stimuli of different nature, stabilizing role during flight or even formation of air reservoirs in some aquatic beetles (Schneider, 1964). According to their morphology, two types of antennae are distinguishable: Segmented and flagellar antennae (Schneider, 1964). Flagellar antennae are recognized by two basal segments (scape and pedicel) and a flagellum composed of several segments with similar shapes (Schneider, 1964). The absence of intrinsic antennal muscles beyond the scape has been defined as one of the synapomorphic characteristics of the class Insecta (Kristensen, 1998). Therefore antennal structure beyond pedicel in Insects s. str., is primarily maintained by non-muscular joints (Zrzavy, 1990).

For insects of the Order Hemiptera the number of antennal segments and intersegmental non-muscular joints increases proportionally to the length of the antenna, preserving a constant level of flexibility along different species (Zrzavy, 1992a). An exception to this pattern is the subgroup of Heteroptera, where the long antenna is divided into a few number of segments (Zrzavy, 1992a). In fact, the antennal ground-plan in Heteroptera consists of a unique configuration of four long and cylindrical segments called scape, pedicel, basiflagellum and distiflagellum (Zrzavy, 1990). Non-muscular joints are located between pedicel and basiflagellum and between the two subdivisions of flagellum (Zrzavy, 1990). Additionally, two sclerotized intersegmental nodules are located in those joints (Zrzavy, 1990) (Fig. 1). These nodules are recognized as an autapomorphy of Heteroptera (Zrzavy, 1992a), and their presence has been suggested as a mechanism to maintain the structure of the long antenna presented by this group of predatory insects (Zrzavy, 1992a).

Little is known about these intersegmental nodules in the antennae. Their presence / absence and their shape tend to be used as morphological traits in phylogenetic analysis and taxonomic classification (Spangenberg et al., 2013). Although there have been some attempts to establish anagenesis of intersegmental nodules, their morphogenesis remains ambiguous (Zrzavy, 1992b).

Considering the origin of the intersegmental nodules, two mechanisms have been proposed (Zrzavy, 1990): in the first mechanism, some parts of the existing articulation gradually develop more sclerotization until they become structural reinforcements called intersegmental sclerites. In that case, the first nodule located between the pedicel and the flagellum is called the preflagelloid and the second one located between the basiflagellum and the distiflagellum is called the intraflagelloid. The second mechanism suggests that a progressive separation of the bases of one antennal segment is the origin of the intersegmental nodules. In this mechanism, the intersegmental nodules are considered very short

6 segments with a function in the respective articulation, often replacing intersegmental sclerites, and their name depends on the segment from which they separated (Zrzavy, 1990).

Zrzavy (1990) studied morphologically the antenna in various species of Heteroptera, and established that the ground-plan in this group includes two intersegmental sclerites, the preflagelloid and the intraflagelloid. In a very small number of families, the presence of bases instead of sclerites was registered. Only the family Hebridae showed co-occurrence of the two structures in the articulation between pedicel and basiflagellum (Zrzavy, 1990). Due to its size, high-resolution microscopy techniques are required to analyze these small nodules located in the insect antennae.

Discrimination between bases and sclerites is not possible based only in morphological traits; then, it highlights the need for developmental studies to track changes in different parts of the antenna in order to better understand their origin, structure and perhaps their function. The origin of structures in the antennae of some insects can take place during embryogenesis while in other insects those changes can happen as part of postembryonic development. For example, in some families like Nepidae, Corixidae and Notonectidae, antennal segmentation takes place during postembryogenesis: The early nymph has only one segment, which undergoes subsequent divisions until the four segments are developed (Zrzavy, 1990). In contrast, development of intersegmental nodules is unknown, and data is still insufficient to tell if there is any part of the process taking place during postembryonic development.

Using data from morphological studies of antennal structure in Rhodnius pallescens and Triatoma infestans, Zrzavy (1990) proposed that the subfamily Triatominae retained the original ground-plan of Heteroptera with four segments in the antennae and two intersegmental sclerites (scape–pedicel– preflagelloid–basiflagellum–intraflagelloid–distiflagellum; in order from proximal to distal) (Fig. 1A). Rhodnius prolixus is a well-known species of the subfamily Triatominae, not only because of its medical importance as a vector as the vector of the Chagas disease (Garcia et al., 2007), but also for being a good biological model, easily raised under laboratory conditions and suited for experimental manipulation (Buxton, 1930). R. prolixus has been widely used in ecological, morphological, behavioral and physiological studies (Pachebat et al., 2013; Reisenman, 2014; Urbano et al., 2015; Vinauger et al., 2013; Zandawala et al., 2015).

Therefore, the aim of this study is to analyze the structure of antennal non-muscular joints in Rhodnius prolixus (Reduviidae - Triatominae), during postembryonic development. This information could be integrated with data of the mechanical characteristics of the cuticle in different parts of non-muscular joints such as rigidity, as a preliminary approach to understand the role of intersegmental nodules in the antennal non-muscular joints of Heteropteran insects. That is because the performance of biological

7 materials is the product of the combination between their mechanical properties and the specific structure at each level of organization (Nikolov et al., 2010).

Atomic force microscopy (AFM) is a very accurate technique capable of characterize topography of biological materials with high resolution and to provide data of mechanical properties (Vinckier & Semenza, 1998). Elasticity measurements are performed by pushing a tip onto the surface of the sample obtaining force-versus-distance curves, then a theoretical model was applied to these curves obtaining the Young’s modulus (Vinckier & Semenza, 1998). This modulus is the mechanical resistance of a material to elongation or compression (Beer et al., 2008). Comparing rigidity among different regions in the non-muscular joint of Rhodnius prolixus antenna is useful to determine which parts are responsible for bending, and if the intersegmental nodules have similar properties to the cuticle in the segments or if they are flexible.

Using high-resolution microscopy techniques we aimed to characterize the structure of intersegmental nodules, determine if there is any change in its shape in different stages of the life cycle, identify their origin, and measure the rigidity of different regions of the non-muscular joint. This information is useful to better understand the bending process of the antenna.

Methodology

Insects Adults of Rhodnius prolixus were used to carry out all the experiments. Insects were maintained at 27 ± 2 °퐶, 75 ± 10 % of relative humidity and artificial light illumination from 6:00 to 18:00 h. Insects were fed every 15 days with bird blood.

Transmitted light microscopy In order to identify details in the non-muscular joints of Rhodnius prolixus antenna, particularly the intersegmental nodules, we used transmitted light microscopy. Two adults were anaesthetized at 4 °C for 5 minutes and one of their antennae was removed with scissors at the level of the scape. A stereomicroscope (ZOOM 2000, LEICA) was used to mount on a glass slide and ventral side upwards the removed antenna with a thin and drying coat of superglue. Pictures of the mounted sample were taken with a digital camera.

Atomic Force Microscopy (AFM) AFM analysis was carried out in order to characterize the topography and rigidity of the different parts in the non-muscular joint between pedicel and basiflagellum. First of all we anesthetized the insects at

4 °C for 5 minutes and removed the right antennae with scissors at the level of the scape. A stereomicroscope (ZOOM 2000, LEICA) was used to mount the removed antenna on a glass slide ventral side upwards. A thin and drying coat of superglue was used to glue the sample to the glass slide. After one hour of drying, antennal sensilla were removed from the antenna by rubbing it gently with a small piece of cotton in the opposite direction to the sensillar axis.

An Atomic Force Microscope (MFP-3D-BIO, Asylum Research) was used to establish the local topography of regions in the non-muscular joint between pedicel and basiflagellum: on the tip of the pedicel, the proximal sub-articulation (between pedicel and the nodule), the intersegmental nodule, the distal sub-articulation (between the nodule and the basiflagellum), the proximal part of the basiflagellum and a deep median groove ventrally located on the pedicel (Fig. 2A). Images were taken in AC mode with an AC160TS probe at an oscillation frequency of 1.38 푁⁄푚 in 30 x 30 µm area. All images were later analyzed with the Software Igor Pro 6.2.3.2 and the tools from the Asylum Research software.

We also used an AC240 TS – R3 (Olympus) probe for indentation in 5 points of the cuticle, (20 repetitions each point) in three regions of the non-muscular joint between pedicel and basiflagellum of one antenna: the pedicel, distal sub-articulation and intersegmental nodule. Indentations were made with a force of 20 nN and 500 nm/s velocity. Young’s modulus was calculated by using the JKR model fitted to a spherical probe of 9 nm diameter made of silicon and 0.3 Poisson ratio for biological samples. We determined the mean and standard error of the mean for the Young’s modulus of each region, and we used a non-parametric Wilcoxon singed rank test to compare Young’s modulus data between the three regions.

Scanning Electron Microscopy (SEM) Sequences of SEM images were used to determine the structure of the antenna and the shape of intersegmental nodules; and also, to track the changes in the non-muscular joint between pedicel and basiflagellum during postembryonic development.

Two adults and nymphs from each stage were anaesthetized at 4 °C for 5 minutes and their heads were removed with scissors. Heads and antennae were mounted ventral side upwards on metal pieces with conductive double-sided sticky tape, under a stereomicroscope (ZOOM 2000, LEICA). The mounted antenna were coated with a thin, uniform layer of fine particles of gold (100 Å) in low pressure conditions (10 – 4 Torr) for five minutes with a sputter-coater (Dentom vacuum Desk IV).

Finally, the specimens were observed with a JSM-6490LV (Jeol) scanning electron microscope at 15 KV. Micrographs were taken from the whole antenna in order to measure the length and width of the segments with magnifications ranging from 10X to 60X. Images of the non-muscular joints were taken

9 ranging from 100X to 2000X. We measured the length and width of the intersegmental nodules and the four segments present in the antennae of Rhodnius prolixus in the five nymphal stages and the adults.

Results

Antenna in Rhodnius prolixus According to morphological analysis of the images obtained with transmitted light microscopy, the antennae of males and females of Rhodnius prolixus seems to have retained the ground-plan of Heteroptera (Fig. 1A). Four segments scape, pedicel, basiflagellum and distiflagellum are present in the antenna. Intersegmental nodules are clearly visible in three joints: Scape – pedicel, pedicel – basiflagellum and basiflagellum – distiflagellum. They were named by Zrzavy (1990) prepedicelite, preflagelloid and intraflagelloid respectively. Only the last two are located of the non-muscular joints.

In both non-muscular joints, three parts are clearly differentiated, the proximal sub-articulation, the intersegmental nodule and the distal sub-articulation. Sub-articulations present a pale color and they appear to be the regions usually subjected to bending stress, according to stereomicroscope observations. On the other side, intersegmental nodules seem to be more sclerotized and also less flexible. On the distal part of the pedicel, we observed a deep median groove located ventrally in the antenna, this groove could be described as an extension of the proximal sub-articulation in terms of color and appearance, and it is also present in all developmental stages. No ventral groove was observed in the distal part of the basiflagellum, but potential mechanoreceptive sensilla were observed at the tip of the basiflagellum.

Mechanical and structural properties of the non-muscular joint between pedicel and basiflagellum Four types of surface textures were registered by AFM in different regions of the joint between pedicel and basiflagellum. An irregular topography with ridges and dips was observed in the long segments (Fig. 2B); a flat surface distally in the intersegmental nodule and a characteristic pattern of irregular ridges on the proximal edge of it (Fig. 2C). Finally, a large number of semi-spherical raised areas of different heights (Fig 2D) that are easily observed on flexible parts such as the ventral groove, the proximal sub-articulation and the distal sub-articulation. Three of these topographies are shown in Fig. 2 B-D.

The Young’s modulus values obtained with AFM analysis are displayed in Fig. 2E for three major areas with their respective standard error of the mean. Results indicate that bumpy areas have greater values of Young’s modulus, while regions with semi-spherical raised areas are more elastic. Young’s modulus of the intersegmental nodule 819.82 ± 349.655푀푝푎 and pedicel 739.01 ± 345.768 푀푃푎 were not statistically different (Wilcoxon signed rank test, 푝 푣푎푙푢푒 = 0.93 ). The distal sub-articulation modulus

10 was 26.64 ± 15.587 푀푃푎 and it was significantly different from the intersegmental nodule (Wilcoxon signed rank test, 푝 푣푎푙푢푒 = 0.008) and pedicel (Wilcoxon signed rank test, 푝 푣푎푙푢푒 = 0.004). Since the AFM images were comparable to the textures observed in SEM microscopy, we identified bumpy and flat areas as rigid surfaces and pointed areas as part of the elastic surfaces.

