Ticks and Tick-borne Diseases 12 (2021) 101690

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Ticks and Tick-borne Diseases

journal homepage: www.elsevier.com/locate/ttbdis

Original article

First neuronal projection from Haller’s organ to the synganglion and

three-dimensional reconstruction of Amblyomma sculptum olfactory lobe
Karolina Martins Ferreira Menezes a, Jaires Gomes de Oliveira Filho a, Lorena Lopes Ferreira a, 1,
Lígia Miranda Ferreira Borges b, *
a
Escola de Veterinária e Zootecnia, Universidade Federal de Goiás – UFG, Goiânia, Goiás, Brazil
b
Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás – UFG, Goiânia, GO, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Because information regarding neural and sensory functions of ticks is scarce, the aim of the present study was to
Amblyomma cajennense complex ascertain the neuronal projection patterns of olfactory sensilla from Haller’s organ to the olfactory lobes of the
Glomeruli synganglion in Amblyomma sculptum adults. Additionally, the aim was to perform three-dimensional (3D)
Neuronal projection
reconstruction of the glomeruli in the olfactory lobes. Unfed female and male adults of this tick species were
Synganglion
Tick olfaction
immobilized, Haller’s organ was sectioned, and the olfactory nerves were labelled with a neuronal tracer (1%
dextran tetramethylrhodamine). The labelled olfactory lobes were observed under a confocal laser scanning
microscope and the images were analyzed using the Reconstruct software. The neuronal projection originating
from the olfactory sensilla of Haller’s organ was found to be strictly confined to the olfactory lobes of the
synganglion, which were organized into glomeruli. Males and females presented corresponding round-shaped
glomeruli in the olfactory lobes. The number of glomeruli ranged from 26 to 30 in the males and 24–29 in
the females. Positive correlations regarding the glomeruli volumes (μm3) intrasexually (females: r = 0.85, P <
0.05) and intersexually (r = 0.81, P < 0.05) were observed. This is the first report on 3D reconstruction of the
olfactory lobes of a tick species.

1. Introduction
Arthropods, including ticks, interact through stimuli called semi­
ochemicals that are responsible for guiding their behavior. These stimuli
in ticks are detected by the sensory organs, such as Haller’s organ, palps
and chelicerae, and are transmitted to the central nervous system
(synganglion). This information will be translated into an appropriate
behavioral response in a given situation. The tick synganglion is
composed of a highly fused nerve mass and peripheral nerves are pro­
jected to innervate appendages, sensory structures and organs (Simo
et al., 2014).
Haller’s organ is involved in olfaction and it is analogous to the insect
antennae in chemosensation, but morphologically different (Carr et al.,
2017). This olfactory organ is found on the dorsal surface of the tarsus of
the first pair of legs of ticks and has olfactory sensilla that detect volatile
molecules (Foelix and Axtell, 1971; Hess and Vlimant, 1982). Moreover,
the olfactory sensilla of ticks present bipolar neurons that project to the
synganglion, where two olfactory lobes are located and the olfactory
information is processed (Simo et al., 2014).
It is important to highlight that chemosensation is better-known in
insects than in ticks (Carr et al., 2017). In relation to harmful insects,
studies on neural mechanisms have been conducted with the aim of
understanding their behavioral response to olfactory cues from the
environment, hosts and mates (Reisenman et al., 2016).
Three-dimensional (3D) reconstructions of the olfactory lobes enable
comparisons of olfaction process within and between species and iden­
tification of glomeruli (i.e. quantity, shape and volume). A variable
number of glomeruli have been reported in different hematophagous
arthropods such as triatomines and mosquitoes (Barrozo et al., 2009;
Ghaninia et al., 2007; Ignell et al., 2005; Ye et al., 2020)
Some studies on ixodid ticks have demonstrated similarities between
the synganglia of males and females of the same species and between

* Corresponding author at: Lígia Miranda Ferreira Borges. Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás – UFG, Rua 235, s/n,
Setor Universitário, CEP 74605-050, Goiânia, GO, Brazil.
E-mail addresses: karolinamartins.f@gmail.com (K.M.F. Menezes), jaires_filho@hotmail.com (J.G. Oliveira Filho), loren4_lopes@hotmail.com (L.L. Ferreira),
ligia@ufg.br (L.M.F. Borges).
1
Escola de Veterinária, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte, Minas Gerais, Brasil.

