1
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
2
in some groups of holometabolous insects (Kamikouchi et al., 2009), graviception could be a functionpresent 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
3
presented in the first manuscript with emphasis in the non-muscular joints. We also evaluated thicknessof 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
4
gravity stimulus to the antenna, and to interpret the obtained results in different stages of postembryonicdevelopment 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.
5
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.
6
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
7
segments with a function in the respective articulation, often replacing intersegmental sclerites, and theirname 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
8
materials is the product of the combination between their mechanical properties and the specificstructure 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 °𝐶, 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
9
4 °C for 5 minutes and removed the right antennae with scissors at the level of the scape. Astereomicroscope (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 𝑁 𝑚⁄ 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
10
ranging from 100X to 2000X. We measured the length and width of the intersegmental nodules and thefour 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 ± 349.655𝑀𝑝𝑎 and pedicel 739.01 ± 345.768 𝑀𝑃𝑎 were not
11
was 26.64 ± 15.587 𝑀𝑃𝑎 and it was significantly different from the intersegmental nodule (Wilcoxonsigned rank test, 𝑝 𝑣𝑎𝑙𝑢𝑒 = 0.008) and pedicel (Wilcoxon signed rank test, 𝑝 𝑣𝑎𝑙𝑢𝑒 = 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
12
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
13
Our findings are of morphological and developmental interest because we are reporting two differentorigins 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
14
& Schmitz, 2011; Vincent & Wegst, 2004). Values around 800 MPa were found in hard cuticular partslike 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
15
Heteroptera, while the formerly called preflagelloid has its origin in the basis of the basiflagellum, so itis 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.
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Zrzavy, J. (1992b). Morphogenesis of antenna exoskeleton in Heteroptera (Insecta): Fromphylogenetic to ontogenetic pattern. Acta Entomologica Bohemoslovaca, 89(3), 205–216. Zrzavy, J. (1990). Evolution of antennal sclerites in Heteroptera. Acta Universitatis
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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 𝜇𝑚. 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.
19
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 𝜇𝑚, D- E. 20 𝜇𝑚. 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
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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.
27
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
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
28
gravity sensing could be one of the basal functions for JO. Some previous behavioral experiments carriedout 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 °𝐶, 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
29
The swinging platform (Fig. 1A-6) changes its position around a central axis, reaching tilt anglesbetween -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 𝛼 angle from the initial baseline 𝛼 value. Pedicel angle 𝛽, for
each experiment, depends on the way the antenna was glued to the head, and insect’s angle 𝛾 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 (∆𝛼) 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 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝛼𝑖 (𝑤ℎ𝑒𝑛 𝛾 = 0°) for each insect.
Values of 𝛼𝑖 > 90° were possible when placing the insect in a vertical position on the platform. We
30
in each of the four orientations (Fig. 1B) and in seven insects in the angle resolved experiment. Weanalyzed 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
31
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 𝜇𝑚 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 𝜆 = 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 𝜇𝑔. 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 (𝛾)
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):
𝛾𝑡= 𝑀 sin(𝑏(𝑡) + 𝑐) (1)
𝛼𝑡 = 𝑁 sin(𝑏(𝑡) + 𝑐) (2)
Where M and N are constants describing the amplitude of the sinusoidal wave, b is a term describing
the period of the wave by the expression: 2𝜋