Postembryonic development of the intersegmental nodules and antennal segments SEM images allowed us to track the postembryonic changes in the two non-muscular joints during Rhodnius prolixus life cycle. As shown in Fig. 3A, the structure of the pedicel-basiflagellum joint in the first nymphal stage is different to the adults. In the first instar, only one articulated zone is visible at the tip of the pedicel, characterized by the presence of semi-spherical raised areas. A characteristic pattern of irregular ridges was observed on the basal part of the basiflagellum, suggesting the presence of the basal part of the future intersegmental nodule (Preflagelloid by Zrzavy (1990)). No evidence of a division between the basal part of the basiflagellum and the distal edge of the future nodule was observed in this stage.

The second and third nymphal stages presented a similar pattern with only one articulated zone visible at the tip of the pedicel, but in these cases, the basal part of the basiflagellum starts to develop a flat cuticular area separating the basiflagellum pattern and the irregular ridges present on the basis of the ground-plan preflagelloid nodule (Zrzavy 1990) (Fig. 3B and C). It is only up to the fourth nymphal stage (Fig. 3D to F) that the distal sub-articulation starts to separate the distal edge of the completely formed intersegmental nodule from the highly structured basal part of the basiflagellum. However, the intersegmental nodule observed in these nymphal stages was actually very different in shape and size when compared to the adult nodule (Fig. 3F). Morphological changes observed during the postembryonic development in the non-muscular joint between the pedicel and the basiflagellum were quantified to evidence how the intersegmental nodule increases its length and width during the insect’s growth (Fig. 4B and C). During the postembryonic development, the proximal section in the basiflagellum increases its width proportionally to the width of the intersegmental nodule known as preflagelloid (Zrzavy 1990), but in the last ecdysis a sudden drop in basiflagellum width was observed (Fig. 4B and Fig. 3E-F). As a consequence, the distal edge of the nodule is wider than the basal part of the basiflagellum in adults (Fig. 3F).

We also studied the postembryonic development of the intraflagelloid nodule located between the basiflagellum and the distiflagellum by using a sequence of SEM images. In this case, all nymphal stages and adults had a very well formed nodule with proximal and distal sub-articulations separating both flagellar segments (Fig. 5A-F). This non-muscular joint undergoes changes in size of its different components during the postembryonic development, but these changes were less dramatic than those observed for the joint between pedicel and flagellum. Interestingly, the four measured parts in the

11 basiflagellum – distiflagellum non-muscular joint increased their width more or less at the same rate, preserving then their proportions until insects reached the fifth nymphal stage. In the adult stage, both the basal part of the distiflagellum and the proximal part of the basiflagellum sharpen until they reached the same width of the distal edge of the intraflagelloid (Fig. 4D-F).

Finally, the SEM micrographs allowed us, to measure the length of the four segments in the antenna during the postembryonic development. The scape increased in length only twice its size in the early nymph. The segment with a higher rate of growth was the pedicel, which increased its length almost ten times from early nymph to adult. The basiflagellum and the distiflagellum increased their lengths too, but only until the insect reached the fifth instar, when they seem to reach an asymptote (Fig. 6). Another important feature was the fact that in the early nymph, both flagellar segments were longer than the pedicel, while in the adult the opposite was true (Fig. 6).

Discussion

Our results confirm that the antennal structure of Rhodnius prolixus resembles the ground-plan of Heteropteran insects described by Zrzavy (1990). Four long segments and two intersegmental nodules in the non-muscular joints were observed (Fig. 1). Nevertheless, previous work specifies that both of these nodules were sclerites originated by a tanning process in the already existent non-muscular joint Zrzavy (1990). Our analysis of each nymphal stage and adult of Rhodnius prolixus lead us to the conclusion that the two nodules had different origins: The distal nodule, called Intraflagelloid had an embryonic origin by sclerotization of the central section of the non-muscular joint (Fig. 5), while the proximal nodule located between the pedicel and the basiflagellum had a postembryonic origin based on a gradual separation from the basal part of the basiflagellum (Fig. 3). Following the nomenclature proposed by Zrzavy (1990) this kind of intersegmental nodule, should be called prebasiflagellite to indicate its postembryological origin.

The implications of these antennal organization could lay in the common usage of the intersegmental nodules as a morphological trait to perform phylogenetic, anagenetic and ontogenetic analysis (Zrzavy, 1992a, 1992b), taking into account that Rhodnius prolixus is known to be part of a derivate clade inside the Heteroptera according to molecular studies (Xie et al., 2008). The Heteroptera group is also part of the most successful radiation in the hemimetabolous insects and the non-solved relationships inside this group complicate the understanding of their evolution (Weirauch & Schuh, 2011). Therefore, more studies of comparative morphology could be useful to understand the evolution of the major clades and their specializations.

Our findings are of morphological and developmental interest because we are reporting two different origins for two almost morphologically indistinguishable antennal structures (compare Figs. 3 and 5). This raises the question about their function. Having in mind that the first nymphal stages lack a fully formed prebasiflagellite and that during the adult stage the same prebasiflagellite has a specific morphology, it is necessary to highlight the interest of understanding the implications of the antennal differences and/or the selective pressures that forced the adult antenna to develop its particular structure.

Topographic patterns in the surface of different regions of insect exoskeleton are usually related to the way cuticular layers are organized (Vincent & Wegst, 2004). This disposition can vary depending on the function of each part of the exoskeleton. Then, it is expected that more resistant and rigid structures often are presenting a particular topography with irregular dips and elevated regions, while more flat surfaces are those which need more movement (Nikolov et al., 2010; Vincent & Wegst, 2004). Our results in Figs. 2B-D are showing irregular dips in rigid antennal segments like pedicel and basiflagellum (Fig. 2B), and more flat surfaces in flexible cuticles like those observed in the prebasiflagellite and the proximal and distal regions of the joint (Figs. 2C and 2D).

Previous reports about cuticular properties have carried out mostly nanoindentation on very thin sections (Klocke & Schmitz, 2011), in this study we analyzed the cuticle attached to the whole structure by indentation. We believe that our macro-level approach is useful to obtain reliable data about mechanical properties in a situation as close as possible to the reality. However, in order to obtain even more accurate data, models to process force-indentation curves are still needing some modifications to include a wider array of parameters; for instance, other probe shapes and some adjustments to analyze samples with adhesion and pile-up materials (Oliver & Pharr, 2004).

The AFM analysis carried out here was effective as a technique to establish the relationship between the topographic patterns observed in different regions of the non-muscular joint and their elastic properties. The AFM analyses were also used as a complement to SEM images, reducing the possibility of artifacts due to dehydratation, sputtering or vacuum. The basal part of the flagellum and the distal part in the pedicel bear a characteristic pattern of dips and higher regions (Fig. 2) which might be related to their higher rigidity (Vincent & Wegst, 2004). We confirmed that the sub-articulations, seen under transmitted light microscopy as clear parts in the non-muscular joint, are in fact the more flexible regions responsible for antennal bending.

On the other hand, the prebasiflagellite had the same Young’s modulus of the tip of the pedicel which means it has a similar degree of tanning and it should allow a limited amount of movement compared to what sub-articulations are able to do (Fig. 2). Our results of the Young’s modulus are in the range of the values reported previously for cuticle with low degrees of tanning: between 10 to 1000 MPa (Klocke

& Schmitz, 2011; Vincent & Wegst, 2004). Values around 800 MPa were found in hard cuticular parts like the pedicel and the nodule, and values close to 10 MPa were measured in the more flexible cuticle forming the joint (Fig. 2E).

Considering the pattern of change in length of the segments during development (Fig. 4), it is possible to interpret that intersegmental nodules could be a kind of structural reinforces to protect the non- muscular articulations from failure (Zrzavy, 1992b). This hypothesis is based on the phylogenetic analysis of the presence-absence of the intersegmental nodules coupled with patterns on feeding habits (Zrzavy, 1992b), and suggest that a notable elongation of the segments in Heteroptera represents an adaptation for increased locomotor and sensory activity required by predatory insects, which triggered development of other apomorphies in order to restore flexibility in the long and oligomerous antenna (Zrzavy, 1992a). This flexibility could be interpreted as a key feature to prevent the antenna from damage but they could also have a function in coupling sensorial information as part of the mechanoreceptor systems.

A possible analysis to consider the structural importance of the non-muscular joints in Rhodnius prolixus antennae is that only the pedicel increases its length significantly more than the other segments (Fig. 6) during postembryonic development, possibly because intrinsic muscles in its base (Kristensen, 1998). Maybe, because muscles attaching the pedicel to the scape, can support a heavier structure than non- muscular joints located more distally. The reason why flagellar segments increased their length at a slower rate might be that the non-muscular joints are not capable of resisting structures longer than two millimeters for the prebasiflagellite and one millimeter for the intraflagelloid (Fig. 6). Further experiments are needed to test the validity of this hypothesis.

Finally, our analyses were focused mainly in the proximal non-muscular joint (prebasiflagellite between the pedicel and the basiflagellum) because in this joint the presence of the mechanosensory receptor called the Johnston’s Organ has been reported (Wigglesworth & Gillet, 1934). Previous reports have pointed out that in the Order Hemiptera the Johnston’s Organ is attached in a ring known as “Skleritring” located in the basal part of the prebasiflagellite (Weirauch, 2003; Zrzavy, 1990).

Conclusions

The structure of the antenna in Rhodnius prolixus is very different in the nymphs and the adults. The two intersegmental nodules in the non-muscular joints were observed and according to the ontogenetic changes we determined that the formerly called preflagelloid and the intraflagelloid have different origins. We confirmed that the intraflagelloid is a sclerite correspondent to the ground-plan of

Heteroptera, while the formerly called preflagelloid has its origin in the basis of the basiflagellum, so it is actually a basis instead of a sclerite and should take the name of prebasiflagellite. . Various changes occur in the non-muscular joints and segments of the antenna during the life cycle of R. prolixus. For example, the length and diameter of the segments and the intersegmental nodules; and the shape and size of the intersegmental nodules and articulated sections showed clear changes. The study of mechanical properties of the cuticle in different parts of the antenna can be correlated with developmental and morphological data founded here in order to better understand the function of intersegmental nodules.

Acknowledgements

We are grateful to Juan Diego Arango for his collaboration in collecting data from AFM and to Daniel Felipe Otálora and Alejandro Castañeda for help us with the AFM data processing. This work was supported by a “Proyecto semilla” approved to Bibiana Ospina-Rozo by the Faculty of Sciences at Universidad de los Andes-Bogotá.

References

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FIGURES Figure 1. General external morphology of the antennae of an adult of Rhodnius prolixus. A. Antennal ground-plan in Heteroptera consisting of four long, cylindrical segments (names on the right). Intersegmental nodules are prepedicelite, preflagelloid and intraflagelloid (arrows). The last two nodules are located in non-muscular joints and are considered an autapomorphy of Heteroptera. B. Transmitted light microscopy of the distal non-muscular joint showing the intraflagelloid (arrowhead) C. Transmitted light microscopy of the proximal non-muscular joint showing the preflagelloid/prebasiflagellite (arrowhead). Clear areas in B and C are more flexible than the darker areas.

Figure 2. Local topography of different regions of the pedicel – basiflagellum joint in the adult antennae of Rhodnius prolixus. A. General organization of the pedicel - basiflagellum joint. B. 30 x 30 µm AFM images showing the irregular topography of dips and higher regions present in the pedicel and basiflagellum. C. 30 x 30 µm AFM images showing irregular ridges present only in the proximal region of the nodule, D. 30 x 30 µm AFM images showing a large number of semi-spherical raised areas on the flexible parts of the articulation present in the ventral groove and distal joint (sub-articulation). Scale of all images ranging from 0 µm up to 4 µm of height where darkest regions are closer to 0. E. Young’s modulus measured for three major regions in the pedicel - basiflagellum non-muscular joint. Error bars represent the standard error of the mean for each antennal region.

Figure 3. Postembryonic development of the intersegmental nodule between the pedicel and the basiflagellum of Rhodnius prolixus. Sequence of SEM microphotographs showing ventrally the intersegmental nodules present between the pedicel and the basiflagellum, from the first nymphal stage to the adult (A - F). All scale bars are 25 휇푚. In the first stage the topographic pattern characteristic of the intersegmental nodule is clearly visible (arrow), but there is no division between the basis of the basiflagellum and distal region of the nodule (dotted arrow). In the adult, the ridge pattern is visible in the basal part of the nodule (arrow) but the distal part has developed a flat pattern. A considerably large division is present between the nodule and the basal part of the basiflagellum (arrowhead) and this is the newly developed distal sub-articulation. The intersegmental nodule appears to be gradually separating from the basal part of the basiflagellum during the life cycle as well as changing its shape.

Figure 4. Size of different segments of the antennal joints during the postembryonic development of Rhodnius prolixus. A. Scheme showing the regions measured at different levels of the pedicel – basiflagellum joint: Pedicel distal tip (Pd), basal edge of the nodule (pn), distal edge of the nodule (dn), basal part of the basiflagellum (Bfl), proximal joint (pj), Intersegmental nodule (preflagelloid/prebasiflagellite) (Pfl), distal joint (dj). B. and C. Width and length variations of the pedicel – basiflagellum joint at different stages in the life cycle of R. prolixus, respectively. D.

Intraflagelloid scheme showing the regions measured at different levels of the basiflagellum – distiflagellum joint: Basiflagellum distal tip (Bfl), basal part of the distiflagellum (Dfl), Intraflagelloid (Ifl). E. and F. Width and length variations of the basiflagellum – distiflagellum joint at different stages during the postembryonic development of R. prolixus, respectively.

Figure 5. Postembryonic development of the intersegmental nodule between the basiflagellum and the distiflagellum of Rhodnius prolixus. Sequence of SEM microphotographs showing ventrally the intersegmental nodules present between the basiflagellum and the distiflagellum, from the first nymphal stage to the adult (A - F). Scale bars: A- C. 10 휇푚, D- E. 20 휇푚. The intersegmental nodule (arrow), as well as the distal joint (arrowhead) are already present in the first nymphal stage and suffered changes in shape and size during the different growth phases.

Figure 6. Length of the four segments of the antenna during the postembryonic development of Rhodnius prolixus. Both flagellar segments seem to enlarge slower than the pedicel. Also, during early nymphal stages both flagellar segments are larger than the pedicel. However, when the insects reached the adult stage, the contrary is true.

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FIGURE 2

FIGURE 3

FIGURE 4

FIGURE 5

FIGURE 6

Biomechanical analysis of the antennae of Rhodnius prolixus as a gravity sensor

Bibiana Ospina-Rozo1; Manu Forero-Shelton2, Jorge Molina3

1. M. Sc. CIMPAT - Departamento de Ciencias Biológicas – Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected] 2. Dr. sc. Grupo de Biofísica - Departamento de Física - Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected] 3. Dr. rer. nat. CIMPAT - Departamento de Ciencias Biológicas – Universidad de los Andes Cra 1 No 18 A – 12 Bogotá, [email protected]

Abstract

Earth gravity is the main stimulus used by insects to monitor their spatial orientation. Rhodnius prolixus insects show a negative geotaxis behavior but the mechanism they use for gravity sensing is not clear. Since flagellar antennae of some insects act as graviceptors, the aim of this study was to determine if the basiflagellum of Rhodnius prolixus could act as a coupling organ, linking gravity force information to the Johnston’s Organ (JO) sensory subunits. We designed a platform to change insect’s angle while video-recording both flagellum and insect´s position simultaneously. Changes in basiflagellum angle are proportional to insect’s angle, but their amplitude is higher when the insect is located vertically (climbing position). Gravity torque is higher at horizontal position and bending directionality was observed in pitch orientation. Bending of non-muscular joint takes place mainly in the proximal sub- articulation between pedicel tip and prebasiflagellite. Histological sections of the antenna confirmed that the anchoring point of the JO in R. prolixus is located in the basal part of the prebasiflagellite. Our data suggested that the movement of the basiflagellum due to gravity force could be transferred to the scolopidia in the JO, supporting its relevance in gravity sensing and spatial orientation in R. prolixus.

Key words: Johnston’s Organ, Gravity sensing, Spatial orientation, Antennal joint, Negative geotaxis.

Introduction

Johnston’s organ (JO) is a chordotonal organ present only in the Class Insecta (Dicondilia) (Yack, 2004), which converts the movement of antennal flagellum with respect to the pedicel apex, into action potentials by specialized neurons called scolopidia (Yack, 2004). This mechanosensory organ has many different functions in holometabolous insects, for instance monitoring flight balance in Lepidoptera (Sane et al., 2007), near-field hearing in Diptera (Kamikouchi et al., 2009) and detection of electric fields in Hymenoptera (Greggers et al., 2013). However, as it is a synapomorphic character for all insects, the JO could have a simpler function in basal groups, and also undergone an exaptation process according to different selective pressures. There is a number of studies describing the structure of the JO in hemimetabolous insects (Toh, 1981; Wolfrum, 1990) and particularly in Hemiptera (Howse & Claridge, 1970; Jeram & Pabst, 1996; Rossi & Romani, 2013), but the physiological role of the JO in these groups is still unknown.

The forces caused by gravity are a very important stimulus for all living organisms affecting mainly their spatial orientation, their locomotion and the disposition of the fluids inside their body (Morey- Holton, 2003). That is the reason why the majority of living organisms have gravity sensors (Morey- Holton, 2003). In insects, there are many strategies to sense the direction of gravity force. For instance, specialized sensilla on the antennae or the cerci (Horn & Bischof, 1983; Walthall & Hartman, 1981) or chordotonal organs in the joints of their legs (Horn & Lang, 1978). Gravity sensing function has been very well described for the JO of D. melanogaster, in terms of its molecular basis (Kamikouchi et al., 2009), developmental process (Eberl & Boekhoff-Falk, 2007), physiological activation and behavior (Matsuo & Kamikouchi, 2013). Although little is known about JO as a gravity sensor in other species, gravity sensing could be the basal function of the JO since earth gravity is an omnipresent stimulus essential and common to all members of the Class Insecta.

Due to their medical importance in tropical regions (Lent & Wygodzinsky, 1979), Rhodnius prolixus (Reduviidae) has been very well studied, and since it can be easily reared and manipulated it has become a biological model organism to carry out a wide variety of studies (Buxton, 1930). In addition, this species is also a good model to study gravity sensing because it is characterized by a marked negative gravitaxis behavior, which is important in their natural habitat where they usually climb and walk on the trunk and branches of palm trees (Gaunt & Miles, 2000).

The Order Hemiptera has a relatively basal position in the Class Insecta, and it is the largest of the non endopterygote Orders (Cranston & Gullan, 2009). Thus, it is relevant to study if the JO is one of the mechanisms used to sense gravity in species from this Order, such as R. prolixus, in order to establish if

27 gravity sensing could be one of the basal functions for JO. Some previous behavioral experiments carried out in our group showed that the climbing behavior of R. prolixus is affected by neutralization of the JO (Rodriguez, 2003).

With the aim to validate previous behavioral experiments, it is necessary to describe the structure of the JO as a gravity sensor. According to previous studies (Kamikouchi et al., 2009; Matsuo & Kamikouchi, 2013) the JO’s scolopidia are able to detect the movement of the flagellum because they are attached to the basis of the flagellum (Todi et al., 2004). In order to asses if the JO of R. prolixus has a role as a coupling organ for the detection of the stimulus of standard earth gravity, we measured the effect of gravity on the pedicel-basiflagellum joint while changing the spatial orientation of the insect’s body.

Methodology

Insects Adults from Rhodnius prolixus species were used to carry out all the experiments. Insects were maintained in our colony at 27 ± 2 °퐶, 75 ± 10 % of relative humidity and artificial light illumination from 6:00 to 18:00 h. Insects were fed every 15 days with bird blood.

General characteristics of the antenna We estimated the average weight of a single antenna by measuring overall mass of 16 antennae with a precision analytical balance (New Classic MS - METTLER TOLEDO). General morphology of the antenna and the details of the non-muscular joint where the JO is located were described and showed in Fig. 1A and 1B of the first manuscript of this thesis.

Angle-changing device With the aim to facilitate changing of insect’s spatial orientation, we designed an accurate device (Fig. 1A) inspired in Walthall and Hartman (1981). Insects were fixed with plasticine in a small chamber (Fig. 1A-1) under a stereomicroscope (Leica ZOOM 2000). We used supercryl to glue the antenna (scape and basal part of the pedicel) to the head of the insect in order to prevent any movement of the antenna by muscle contraction. With the antenna in this position we used a webcam (V-UCR45- Logitech, Fig. 1A-3) with an altered lens to permanently record the changes in the angle of the basiflagellum with respect to its initial position. Distiflagellum displacement was not considered. A platform (Fig.1A-4) supported the chamber with the insect and the webcam, and allowed us to switch the position of the insect from horizontal to vertical orientation by using a rotation disc (Fig. 1A-5).

The swinging platform (Fig. 1A-6) changes its position around a central axis, reaching tilt angles between -30° and +30°, which can be achieved manually or automatically with a DC motor powered with 2 V. This motor transformed rotation in vertical movement by a not centered rod (Fig. 1A-9). The platform oscillation was recorded with a video camera (VixiaHf G30 Hd – Canon) and monitored when needed by tracking a signaling point (Fig. 1A-7). Steps of 10° were produced manually by aligning the signaler with the black infra-red sensors installed in the lateral support, which were part of an electronic circuit to monitor platform position (Fig. 1A-8). A laser beam (Fig. 1A-2) was projected in a white background where it could be recorded at the same time by the webcam focused on the antenna and the video camera used to record all movements of the platform.

Angle measurements We tracked the magnitude of the angles between the insect, the pedicel and the basiflagellum in relation to the horizontal (γ, β and α angles in Fig. 1B) by processing the videos with the software Tracker 4.8x. We obtained real-time variations of the 훼 angle from the initial baseline 훼 value. Pedicel angle 훽, for each experiment, depends on the way the antenna was glued to the head, and insect’s angle 훾 depends on the tilt of the platform. Four orientations were analyzed: pitch (horizontal and vertical) and yaw (horizontal and vertical) (Fig. 1B).

Angle resolved experiments and constant oscillation experiments To test the effect of gravitational acceleration on the angle of the basiflagellum in static orientations of the insect, we operated manually the angle-changing device. In order to carry out an angle resolved experiment we changed the angle of the platform between -30° and +30° in 10° steps, each step lasting for 5 to 10 s approximately. The beginning of each step was registered by projecting a laser beam into the webcam video of the antenna. We analyzed flagellum movement (∆훼) of seven insects in all orientations (Fig. 1B).

To test the effect of constant oscillations of the insect at 1 Hz between -30° and +30° on the angle of the basiflagellum, we tracked the position of the platform with the video camera and we used the laser beam (Fig 1A-2) to synchronize this video with the webcam video of the antenna. Both videos were analyzed with the software Tracker 4.8x. We carried out this experiment with six insects only in the pitch horizontal orientation.

In order to study the factors that determine the relationship between the change of the position by the insect and the movement of the flagellum, we analyzed mainly two characteristics of the movement of the basiflagellum: its total amplitude and symmetry around the 𝑖푛𝑖푡𝑖푎푙 훼𝑖 (푤ℎ푒푛 훾 = 0°) for each insect.

Values of 훼𝑖 > 90° were possible when placing the insect in a vertical position on the platform. We evaluated the amplitude of the movement of the basiflagellum in a five oscillation cycle, in 12 insects 29 in each of the four orientations (Fig. 1B) and in seven insects in the angle resolved experiment. We analyzed the symmetry of the movement in the pitch horizontal and vertical orientation for 18 insects. Additionally, the effect of the starting angle of the pedicel on basiflagellum displacements was also established in these experiments.

Modeling We fitted data of the movement of the basiflagellum when changing insect’s orientation, to a sinusoidal model and we did the same with the platform oscillation in order to evaluate if both movements were in phase. This was important to ensure that the only stimulus applied to the antenna is gravity and that there are no inertial effects in the oscillating experiment.

With the aim of describing the effect of gravity on the basiflagellum, we carried out second-order polynomial regressions for data of total amplitude and symmetry, in order to observe how this two properties change according to initial basiflagellum angle in pitch and yaw orientations, and relate this with the torque generated in the antenna by standard gravity force.

Finally, to determine the relevance of pedicel angle in the response of the basiflagellum to changes in insect’s orientation, we used linear regressions between pedicel and flagellum angle when the insect was located horizontally and remained static. We also established a proportionality constant between the insect angle and the basiflagellum angle, by using a second-order polynomial regression. We described the role of the pedicel angle in determining the magnitude of this constant. All of the statistical analyses were carried out with the software R x 64 3.0.3.

Experiments with non-muscular joint flexible regions Since the movement of the basiflagellum is the key factor to propose the antenna as a coupling organ for gravity information detected by the JO, we carried out experiments to determine which part of the non-muscular joint between pedicel and basiflagellum affected mostly the movement. Two flexible parts can be found in this joint: a proximal-one located between pedicel and prebasiflagellite; and a distal-one located between prebasiflagellite and basiflagellum.

In eight insects we evaluated the movement of the basiflagellum in the way described above but only for pitch orientation. Later, one of the two sub-articulations was glued with a very small drop of superglue and tested again in the platform. Finally, both sub-articulations were glued and tested. We quantified the reduction in the amplitude of the basiflagellum displacement with one or both treatments. Distal sub-articulation was glued first in four insects; while proximal sub-articulations was glued first in other four insects.

Anchoring of Johnston´s organ With the aim of determining the anchor point of the JO scolopidia, the left antenna was removed from the head capsule of two insects and fixed at 4 °C overnight in a solution of glutaraldehyde 2.5 %. Afterwards, longitudinal and transversal slides of 1 휇푚 were obtained with an ultramicrotome. Slices were dyed with toluidine blue stain. Images were obtained with an Olympus FluoView TM FV1000 confocal laser microscope stimulating with a laser of 휆 = 405 nm.

Results

General characteristics of the antenna The calculated average mass of one antenna (from the basis of the pedicel to the end of the distiflagellum) was approximately 54.37 휇𝑔. Taking into account the measurements of the size of different parts of the antenna obtained by SEM imaging in the first manuscript of this thesis it can be calculated that the mass of the flagellum must represent less than 50% of the estimated mass of the antenna.

Angle resolved experiments Overall, changes in the angle of the basiflagellum were caused by changes in the pitch of the insect (훾) in steps of 10° (Fig. 2A). Three types of responses were observed in the experiments: a full response in which the basiflagellum accurately followed the changes in insect pitch step by step a limited response in which he flagellum (under certain conditions) was not able to respond to changes in insect’s pitch; and an asymmetric response when the antennae only followed the upward movements (Fig. 2A).

Constant oscillation experiments In an oscillating scenario where the insect changes its angle between -30° and +30° during one second, the basiflagellum was able to follow the changes in insect angle (Fig. 2B). In order to demonstrate that both movements were in phase, data from the platform’s movement were fitted to a sinusoidal function (1) as well as the data from flagellum (equation 2):

훾푡 = 푀 sin(푏(푡) + 푐) (1)

훼푡 = 푁 sin(푏(푡) + 푐) (2)

Where M and N are constants describing the amplitude of the sinusoidal wave, b is a term describing 2휋 the period of the wave by the expression: , and c represents the phase shift. The average amplitude M 푏

31 for the movement of the platforms was 30.407 ± 0.983 ° and average period was 1.078 ± 0.061 s. The amplitude of the flagellum movement N was dependent on several factors discussed below, and was calculated for each curve independently. The value of the period for flagellar movement was calculated for each insect and compared with the period of oscillation of the respective platform. The average difference between the two periods was 0.0195 ± 0.013 s (n=6), meaning that the flagellum moves in accordance to the change in the platform’s angle.

In order to calibrate the movies of the platform and the flagellum that were taken with two different cameras, we calculated the duration of a laser beam projection shining for a short period of time (2 seconds approximately) in both videos for each insect. Average differences in laser beam duration between the two videos were 0.028 ± 0.017 s. We attributed this difference to the fact that frame rate of the webcam recording the antenna was 15 frames per second while video camera recording platform position had 23 frames per second. Since the difference originated by the frame rate of used cameras is higher than the difference calculated for the periods of the sine functions, differences in periods can be neglected.

In order to calculate if there was a phase shift between the movement of the flagellum and the movement of the platform, we calculated the time between the first appearance of the laser beam and the moment when the first evidence of movement was observed. Differences in time calculated from the two videos were 0.0286 ± 0.066 푠. Again, these differences can be attributed to the differences in frame rates of the two cameras.

In conclusion, both the change on insect’s angle and the change on the basiflagellum angle are synchronized, which is why it is correct to assert that even in oscillatory experiments, gravity is the only force causing the flagellum to change its angle. There are no inertial effects at this scale, because the speed of oscillation is low (1 Hz) and also because the antenna has a low mass.

Under certain conditions the flagellum was seen to move upwards. This can be explained because the amount of torque caused by the weight of the flagellum depends entirely on the weight component perpendicular to the longitudinal axis of the flagellum, which reduces its magnitude when the flagellum angle increases (Fig. 2C). With a constant value of gravity acceleration (9.8 푚⁄푠2), when the basiflagellum angle increases, the weight’s ability to make it rotate around the tip of the pedicel decreases. Under this conditions, gravity exerts a smaller effect on the basiflagellum than when the antenna was placed horizontally (훼 = 0), enabling the flagellum to move upwards. Effect of the initial basiflagellum angle and pedicel position on basiflagellum displacements caused by gravity

In order to establish the key elements defining basiflagellum displacement, we decided to study the properties of the basiflagellar response either to oscillations or to angle resolved changes of insect’s position in pitch and yaw: we evaluated the amplitude and the symmetry of the flagellar response as a function of the initial angle of the basiflagellum (Fig. 3).

When the initial basiflagellum angle (훼𝑖) was negative or close to 0°, the amplitude of the movement was less than 1° (Fig. 3A) and it can not copy the changes in insect position. Under these conditions, the basiflagellum had a limited response because gravity force acts preventing it from moving, since the value of the weight’s perpendicular component reaches its maximum value (Fig. 2C).

When 훼𝑖 was positive but smaller than 50° the basiflagellum positive amplitude (against gravity direction starting from 훼𝑖) tends to be higher than the negative amplitude (according to gravity direction starting from 훼𝑖) and it can reach values near to 4 times the negative amplitude. It means, that in this angle range and its counterpart greater than 90° (130° to 180°) the basiflagellum can easily goes upwards when insect’s position is increasing to the horizontal (Fig. 3).

On the other hand, when initial basiflagellum angle (훼𝑖) was equal or close to 90°, the value of the torque was very small or even 0. At this position, the basiflagellum was not affected by the gravity force, so it can accurately copy the movement of the insect. As gravity force action becomes more subtle, the amplitude of the basiflagellar movement increases as well as the symmetry index approaches to 1. A reduction in amplitude and alteration of the symmetry are both evidences of the gravity force acting on the basiflagellum.

The simpler way to describe how the amplitude and the symmetry are related to the initial basiflagellar angle is to adjust data to a second-order polynomial regression (Fig. 3). The following equations correlate total amplitude (from minimum value to maximum value) with the initial angle of the flagellum for pitch orientation (equation 3) (푝 = 4.104 × 10−10; 푅2 = 0.693), and yaw orientation (equation 4) (푝 = 2.2 × 10−16; 푅2 = 0.9107); while, symmetry of the movement for insects in both orientations pitch and yaw was correlated with the initial angle of the basiflagellum by equation 5 (푝 = 7.896 × 10−7; 푅2 = 0.331).

−4 2 −2 퐴푡표푡(푝𝑖푡푐ℎ) = −3.718 × 10 (훼𝑖) + 7.519 × 10 훼𝑖 + 1.394 (3)

−4 2 −2 퐴푡표푡 (푦푎푤) = −1.625 × 10 (훼𝑖) + 6.450 × 10 훼𝑖 + 1.784 (4)

−4 2 −2 푆𝑖푛푑푒푥 (푝𝑖푡푐ℎ) = 1.947 × 10 (훼𝑖) − 3.520 × 10 훼𝑖 + 2.712 (5)

The amplitude data of the basiflagellum displacements in pitch and yaw orientation were fitted to a parabolic equation, consistent with the physical principles involved in this phenomenon. In both kinds of movement, the amplitude was higher in angles close to 90°, however it seems that amplitude of movement was higher in the yaw orientation. By finding the vertex of the parabola for pitch orientation based on the equation 3 (5.195°), and then replacing its respective 훼𝑖 (101.116°) value into the yaw equation (4), we determined that the difference in maximum amplitude between these two polynomial regressions at that specific angle was 1.449°. Based in our data, movement of the basiflagellum was more restricted by gravity force in pitch orientation. Symmetry index pattern was similar for pitch and yaw orientations, so both sets of data were combined and correlated to the initial angle of the basiflagellum in order to obtain the parabolic equation 5. This equation showed that the gravity force was acting on the basiflagellum making its movement more asymmetric when higher values of torque are produced by the perpendicular component of the gravity force vector.

The starting position of the pedicel had also an effect on the responses of the basiflagellum to the movement of the insect (Fig. 4A). In this case, an increase in the position of the pedicel angle results in greater amplitude in the response of the basiflagellum. First, we evaluated if there is a pre-determined starting angle of the basiflagellum for each starting angle of the pedicel when an insect was not moving. Under this condition, we measured the values of 훼 and 훽 with insects in horizontal position (훾 = 0) in pitch and yaw orientations. We found a positive correlation between the two angles described as follows by the linear regression in equation 6 for pitch orientation (푝 = 2,2 × 10−16; 푅2 = 0.998) and for the linear regression in equation 7 with yaw orientation (푝 = 3,6 × 10−16; 푅2 = 0.997).

훼푝 = 1,085 훽 − 8,475 (6)

훼푦 = 1,102 훽 − 2,507 (7)

The angle of the pedicel has an effect on the basiflagellar angle in pitch and yaw orientations in a similar way since the slope in both equations was very similar. However a difference of approximately 6° in the intercept was found between the two orientations. That means that there is certain degree of directionality in the antenna. When the pedicel is at 0° to the horizontal and the insect is not moving, the basiflagellum goes more degrees downwards during the pitch orientation than in the yaw orientation.

Afterwards, we explored the relationship between the three studied angles (훼, 훽, and 훾) (Fig. 1B) only for pitch orientation because of the availability of the data from a wide range of initial angles in the basiflagellum, which was difficult to obtain in a yaw orientation because of the separation between the

34 antenna and the head of the insect. As we have already mentioned, the movement of the antenna is in the same phase with the movement of the platform. In this situation, it is possible to establish a mathematical relation between both for a given time instant as in the equation 8. By replacing 훼 and 훾 for the correspondent sinusoidal functions described in equations 1 and 2 respectively, we obtained equation 9.

훾 퐾 = 훼 (8)

푁 sin(푏(푡)+푐) 퐾 = (9) 푀 sin(푏(푡)+푐)

Our results from the constant oscillation experiments, demonstrate that there is no phase shifting or difference in period between the basiflagellum and the movement of the platform (Fig. 2B). Then equation 9 can be simplified as follows:

푁 퐾 = (10) 푀

Therefore, for the 18 insects tested in the oscillation experiments in pitch vertical and horizontal orientations we calculated K constant by dividing the amplitude of the sinusoidal function fitted to data to the average amplitude for the platform movement (30.407°). Something important to consider is that the amplitudes in this analysis were different than the “total amplitude” mentioned above, because they were not calculated from peak to peak but from zero to the values of peaks and troughs in the fitted sinusoidal model. Also, this sinusoidal fitting does not consider any change in symmetry of the movement.

There was a correlation between the amplitude of the flagellum movement in response to changes in insect’s orientation (platform movement) given here by the proportionality constant (equation 10) and the pedicel angle (Fig. 4B) described by the polynomial regression in equation 11 (푝 = 5.460 × 10−5 ; 푅2 = 0.4809).

퐾 = −8.948 × 10−6훽2 + 1.503 × 10−3훽 − 1.836 × 10−3 (11)

Other factors could be considered to determine the causes of the variation in data, however this pattern is a valid explanation for the constant of proportionality K, and it highlights the importance of pedicel angle. These results suggest that there is a specific range of optimal pedicel angles where the resolution in gravity information is increased. This hypothesis should be tested in behavioral experiments by

35 calculating pedicel angle while walking of climbing and in electrophysiological experiments to test patterns of codifying gravity information with different pedicel angles.

Experiments with non-muscular joint flexible regions A reduction in the amplitude of the basiflagellum movement was observed when any region in the non- muscular joint was neutralized (Fig. 5A). When we compared the amplitude of the oscillations of the basiflagellum, we measured a 24.750 ± 9.702 % reduction in the oscillation response of the basiflagellum when the distal sub-articulation was glued (Fig. 5B). While a reduction of 82.913 ± 17.165 % was found when the proximal sub-articulation was glued (Fig. 5C).

Anchoring of Johnston´s organ Scolopidia of the JO were observed in longitudinal sections of the antenna (arrow Fig. 6A). The presence of the ventral groove on the pedicel allowed us to identify ventral side in the slices (Fig. 6B). Scolopale sub unities were observed in dorsal side of the antenna in all sections, but it is still not clear whether the structures under the ventral groove are modified scolopale sub-unities attached to the pedicel tip more proximally due to the presence of the ventral groove itself, or if they are different structures (Figs. 6A and 6B).

Prebasiflagellite bears an “L” shaped cuticle projection below the flexible cuticle of the proximal sub- articulation, only in the dorsal side of the antenna. Scolopidia are anchored to the base of the “L” shaped cuticle projection (Fig. 5A).

Scolopidia were observed in transversal sections only in the dorsal side of the antenna. The ventral groove is seen as a straight line causing the antenna to be less symmetric (Fig. 5B). Approximately 50 scolopidia were counted in the periphery of antennal lumen and none of them was observed below the ventral groove (Fig. 5B).

Discussion

Despite the difficulties when measuring antennal weight, a better estimation of this amount of mass could be used as a term in the torque equation to determine the theoretical value of the effect of gravity force on the antenna and then compare it to our experimental results.

Standard earth gravity has a measurable effect on the antennae of Rhodnius prolixus (Fig. 2). When changing insect´s position with respect to the gravity vector direction, the basiflagellum changes the magnitude of the angle with respect to the horizon. When the insect was oriented in a horizontal position

36 the gravity effect was higher, meaning that the movement of the antenna was more limited. When the insect was located at 90° to the horizon, the effect of gravity force can be neglected. In these conditions, a small change in insect’s angle causes a significant change on the angle of the basiflagellum. In spite of the fact that magnitude of gravity force is always constant, its effective component was modified by the changes in insect´s orientation.

This phenomenon has two implications: on one hand, segments in the antenna do not need to detect changes in magnitude of gravity force (Horn & Kessler, 1975) to help the insects to determine its spatial orientation. It is possible that a system of spatial orientation in insects included a comparative process by sensing flagellum restriction when the insect is more horizontal with respect to an initial position. In order to explore physiological responses to gravity stimulus, considering both magnitude of the flagellum angle and changes in this magnitude, electrophysiological recording experiments are needed. On the other hand, if a climbing insect can detect differences in movement of the antenna close to 90°, it would mean that its antenna is optimized to work in this orientation because they would give more information of any small change in any position different than at 0° (walking position). This ability could be particularly relevant to Rhodnius prolixus if we have in mind that they spend a significant part of their life climbing palm trees and exposed to a strong negative gravitaxis (Gaunt & Miles, 2000). Electrophysiological studies are also needed to determine the way this information is processed by insects of this species, which depends on the way transduction system works: if it is a tonic or a phasic receptor (Sun et al., 2009).

Following the same line of thinking, the deep ventral groove located on the pedicel (Fig. 6) could be responsible for giving directionality to the movement but not in the expected way considering its position. It does not give more flexibility for movements in the pitch orientations; conversely it causes the basiflagellum to be always in a smaller angle with respect to the horizon and forced to be under a higher effective component of the gravity force. Insects could interpret this information too, making them easily identifiable when they are in vertical or horizontal positions and moving in pitch or yaw orientations. Although this ventral groove has been mentioned in previous studies (Carbajal De La Fuente & Catalá, 2002; Weirauch, 2003), this is the first time a function is proposed for this structure in Rhodnius prolixus.

Results of both experiments carried out changing insect’s orientation (angle resolved and oscillating movement of 1 Hz) showed to be appropriate to study the gravity force effect on the antennae of Rhodnius prolixus. It would be desirable to use an angle –change device such as the one we used to carry out similar experiments while recording the physiological activity of the JO neurons, because in this way, it is possible to apply the real gravity stimulus, calculate its effects experimentally and not only theoretically (Kamikouchi et al., 2009), and also study the adaptation of the JO neurons.

The frequency of oscillation is a very important factor; its reduction to less than 1 Hz may cause the presence of inertial effects, which can also be studied for invertebrates conducting the same experiments with different frequencies of the platform oscillations. In fact, our results do not allow us to refuse the possibility that the JO in Rhodnius prolixus could detect acceleration changes to give information about equilibrium. However, when making a comparison between invertebrate and vertebrate animals, inertial effects are more important for equilibrium sense in vertebrates (Barrett et al., 2010; Lockhart & Ting, 2007). We demonstrated that coupling of gravity information by the basiflagellum can take place during very short scales of time. In many studies the JO has been proposed to be integrated by phasic neurons (French, 1986; Howard & Bechstedt, 2004). This data, together with the coupling mechanism described here, points out to a system which can obtain a continuous set of information about the changing in position of insect along time.

Still, our experiment is appropriate for studying gravity force action on any insect receptor (Walthall & Hartman, 1981), and it is desirable to use an appropriate device to ensure that magnitude and other characteristics of the applied stimulus are as close as possible to reality. Traditionally, gravity detection has been studied with different methods (Matsuo & Kamikouchi, 2013; Sun et al., 2009) less accurate than the one proposed here, since they are unable to reproduce the amount of movement of the basiflagellum in reality or they are not able to measure the magnitude of the provided stimuli.

This study established the important role of the pedicel in the response of the basiflagellum to insect’s movement (Fig. 4). According to our results, R. prolixus should be able to control the pedicel angle when they need information of gravity direction. Also, based on observations of these insects raised in our colony, we can say that when walking and climbing the pedicel angle is equal to 0°. The antennae in R. prolixus are usually lifted up in a certain angle. Control of this angle has been reported for other insects, for instance, in Calliphora erytrocephala it has been shown that they can monitor their body orientation when walking or before starting to flight by ensuring the optimal angle for the pedicel with specialized sensilla in the dorsal side of scape (Krishnan et al., 2012).

Our histological results are similar to those obtained for the JO of Nezara viridula by Jeram and Pabst (1996) who used TEM to report a basement membrane surrounding the scolopidia and connecting them to each other and to the epidermis. This base membrane is produced by the accessory cells (Jeram & Pabst 1996) and it is the only structure of the scolopidium visible with transmitted light microscopy because of its size. In our case, this could be the structure observed with optical microscopy. But with this resolution it was possible to see that the anchoring point of the scolopidia of the JO in R. prolixus is in the basis of the prebasiflagellite (Fig. 6A).

The “L” shaped projections where the scolopidia seem to be attached, are similar to the structures called “prongs” previously reported in the JO of mosquitoes (Avitabile et al., 2010). In mosquito males, near- field sound hearing is very important for mating, so their JO has a very high number of scolopidia, near to 7500 and a high level of organization (Boo & Richards, 1975). A series of various scolopidia attach lengthwise each prong and they have slightly different roles in the process of hearing according to a mathematical modeling developed for the JO of Toxorhynchites brevipalpis (Avitabile et al., 2010). In the case of Rhodnius prolixus the scolopidia attach only to the vertex of the “L” shaped prong revealing a simpler organization. However, both mosquito and R. prolixus prongs seem to be a geometrical strategy to amplify the effect of flagellar movement on the scolopale units.

That morphological information combined with our gluing experiments, where we demonstrated that the movement of the basiflagellum was mainly due to the characteristics of the proximal sub- articulation, suggests that the movement allowed by the proximal sub-articulation (Fig. 5) could be easily transmitted to the scolopale cells attached to the intersegmental nodule. In order to obtain more data about the ultrastructure of the JO scolopidia and the way they attach to the flagellum, more detailed TEM microscopy images are needed. Results presented here seem to agree with the proposal that the JO is located in the tip of the pedicel and anchored to the basal part of the prebasiflagellite, called “Skleritring” by Zrzavy (1990) and showed previously for R. prolixus (Wigglesworth & Gillet, 1934).

According to previous studies the number of scolopidia in the JO of Hemiptera remains quite constant among the species and never exceeds 50 which is considered a very small number when compared to the JO in holometabolous insects (Rossi & Romani, 2013). Our results show a preliminary number of 48 scolopidia in the JO of R. prolixus arranged in a radial disposition in the periphery of the antenna. This is a very high number for a member of the Order Hemiptera. Rossi and Romani (2013) proposed that higher numbers of scolopidia in the JO and higher levels of organization in their array are presented by insects with a strong negative gravitaxis and with very active habits including climbig, jumping and walkig for relatively long distances. According to all this information, the JO in R. prolixus can be considered a very important sensorial structure for its ecological habits.

Conclusions

We determined here that the gravity force has a measurable effect on the basiflagellum of R. prolixus adults by changing its angle according to insect’s orientation. This effect is given by the perpendicular component of gravity force on the basiflagellum, which is higher in the horizontal pitch orientation, maybe because of the action of the deep ventral grove observed on the pedicel. However, when the insect is located in vertical orientation, gravity force exerts a very small torque over the basiflagellum,

39 allowing it to move and copying with higher amplitude any changes in insect’s angle. The movement of the basiflagellum is possible, mainly because of the flexible proximal sub-articulation observed between the pedicel and the prebasiflagellite. Therefore, anchoring the scolopidia of the Johnston’s organ in R. prolixus to the prebasiflagellite could be interpreted as the optimal disposition to codify basiflagellar movements caused by the action of the gravity force. Our methodology allowed us to study graviception in a proper range of the stimuli very close to the reality, and also allowed us to establish that from a biomechanical point of view, the pedicel-basiflagellum joint in R. prolixus may be well suited for gravity sensing and transducing it with the Johnston’s organ anchored to the prebasiflagellite.

Acknowledgements

We are very grateful to David Gerardo Rozo for his professional assistance in designing the device to change angles in the body orientation of Rhodnius prolixus. We also want to thanks César Quintana for his technical assistance to glue the antennal sub-articulations and to Camilo Andrés Espejo for his orientation in the analysis of mechanics of the antenna. Finally we thank Daniel Felipe Otálora for his help with confocal imaging. This work was supported by a “Proyecto semilla” approved to Bibiana Ospina-Rozo by the Faculty of Sciences of Universidad de los Andes.

References

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FIGURES

Figure 1. Device to produce basiflagellar displacements with different body orientations. A. Main components of the device are insect chamber (1), laser beam (2), webcam (3), insect´s platform (4), rotation disc (5), swinging platform (6), signaling point (7), infra-red sensors (8), and connecting rod (9). B. Drawing showing the angles measured and the orientations evaluated in this study.

Figure 2. Changes in the basiflagellar angles due to the action of the standard earth gravity. A. Angle of the basiflagellum was measured for changes in steps of 10° (rough data). Three types of responses were identified, black line was considered a complete response (accurately copying changes in insect position), gray line was considered a limited response (no relationship with the changes in insect’s position) and blue line was an asymmetric response (antennae can only go upwards). B. In an oscillating scenario (1 Hz) the basiflagellum was able to copy the information of changes in the insect position. As both movements are in phase, gravity was the only force causing the flagellum to move. C. Comparison of the torque originated by gravity force over the basiflagellum when flagellum angle 훼 is near to 0° (left) and between 0° and 90° (rigth). The term “mg” is the flagellum weight, r is the ratio from rotation point (pedicel tip) to mass center.

Figure 3. Effect of the initial angle of the basiflagellum on its own response to body movements. A. Amplitude of the basiflagellar oscillating movement was higher when initial basiflagellum angle (훾 = 0) was near to 90°. Continuous lines are representations of the polynomial regression for amplitude in pitch (black) and yaw (gray). B. Basiflagellar movement was more symmetric when the angle was near to 90°, in contrast, when the angle was closer to the horizontal, the movement against gravity direction was up to 4 times the movement in the same direction of gravity force. The polynomial regression applied to data from pitch and yaw orientations combined is showed with a red line.

Figure 4. Relevance of the pedicel angle in the blasiflagellar response. A. An example of rough data with clear effect of pedicel angle on the amplitude of the movement of the basiflagellum. In order to quantify this effect, we calculated a constant of proportionality (K) between the insect angle (훾) and the basiflagellum angle (훼) during five oscillation cycles. B. Representation of the constant of proportionality (K) calculated with a polynomial regression for pedicel angle (훽) (red line).

Figure 5. Regions of the non-muscular joint regulating basiflagellar movement. A. General scheme of the non-muscular joint between pedicel and basiflagellum. Three regions are clearly visible, the prebasiflagellite in the middle of the articulation (Pf), proximal sub-articulation (px) between pedicel and prebasiflagellite, and distal sub-articulation (ds) between prebasiflagellite and basiflagellum. B. and C. are examples of experiments before and after gluing one of the sub-articulations. Black lines are flagellum response to oscillations with intact joint; blue lines are the response of the same antenna with

43 distal sub-articulation glued (B) and proximal sub-articulation glued (C); gray lines are the response of the same antenna with both sub-articulations glued.

Figure 6. Thin sections of the non-muscular joint between the pedicel and the basiflagellum in Rhodnius prolixus antenna. A. Longitudinal section showing the tip of the pedicel (Pd), prebasiflagellite (Pf) and basal part of the basiflagellum (Fl), deep ventral grove (V), antennal nerve (AN) and a scolopidium from the JO located dorsally (arrow). B. Transversal section at the level of the proximal sub-articulation near to the basis of the prebasiflagellite. Ventral side is identifiable by the presence of the ventral groove (V). Trachea (T) and antennal nerve (AN) are also visible. The region with the aspect of a concentric ring (SK) in the middle is the basis of the prebasiflagellite. An example of anchor of the scolopidia is marked with an arrow. Several of these structures are arranged in the periphery of antennal lumen. Some of them are not visible, but their envelope remains (arrowhead). Bar- scale in both images is 30 휇푚.

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Finite element analysis applied to understand the role in gravireception of the antennal non-muscular joints of Rhodnius prolixus

Bibiana Ospina-Rozo1; Manu Forero-Shelton2, Jorge Molina3

Abstract

Non-muscular joints in insect flagellar antennae allow it to be flexible and in some cases to couple mechanical cues. Heteropteran antenna has only two of these joints in its long flagellum, which makes it stiff compared to other Hemipterans. What might be the structural trait that provides flexibility to this kind of antenna in order to couple mechanical information, without compromising its structural stability? To asses this question, we simulated strain patterns caused by gravity force in different structural variations of the ground-plan in the antennae of Rhodnius prolixus (Heteroptera) with Finite Element Analysis (FEA). We found that the prebasiflagellite is not a structural reinforce, but a stress raiser to increase flexibility and intensify the effect of gravity on the non-muscular joint. Conversely, both the intraflagelloid and the deep ventral groove reduce strain in non-muscular joints. Regions with higher strain are located near to the anchoring point of the Johnston’s Organ. Finally, we compared FEA modeled antenna of first and fifth nymphal stages with those of the adult of Rhodnius prolixus, including all structural traits as close as possible to reality. Cuticle thickness was calculated with SEM imaging. Patterns of normal strain were very different between developmental stages. Further studies are needed to evaluate the biological relevance of these differences.

Keywords: Finite Element Analysis, non-muscular joints, prebasiflagellite, intraflagelloid, graviception.

Introduction

Antennae in insects are complex organs which can detect a wide variety of sensory cues such as chemical substances, humidity, temperature and mechanical stimulation (Schneider, 1964). Mechanical stimuli can be detected by either antennal sensilla or chordotonal organs located in the antenna such as the mechanoreceptive Johnston’s organ (JO) (Schneider, 1964). In order to activate these sensors, the antenna has the property of bending in response to external forces, allowing for example flight control (Gewecke & Niehaus, 1981), walk monitoring (Dürr et al., 2001), among other functions in different groups of insects.

Insects have protective and structural cuticle all over their body including the antenna, which is well sclerotized with the exception of the intersegmental membranes (Schneider, 1964). Bending is possible only in these joints with small degrees of tanning, resulting in a more elastic surface, as it was demonstrated in the first manuscript of this thesis. Antennal joints beyond the scape, without muscular attachments are thought to be simple folds of the cuticle (Snodgrass, 1935). These antennal joints are considered a synapomorphy of the Class Insecta (Kristensen, 1998).

On the other hand, the antennae of many species of insects are very long, thin and segmented not only to increase the surface and to bear more sensilla, but also because they are used literally as sensory feelers (Schneider, 1964). Structural design of a mechanoreceptive sensor with the properties mentioned above, is clearly an engineering challenge. In some groups like crickets (Loudon et al., 2014) and stick insects whose antennae can be as long as 100 times its diameter at the base (Dirks & Dürr, 2011), structural design has been studied, because it prevents the antenna from breaking, allows it to keep its optimal orientation, and makes it flexible enough to detect mechanical information (Dirks & Dürr, 2011).

Insects from the Heteroptera group have long antennae too, but they have a very different ground-plan consisting of only four long cylindrical segments (Zrzavy, 1990). As a result, the two non-muscular joints distal to the pedicel are responsible to maintain structure and give flexibility to the antenna, possibly with the help of two intersegmental nodules (Zrzavy, 1992). However, the function of these nodules remains unclear since it is difficult to find mutants with absent nodules and it is impossible to remove the nodules without impairing the antenna. Functions of other interesting structures in the antennae such as the deep ventral groove on the pedicel are difficult to study because of the same reason. As a result, the usage of computerized modeling is a good option to evaluate the role of the different structural features in the function of the antennae in insects.

Gravity force, is considered a very strong selective pressure molding life on earth, as it gives weight to objects (Morey-Holton, 2003). Therefore, terrestrial species have developed systems to keep postural stability and structural support (Morey-Holton, 2003). It has also influenced the evolutionary development of unique structures to amplify its effect and function as sensors required for orientation, balance and effective movement (Morey-Holton, 2003). Therefore, this stimulus is the best to test at the same time, if the structure of the antenna has been developed to protect it from bending or breaking because of its weight or if conversely it gives flexibility to detect mechanical stimuli.

In the second manuscript of this thesis, we used Rhodnius prolixus species as a model to study Heteropteran ground-plan, establishing that the JO scolopidia are attached distally to the basis of the prebasiflagellite, and possibly sense the movement of the basiflagellum originated by a mechanical stimulus such as gravity force. Additionally, in the first manuscript of this thesis, we determined the changes in shape and dimensions of the prebasiflagellite during the life cycle of R. prolixus. The reason of this change can be afforded once its function is determined. The two possibilities are that either both nymphs and adults are under different selective pressures with respect to mechanical information codified by the JO; or other variations in antennal structure combined, produce the same sensibility in the JO in every stage of development.

Here we wanted to combine both group of results with finite element analysis in order to understand the role of the main structural features observed in the antennae of R. prolixus. We are especially interested in the possible mechanism of processing mechanical information such as earth gravity direction, by simulating different structural scenarios, focused in the proximal sub-articulation that communicates the pedicel with the prebasiflagellite. Secondly we wanted to compare the effect of earth gravity on the antennae of the first and fifth nymphal stages and the adults of R. prolixus.

Methodology

Parameters for modeling the antenna with finite element analysis (FEA) We studied different scenarios changing the structure of the antenna and evaluating its deformation by the effect of the standard earth gravity. FEAs were carried out with the software ANSYS Workbench 14.5. The software calculated changes in vertical displacement, normal strain, shear strain and equivalent stress all along the geometry of the antenna, based on the mechanical properties allocated to materials and the geometry on each of the structures.

We recreated the geometry of Rhodnius prolixus antenna as close as possible to the real structure, based on external measurements carried out with the software Image J of the segments (length and diameter

52 reported in the first manuscript) and the radial extension of the ventral groove and thickness of internal structures estimated from longitudinal sections of the adult antenna (showed in the second manuscript).

We used two kinds of materials, one for flexible regions in the antenna and another one for rigid bodies. These materials differ only in their Young’s modulus but not in their density which was fixed at 퐾𝑔 1250 ⁄ because chitin has the ability of having great changes in properties with a more or less 푚3 constant value of density (Klocke & Schmitz, 2011; Vincent & Wegst, 2004). For flexible joints we used a 40 푀푃푎 Young’s modulus and 0.48 Poisson’s ratio, while for more sclerotized parts such as flagellar segments, pedicel, and intersegmental nodules we used 530 푀푃푎 Young’s modulus and 0.3 Poisson’s ratio. Young’s modulus values were obtained for Rhodnius prolixus in a previous study (Arango et al., 2015) and confirmed with our measurements carried out with the AFM indentation experiments reported in the first manuscript. Values of Poisson’s ratio were fixed according to previous reports for arthropod chitin (Nikolov et al., 2010).

−5 푚 All FEA results for both normal and shear strain were obtained in a scale of X10 ⁄푚 but are presented here without that suffix to simplify notation. We evaluated the two strain parameters since normal strain is defined as a change in the longitude due to certain load (Roylance, 2008), while the shear strain is a deformation of the material due to change in its angles (Roylance, 2008). We established a fixed support in the proximal face of the cylinder representing the pedicel and we fixed its spatial position at 0° in the pedicel angle. Gravity force was applied in vertical directions and perpendicular to the antenna in the mass center determined automatically by the software.

Effect of the prebasiflagellite on the response of the antenna to the gravity force The first three scenarios were designed to evaluate if the presence of the prebasiflagellite represents an advantageous trait for the pedicel-basiflagellum non-muscular joint to deal with levels of strain caused by gravity force. We generated the geometry of a very simple antenna consisting of two cylinders made of highly sclerotized chitin in accordance with the size of pedicel and the basiflagellum and distiflagellum considered as a single unity. In addition, the pedicel-basiflagellum non-muscular joint had always the same length, but was modeled with differences in shape. In the first one, it was a simple conic joint made of flexible chitin joining together the two cylinders. In the second one we included a very simple cylindrical intersegmental sclerite of rigid chitin with length equal to the prebasiflagellite in the adult stage and an average diameter resulting from the diameter of the two sub-articulations. Finally, in the third one we included the prebasiflagellite with its original size and shape. For each model we considered the Normal strain, Shear strain and vertical displacement in Y-axis.

Effect of the intraflagelloid and the ventral groove on the response of the antenna to gravity In order to determine if the modular condition in the flagellum of the antenna generated by the presence of intraflagelloid was a strategy to reduce the amount of strain in the non-muscular joint, we compared and antenna with basiflagellum, distiflagellum and the prebasiflagellite generated previously with a new antenna including the intraflagelloid (with its original measurements in the adult stage) between the basiflagellum-distiflagellum joint.

Additionally we wanted to find out the function of the deep ventral groove observed on the pedicel. For that we generated a new antenna including both intersegmental nodules and the deep ventral groove with its real extension on the distal part of the pedicel tip and an estimated angular extension, but with a squared shape instead of its original shape.

For both models, we considered the two components of Normal strain (x- and y-axis) in the distal and proximal region of the external surface of the proximal sub- articulation, since this is the joint where the JO is located and is also presenting the higher amount of movement in the antenna of adults (see second manuscript).

Regions of strain concentration In order to determine the region in the pedicel-basiflagellum non-muscular joint which undergoes the highest amount of strain when experiencing the action of gravity force, we studied patterns of normal strain in the flexible internal face of the proximal non-muscular joint (see second manuscript). For this analysis, we compared the antenna without intersegmental nodules; the antenna with both intersegmental nodules; and finally, the antenna with both intersegmental nodules and a ventral groove. We also measured the strain responses in the antenna with a ventral groove oriented vertically and trying to simulate an insect climbing on a palm tree (see second manuscript).

Effect of the ontogenetic changes on the response of the antenna We wanted to consider also the effect of changes in size and shape of the pedicel-basiflagellum non- muscular joint on the response of the antenna during the postembryonic development of Rhodnius prolixus. In in this case we replicated the ground design and size of the antenna in the first and fifth nymphal stages but omitting the presence of a deep ventral groove on the pedicel. We selected this two nymphal stages because, they are representative of the major structural changes in antennal during the postembryonic development (see first manuscript).

From our previous work (see first manuscript), we already had some parameters about the geometry of the antenna in different stages such as length of the segments, diameter, and shape. But the thickness of

54 the cuticle was unknown for the nymphal stages. We used SEM microscopy in order to obtain these measurements.

Four adults and nymphs of first and fifth nymphal stages we anaesthetized four insects at 4 °C for 5 minutes and removed their right antenna with scissors. The antennae were then cut with a stainless blade under a stereomicroscope (ZOOM 2000, LEICA) at three different levels: pedicel, prebasiflagellite, and basiflagellum. Each of these sections was fixed with double-sided sticky tape on one side of a metallic cube. The segments with their cut edges were aligned on the top of the cube and coated with a thin, uniform layer of fine particles of gold (100 Å) in low pressure conditions (10 – 4 Torr) for five minutes with a sputter-coater (Dentom vacuum Desk IV).

Afterwards, these sections were observed with a JSM-6490LV (Jeol) Scanning Electron Microscope at 15 KV. Micrographs were taken at 20 KV with magnifications ranging from 550X to 1600X, carefully rotating the samples to orient them with their surface upwards, in order to take images of the thickness of the cuticle.

The pictures obtained were analyzed with Image J to measure the area of the external side and internal side of the cuticle. From these measurements we calculated the diameter of both circles and the thickness of the cuticle (Percentage of the total diameter equivalent to cuticular walls).

With this information we generated a model of the antenna with ANSYS software for each nymphal stage and a new model for the adult stage considering data from SEM images. We used the same properties and materials assignment for the adults and nymphal stages. We compared the normal strain in y-axis in the dorsal view for the three models, and the normal strain in x-axis for the internal surface of the proximal sub-articulation in order to determine the changes of strain for each developmental stage.

Results

Due to FEA aims to obtain data for each cell produced in the meshing process and then present them in a graphical way; we selected some data obtained from each model in order to make our comparison reliable and simple. Normal strain, Shear strain and directional displacement were obtained for each of the models generated.

A comparison of the Shear and Normal strain showed patterns of responses very variable. Shear strain is produced by internal forces in the material (Roylance, 2008) and it is very relevant when considering

55 the possibility of failure in the material. Thus, we only considered this kind of deformation in the models to establish if the prebasiflagellite is a structure used as reinforce for the antenna (Fig. 1C).

Normal strain, on the other hand, indicates a change in the length of materials (Roylance, 2008). This change can happen only in one direction when a load of tension is applied to the object, or as in our case, when the load causes a bending in the object. Normal strain has two components one in x-axis (longitudinal axis of the antenna) and the other one in y-axis (perpendicular to the antenna), so it behaves like a vector with magnitude and direction playing a role. This happens because the strain is being produced by the torque which causes an angled movement of the flagellum in +x and –y direction. We found out that there is a relationship between the two components (Fig. 2), despite they can have different magnitudes, they both experiment stretching or compression for a certain region in the antenna. So we only studied both of them in the second experiment to test the effect of the intraflagelloid and the deep ventral groove on the response of the antenna (Fig. 2).

Since FEA gives results of strain concentration in the three dimensions of the object, the models are difficult to compare without studying them by sections. In the first place, we can analyze the strain in the external or internal surfaces of the materials. We can also study the dorsal or ventral view of the antenna. In order to evaluate the effect of the prebasiflagellite on the antennal response we observed the external dorsal view. To evaluate the function of the intraflagelloid and the ventral groove we considered the external surface of the proximal sub-articulation with its dorsal, ventral and lateral sides. We observed patterns of deformation in the internal surface of the proximal sub-articulation in order to compare the effect of key features on the structure of the antenna in the response of the flagellum to gravity force and also on the comparison between nymphal stages. We also considered the external dorsal view to compare between the different developmental stages in the life cycle of Rhodnius prolixus.

Presence of the prebasiflagellite Figs. 1B and 1C show the maximum value for Normal and Shear strain respectively for 11 discrete points in the dorsal view of the antenna: tip of the pedicel; proximal, medial and distal area of the proximal sub-articulation; proximal, medial and distal area of intersegmental nodule (prebasiflagellite); proximal, medial and distal area in the distal sub-articulations; and finally, the basis of the flagellum.

When the prebasiflagellite was absent, the normal strain was observed all along the flexible material, but its magnitude increases at the distal edge of the non-muscular joint in both components +x (Fig. 1A - B) and -y (not shown). Indicating that this part of the articulation is suffering a tension and stretches downwards all along the longitudinal axis. The opposite happens in the ventral view (not shown), where

56 the distal part of the articulation is undergoing a compression in both x- and y-axis. However, the shear deformation seems to be constant along the joint.

The presence of the prebasiflagellite nodule caused Normal and Shear strain to increase (Fig. 1B - C). Strain patterns in the non-muscular joint were modified too. Distal sub-articulation experienced higher levels of Normal strain than proximal sub-articulation. The middle part of the proximal sub-articulation is stretching in the x-axis; however, the distal part is experiencing the opposite phenomenon in comparison to the situation when the nodule is absent. In this case, the dorsal part of the flexible material is being compressed due to the presence of the prebasiflagellite nodule.

It is also clear that the shape of the prebasiflagellite nodule can determine the pattern and magnitude of the Normal strain. With a conic nodule such as the prebasiflagellite, both Normal and Shear strain were increased in the proximal sub-articulation, and reduced in the distal one (Fig. 1 B-C). However, in the proximal sub-articulation the pattern of tension and compression in the proximal and distal section respectively was the same as the one with a cylindrical nodule. These results show that the prebasiflagellite is able to concentrate the deformation in specific parts of the proximal sub-articulation (Fig. 1A).

Overall, the Shear strain is almost twice the values of the Normal strain for the antennae with nodules and the patterns in which they are produced also are different. We also calculated the amount of vertical displacement under these three conditions (Fig. 3E). The total displacement of the tip of the flagellum of the antenna with prebasiflagellite was almost twice the value of the antenna with absent nodule which was possible because the conic nodule causes Normal strain in both components to increase.

Effect of the intraflagelloid and deep ventral groove on the response of the antenna to gravity In order to better understand the patterns of deformation in the proximal sub-articulation (between the tip of the pedicel and the basal part of the nodule), we observed the two components of Normal strain all around the external surface of this flexible material. We compared the transversal view of this joint between the models with prebasiflagellite, with both nodules and with both nodules and a ventral groove, by establishing a 360° circumference and taking the data of Normal strain for angles from 0° to 330° in 30° steps, being 90° the dorsal side of the antenna, and 270° the ventral side of the antenna. We registered the level of Normal strain in the proximal section closer to the pedicel and the distal section closer to the nodule according to the reference system (Fig. 2A).

For the antenna with only the prebasiflagellite, in the dorsal side of the proximal section, the flexible cuticle presented a positive deformation in x and a negative deformation in y direction (Fig. 2B). That means that this part is stretching parallel to longitudinal axis of the antenna and in accordance to the

57 direction of the gravity force vector. In the ventral side of the proximal section, the opposite is happening. The cuticle is being compressed against the direction of gravity force (+y) and closer to the pedicel (-x).

For the same antenna in the distal region of the proximal sub-articulation (Fig. 2C), the dorsal part of the flexible cuticle is compressed in both directions (-x and +y) and the ventral part is being stretched. This could be related to the sharp edge, shape and rigidity of the prebasiflagellite nodule.

The presence of a intraflagelloid nodule between the basiflagellum and distiflagellum reduces the amount of normal strain in every studied region from the non-muscular joint in average 40.89 %, but it does not change the pattern of Normal strain in the proximal sub-articulation (Fig. 2).

The presence of a deep ventral groove on the pedicel changes entirely the normal deformation pattern in the proximal sub-articulation, and it reduces its magnitude (Fig. 2). For example for the x component, maximum magnitude of normal strain falls to 4.95 × 10−5 푚⁄푚 of positive deformation (stretching) and −2.51 × 10−5 푚⁄푚 of negative deformation (compression).

We also noticed that total displacement of the tip of the flagellum was reduced approximately in 1 휇푚 with the presence of the intraflagelloid. Taking into account the vertical displacement of the tip of the basiflagellum, we can establish that more than 65% of the vertical displacement of the antenna towards the gravity force direction happens because the non-muscular joint between pedicel and basiflagellum, while the rest happens because of the flexibility given by the presence of the intraflagelloid. Also, with the presence of a ventral groove the Normal strain in the flexible parts was reduced, and as a result, the vertical displacement was reduced too, in this case to less than what the joint can allow without bearing any of the intersegmental nodules (Fig. 3E).

Regions of strain concentration In order to determine if the higher values of normal strain take place near to or in the anchoring point of the scolopidia making part of the JO, we compared the x component of Normal strain in the internal surface of the non-muscular joint (Fig. 3A-D). When the antenna lacks both intersegmental nodules, the dorsal region undergoes a 4.93 × 10−5 푚⁄푚 stretching and ventral side a 6. 14 × 10−5 푚⁄푚 compression. Deformation takes place all over the joint (Fig. 3A).

In contrast, when antenna has the two intersegmental nodules Normal strain increases in range from −9.93 to 9.78 × 10−5 푚⁄푚. There is also a polarization in zones undergoing stretching and compression being the former dorsally located and the second ventrally located (Fig. 3B). When a deep ventral groove was added to the antennal model, a noticeable change in the pattern and magnitude of 58

Normal strain was observed (Fig. 3C). The highest stretching was located dorsally and it goes back to a similar maximum value of strain as the one in the antenna with absent nodules: 4.95 × 10−5 푚⁄푚. At the same time, compression in the ventral side of the antenna is ranging from 0.38 to 2.51 × 10−5 푚⁄푚. Also, the basis of the prebasiflagellite (central region in the scheme) experienced more normal strain than regions of proximal sub-articulation closer to the tip of the pedicel.

Finally, when the antenna was placed at 90° to the horizontal, the proximal joint experiences a general symmetric compression in y-axis ranging between 0.005 푎푛푑 0.06 × 10−5 푚⁄푚 (Fig. 3D). The distal sub-articulation between the prebasiflagellite and the basiflagellum also experienced a compression of the same magnitude (data not shown). In the x-axis, Normal strain pattern was different with a stretching in the lateral parts of the joint and a compression in the ventral part, where the ventral groove is located (data not shown).

Ontogenetic changes A comparison of the transversal sections of the pedicel in the three postembryonic developmental stages studied is shown in Fig. 4A. The percentages of the total diameter of the cuticular walls in the transversal sections measured in the antenna were very similar irrespectively from the segment in first and fifth nymphal stages (Fig. 4B). However in the adult, the cuticle forming the wall of the antenna increases its thickness significantly when compared to the nymphs by 19.65 % in the pedicel, 13.53 % in the flagellum and 11.82 % in the intersegmental nodule (prebasiflagellite). Taking into account these values, we designed a very different antenna for each postembryonic developmental stage. We used the same thickness for both the basiflagellum and the distiflagellum and half of the prebasiflagellite thickness in the walls of intraflagelloid of each modeled antenna.

Vertical displacement of the flagellum for the three postembryonic developmental stages was obtained from the model and is shown in Fig. 4C. Displacement of the tip of the basiflagellum, determined by the properties of the prebasiflagellite joint, was smaller for the first nymph, while it was almost the same (2.2 휇푚) for the adult and the fifth nymphal stage. On the other hand, in every stage the intraflagelloid joint allowed at least half of the total displacement of the distal tip of the flagellum. The amount of movement of the tip of the flagellum was higher in the fifth nymphal stage than in the adult.

In Fig. 5A we showed the patterns of Normal strain in y-axis from dorsal view, for the three different developmental stages. The patterns and magnitudes of the Normal strain were very different; however the fifth nymphal stage and the adult model presented a negative deformation, which means stretching in the same direction of gravity, higher in the distal sub-articulation. The first nymphal model suffered a very small deformation in y-axis in all parts of the antenna, except in the distal region of the proximal sub-articulation where it presented the same pattern as the adult: a small compression in the contact 59 region between the proximal sub-articulation and the basis of the nodule (adult)/ basiflagellum (first nymphal stage).

We also compared the patterns of normal deformation in x direction in the internal surface of the proximal sub-articulation for the three postembryonic developmental stages. Higher amounts of Normal strain were located close to the proximal edge of the prebasiflagellite in the three cases. Maximum values of Normal strain positive in x direction was more than 50% higher for the first nymphal stage and 70% higher for the fifth nymphal stage when compared to the adult.

Discussion

We studied both, the Normal and the Shear strain in different parts of the antenna of R. prolixus with the gravity force as stimulus. Both strains should be considered in order to evaluate material failure (Roylance, 2008). We think that only the Normal strain can be linked to the scolopidia of JO. This kind of deformation causes a displacement of the prebasiflagellite in both x- and y-axis, which means a tension in the anchoring mechanism of the scolopidia. Therefore, Normal strain can cause a stretching of the modified cilium of the neurons according to the mechanism described by Yack (2004). It is still unknown whether the cilium of the neuron can be moved only in its longitudinal axis (x-axis for our reference system) or in any of the three dimensions (Todi et al., 2004). So, we still do not know if the scolopidia could process Normal strain in both x-axis and y-axis, because the mechanism of transduction of this stimulus is not clear enough. For instance, it is well known that the basis of the modified cilium has a very intriguing structure called the distal basal body, which consists in a small twist in the axis of the cilium (Yack, 2004). It has been proposed that bending of the cilium due to external forces acting on the flagellum, occurs next to this structure (Yack, 2004). If that is the case, the transduction process could be more complex than a simple transference of tension in only one direction and a bending mechanism with the two components of normal strain (in x-axis and y-axis) could be detected by the scolopidia in the JO.

In the antenna without intersegmental nodules we observed that the dorsal surface in the proximal sub- articulation is being stretched and ventral surface is being compressed. This phenomenon is consistent with the behavior of a symmetric element under the effect of a bending load: Dorsal region is expected to get longer and ventral region was expected to get shorter because of the experienced strain (Beer et al., 2008). This same pattern was produced in the proximal section of the articulation between pedicel and prebasiflagellite when the intersegmental nodules are present. However, the opposite pattern is produced in the distal part of the same joint. This may be due to the action of the weight of the flagellum and the sudden transition from one material to another produced in the model. Gravity force causes the

60 antenna to move downwards, but at the same time, the intersegmental nodule exerts a compression force on the membranous sub-articulation, due to its diameter and rigidity.

The presence of the prebasiflagellite nodule increases the deformation and the vertical displacement. Therefore, it should not be considered a mechanical stabilizer preventing any kind of failure in the flexible material caused by the weight of the antenna (gravity). Due to the very high values of tensil strength associated to chitin, the prebasiflagellite also should not be interpreted as a structural reinforce to prevent the collapse of the non-muscular joint (Vincent & Wegst, 2004). Some other structural characteristics of the antenna can prevent it from unusual bending and breaking, such as the internal trachea and the hemolymph.

On the other hand, the prebasiflagellite nodule was confirmed to be a mechanism to increase flexibility in the non-muscular joint. This finding agrees with the phylogenetic hypothesis proposed by Zrzavy (1992). This intersegmental nodule can concentrate the strain in certain points of the proximal sub- articulation causing higher displacements of the flagellum. Therefore, this structure is significantly important to process any kind of mechanosensory stimulus causing a displacement of the flagellum and activation of the JO.

The prebasiflagellite acts in two ways to increase flexibility of the antenna: It increases the weight of the antenna, because it is thicker than the flexible parts, and its shape turns it into a stress raiser. When certain structural element has a geometric discontinuity such as a sudden change in its transversal section, the concentration of stress will be increased in the regions next to that discontinuity (Beer et al., 2008). Since the transversal area of the distal part of proximal sub-articulation (where it is in contact with the prebasiflagellite) is very small in comparison with the diameter of the distal edge of the prebasiflagellite, here is where the majority of the stress is actually concentrated; consequently here is where the majority of the strain is being produced.

The problem with this design for a mechanoreceptor coupling organ, is that the place where stress is being concentrated is more likely to suffer failure (Beer et al., 2008). Some strategies to avoid failure or cracks of the material is usually to include smooth and curve transitions between the two materials or joint together the two structures by a very small transversal area (Beer et al., 2008; Shigley & Mischke, 2008). This could be the case of the antennal joints, as it can be observed in longitudinal sections of the antenna (see second manuscript), where flexible parts of the cuticle are communicated with rigid and tanned ones by a continuous and smooth transition especially in the distal sub-articulation. This transition is less gradual between the proximal sub-articulation and the basal part of the prebasiflagellite which is also why stress concentrates in this point causing more Normal and Shear strain.

On the other hand, the presence of the intraflagelloid caused a reduction in the concentration of Normal strain. The modular condition in the ground-plan of the antenna could then be considered an advantage to reduce the stress in the non-muscular joints when segments are very long, which is the case of Heteroptera group. This could be the explanation to develop intersegmental nodules when the antenna increases its length and weight such as proposed by Zrzavy (1990) based on phylogenetic comparisons.

Finally, the function of the deep ventral groove on the pedicel was assessed in our models, but our results were inconsistent with the experimental data obtained for pitch and yaw orientations (see the second manuscript). Experimentally, we found that the effect of gravity force on the antenna causing a vertical displacement was higher in pitch orientation, when the groove is located ventrally than in yaw orientation when the symmetric lateral side of the antenna was oriented in accordance to the gravity force. However, in our model of antenna the ventral groove causes a decrease in the amount of movement in the y-axis orientation caused by the gravity force. We think that the differences between both results could be caused by the geometry used to create the ventral groove in the model. Here we kept the shape of the tip of the pedicel and changed only the material in its ventral side, while the real structure reduces the transversal area of the tip of the pedicel in an asymmetric way (see transversal sections of the antenna in SEM pictures from first and second manuscripts). The change in this shape would certainly give very different results. However, the effect of changing the material of the ventral groove for a more flexible was a reduction of the stress on the proximal sub-articulation. Since the ventral groove is acting as an extension of the proximal sub-articulation of the joint, and it is well known that the stress in certain material tends to achieve a uniform distribution all along the material (Shigley and Mischke, 2008), we will expect a reduction of its local magnitude on each small region from the total area. In order to confirm our hypothesis a new model should be generated with the real shape of the ventral groove in order to evaluate if the combination between material properties and shape are able to generate small values of strain deformation and a greater displacement in response to the gravity force.

Taking into account the definition of normal strain (Roylance, 2008), we determined that our results point out to a dorsal stretching of membranous chitin of approximately 0.1 휇푚 in x-axis. This tiny deformation could be detected by the scolopidia in the JO due to their location. Also, deformation required by an animal cell to open its ion channels seems to be very small as it has been estimated to range between 0.1 and 0.5 휇푚2(Sokabe et al., 1991). However, it is necessary to keep in mind that this range is not easy to measure and many factors can alter its estimation (Sokabe & Sachs, 1990). On the other hand, vertical displacements are all between 1 and 3 휇푚, which is close to the theoretical value of 1 휇푚 vertical displacement imposed by earth gravity to the antennae of Drosophila melanogaster (Kamikouchi et al., 2009). We are expecting for R. prolixus a higher value in the vertical displacement

62 of the antennae because they are heavier than those of Drosophila and also because our model is not including the effect of the antennal sensilla on the geometry.

When our model of the antenna was placed at 90° (in the opposite direction to gravity force), the y component became more important than the x. Our data indicate that gravity force can make flexible parts in the non-muscular joints to move in the y- direction and highlight the importance of the y- component of Normal strain. Based on our biomechanical analysis, Normal strain in y-axis is expected to decrease in a reason determined by 푠푒푛 (훼𝑖) (angle of the basiflagellum) which is what we observed in our model of the antenna. However, a new model with the antenna oriented in an initial angle different from 0° or 90° could be useful to verify this assumption. There is no evidence to say that the scolopidia of JO can sense this information, but that doesn’t seem to be the case, because it is one magnitude order smaller than strain caused by gravity at horizontal position. This happens because the torque exerted by weight of the antenna is very small when the antenna is 90° to the horizontal. Interestingly, in this position of the antenna, the ventral groove causes a pattern in the x-component of the Normal strain indicating that in spite of the pedicel is located at 90° to the horizontal, the flagellum tends to be slightly oriented in the direction of ventral groove.

Finally, the general geometry of the antenna, the thickness of the cuticle and in the number of sensilla were different between the nymphal stages and the adults. While the general morphology of the antenna changes gradually during the postembryonic development (see first manuscript), the other two characteristics are presumed to be more or less constant during the five nymphal stages and to change only in the last moult. Our results showed that first and fifth nymphal stages have all the same thickness in the cuticle but more layers of cuticle were added to the walls of the antenna in the last moult (Fig. 4). Also a considerably high number of antennal sensilla are added with the ecdysis to adults (Gracco & Catalá, 2000). These two facts could be related because adding more sensilla to the antenna would mean that a stronger structure is needed to bear them, otherwise the long cylinders forming the segments in the antennae could collapse. Besides, if this thick cuticle is not needed by the nymphal stages, it would be a great waste of energy to create a new thick cuticle on each mold, since a complex metabolic cascade is involved in the process of degrading the older cuticle without affecting the new one (Chaudharia et al., 2011). Our results arise many new questions considering the biological meaning of the changes that are taking place only in the antennae of the adults.

Differences in the patterns of Normal strain in x- and y-axis, and their displacements could be related either to the different morphology of the antenna on each developmental stage or to their sizes. The antenna in the first nymphal stage displays a very subtle and uniform normal deformation along the antenna, only altered in the distal section of the proximal sub-articulation. The main difference between the adult and fifth nymphal stage is the pattern of Normal strain in y-axis in the proximal sub-articulation

63 of the pedicel-basiflagellum joint: the flexible cuticle of the fifth nymphal stage is compressed in the middle and stretched in the distal part close to the prebasiflagellite, while in the adult the opposite is true. Changes in the x- component of the Normal strain could be attributed to a heavier flagellum in the adult than the fifth nymphal stages or to longer joints, allowing the segment to have a greater displacement in the y-direction even when same stimulus are presented. More experiments and simulations are needed to establish the biological relevance of the structural changes observed from the first nymphal stages to the adult stages.

Conclusions

Our experiments with FEA allowed us to generate and modify the general structural ground-plan of the antennae in Rhodnius prolixus in order to establish their possible function. We found that in spite of the restrictive ground-plan of Heteroptera, the structure of the antenna is designed to have great flexibility in the non-muscular joints, especially the proximal sub-articulation of the pedicel-basiflagellum joint. This flexibility makes us consider that the joint could function as a mechanoreceptor and particularly as a gravity sensor. In addition, there are some traits in the antenna such as the prebasiflagellite, the ventral groove and the smooth transitions between the materials (possibly to reduce deformation) suggesting that the JO of this antenna could be optimized to detect very subtle changes in the proximal sub- articulation of the pedicel-basiflagellum joint. In addition, many changes in the structure of the antenna are taking place during the postembryonic development from the first nymphal stages to the adults. More experiments are required to determine the biological relevance of these structural differences observed in the different stages in the life cycle of Rhodnius prolixus.

Acknowledgements

We are grateful to Dery Esmeralda Corredor, for her help obtaining the SEM pictures, and David Gerardo Rozo for his orientation in the analysis of FEA results. This work was supported by a “Proyecto semilla” approved to Bibiana Ospina-Rozo by the Faculty of Sciences of Universidad de los Andes.

References

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FIGURES

Figure 1. Effect of the intersegmental nodule on the non-muscular joint between pedicel and flagellum. A. An example of the results obtained with FEA, showing intervals of Normal strain (longitudinal axis) for the dorsal side view of the three models simulated (from top to bottom): absent nodule, cylindrical nodule and conical nodule (prebasiflagellite). Colored scale bars are 푥 ∗ 10−5 푚⁄푚. B. Maximum values of Normal strain for the three scenarios registered in the tip of the pedicel (Pd), proximal sub-articulation (prox s-a), intersegmental nodule, distal sub-articulation (dist s-a) and the base of the flagellum. Regions in the non-muscular joints were divided into proximal (p), medial (m) and distal (d). C. Maximum values of Shear strain for the three scenarios registered for the same regions than in B. Conical shape increases both kinds of strain in the non-muscular joint, especially in the proximal sub-articulation.

Figure 2. Effect of the prebasiflagellite and the ventral groove on Normal strain values of the external surface of proximal sub-articulation. A. Reference system. Top: x-axis is parallel to the antennal longitudinal axis. Gravity force (푚𝑔) acting in the y- direction on the mass center of the antenna. We compared sections of the proximal sub-articulation between pedicel and the prebasiflagellite. Bottom: transversal view of the proximal sub-articulation. Proximal section (periphery) is closer to the pedicel and distal section (center) is closer to the prebasiflagellite. We registered maximum values of normal strain in steps of 30°, where 90° represents the dorsal side and 270° represents the ventral side of the antenna. These angles are marked in dotted lines in the graphs. B. Data for the proximal section. Normal strain in x-axis (top) and y-axis (bottom). C. Data for the distal section. Normal strain in x-axis (top) and y-axis (bottom).

Figure 3. Normal strain in the internal surface of the proximal sub-articulation and vertical displacement. Transversal section at the level of the pedicel to visualize the Normal strain in x-axis in the internal surface of the proximal sub-articulation of the simulated antenna with: A. Two long cylinders and absent nodule. B. Conic nodule (prebasiflagellite) and intraflagelloid. C. Both intersegmental nodules and the ventral groove on pedicel. D. Normal strain in y-axis in antenna with the same geometry than in C and located vertically (90° to the horizontal). Each of the four images has its independent color scale in order to observe both magnitude and pattern. E. Total amount of vertical displacement (휇푚) according to gravity force direction of the tip of the flagellum and the tip of the basiflagellum.

Figure 4. Cuticle thickness in the antenna of different postembryonic developmental stages of Rhodnius prolixus and vertical displacement of the antenna. A. Examples of SEM images of sections in the pedicel of three different postembryonic developmental stages of R. prolixus (from top to bottom): 67 first nymph, fifth nymph and adult. All bar scales are 20 휇푚. B. Comparison between postembryonic developmental stages of the percentage of the entire diameter of the different segments equivalent to cuticle wall. C. Vertical displacement of the tip of the basiflagellum and the tip of the distiflagellum in the modeled antenna of the three different postembryonic developmental stages.

Figure 5. Patterns of Normal strain for the antennae of the three developmental stages exposed to the effect of the gravity force. A. Maximum values of Normal strain in the y-axis in different parts of the dorsal side view of the antenna for the first nymph, fifth nymph and the adult. Regions in abscise axis are the same as in Fig. 1B. Negative values indicate stretching in accordance to gravity force direction. Patterns of Normal strain in x- component for flexible cuticle in the internal surface of the proximal sub-articulation for each stage. B. First nymphal stage. C. Fifth nymphal stage. D. Adult. Each of the three images has its independent color scale in order to observe both magnitude and pattern.

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