https://doi.org/10.1016/j.ttbdis.2021.101690
Received 9 July 2020; Received in revised form 5 February 2021; Accepted 10 February 2021
Available online 20 February 2021
1877-959X/© 2021 Elsevier GmbH. All rights reserved.
K.M.F. Menezes et al.
different stages of life. In all life stages of Rhipicephalus sanguineus sensu
lato and other ticks, the synganglion is a mass of fused nerves forming a
single structure ventromedially located in the third anterior of the body,
between the genital pore and the gnathosoma, approximately between
the first and second pair of legs (Prullage et al., 1992; Roma et al.,
2012b; Simo et al., 2014). It is surrounded by the neural lamella or
neurilemma, constituted by several layers of finely granular homoge­
neous material, and the perineurium, formed by glial cells, located right
below the neural lamella. Internally, the synganglion is subdivided into
an external cortical region, which contains the cellular body of the
nervous cells, and an internal neuropile formed by a set of neural fibers
(Roma et al., 2012b, 2012a; Simo et al., 2014). Backfills from Haller’s
organ in the tick Dermacentor albipictus revealed serotoninergic neuronal
processes surrounding the glomeruli in the olfactory lobes (Hummel
et al., 2007). On the other hand, in Amblyomma americanum, neuronal
arborizations from Haller’s organ were confined to the olfactory lobes
(Borges et al., 2016).
The tick Amblyomma sculptum belongs to the Amblyomma cajennense
complex, which can be found from Texas (United States) to the northern
part of Argentina. It parasitizes not only domestic and wild animals but
also humans. Furthermore, it is responsible for transmission of Rickettsia
rickettsii, the causal agent of spotted fever, to humans (Brasília, 2017;
Labruna, 2009).
The present study was developed in the light of the importance of
A. sculptum for veterinary medicine and public health and the limited
knowledge regarding neural and sensory function in ticks. It consisted of
2
Ticks and Tick-borne Diseases 12 (2021) 101690
Fig. 1. Representation of the technique of
anterograde filling of olfactory nerves to the
synganglion in Amblyomma sculptum adults. (A)
Unfed females immobilized and positioned
dorsally using double-sided sticky tape with the
first pair of legs extended in dental wax and the
other pairs of legs fixed with sticky tape. (B)
First pair of legs in detail; the red traces indicate
the place of sectioning. (C) Dye (drop of pink
color) consisting of 1% TMR at the site of the
leg sections. (D) Slide with the prepared ticks
with sectioned site covered with liquid vaseline
to prevent evaporation of the dye, stored in a
Petri dish. (E) The female tick positioned
dorsally in Petri dish with PBS, for synganglion
dissection. Legend: 1- double-sided sticky tape;
2- sticky tape; 3- first pair of legs; 4- dental wax;
5- dye; 6- vaseline. (For interpretation of the
references to colour in this figure legend, the
reader is referred to the web version of this
article).
an experiment that had the following aims: to track the neuronal pro­
jection from Haller’s organ to the synganglion; and to make a 3D
reconstruction of the olfactory lobes of A. sculptum adults.
2. Materials and methods
2.1. Local of the study
The colony of ticks and the anterograde fillings of olfactory nerves to
the synganglion were developed at the Laboratory of Chemical Ecology
of Ticks while the evaluations of the slides were made at a fluorescence
microscope on the Laboratory of Molecular Genetics and Human Cyto­
genetics. Both laboratories belong to the Federal University of Goiás,
Goiânia, Goiás, Brazil. The microscopy and three-dimensional compu­
tational analysis were developed at the Microscopy Platform of the
Gonçalo Moniz, Oswaldo Cruz Foundation (Fiocruz Bahia), Salvador,
Bahia, Brazil.
2.2. Ticks
Unfed female and male adults of A. sculptum were obtained from a
colony of ticks. The use of animals was approved by the Committee on
Ethical Animal Use of the Federal University of Goiás (CEUA/UFG),
under protocol number 024/2014. All the unfed adult ticks were
maintained inside a several plastic syringes with the top cut off, which
were sealed with hydrophilic cotton. These syringes were kept in a
K.M.F. Menezes et al.
controlled-climate chamber under total darkness (T = 27 ± 1 ◦ C and RH
≥ 80 %; Eletrolab, São Paulo) before the experiments.
2.3. Anterograde filling of olfactory nerves to the synganglion
The methodology used was adapted from Borges et al. (2016). The
ticks (n = 200) were positioned dorsally on glass slides and were
immobilized using double-sided sticky tape (Scotch 3 M) and dental wax
(DentBras). To track the olfactory lobes, the first pair of legs was
stretched forward over the dental wax and the other legs were stretched
laterally, to each side of the tick’s body (Fig. 1A). After that, the first pair
of legs was cut using a Vannas scissors (9 cm) immediately posterior to
Haller’s organ (Fig. 1B). The sectioned site received one drop of
deionized water (ddH2O) for 30 s. The water was then removed using
Kimwipe-type paper (Kimberly-Clark, Dallas, Texas, United States) and
was replaced with a drop of 1% dextran tetramethylrhodamine (TMR
3000 MW fixable anionic lysine, D-3308, Invitrogen, Carlsbad, Califor­
nia, United States), in ddH2O (Fig. 1C). The sectioned site was covered
with liquid Vaseline to prevent evaporation of the dye. The slides were
then placed in Petri dishes (11 cm) containing a damp paper and were
maintained at 4 ◦ C for 48 h to allow diffusion of the dye through the
nerves to the synganglion (Fig. 1D). Subsequently, the ticks were indi­
vidually positioned in a dissection Petri dish (DD-50-S-BLK-3PK; Pyrex
Bottom), for the synganglion to be dissected (Fig. 1E).
The dissection was accomplished in saline (PBS) and then the syn­
ganglion was fixed in 4% paraformaldehyde in PBS at 4 ◦ C for 12 h. After
fixation, the synganglion was washed in saline and 0.5 % triton X-100
(PBST) six times, for one hour each, in a shaker at room temperature.
Next, each synganglion was washed in PBS for 30 min, in a shaker in the
3
Ticks and Tick-borne Diseases 12 (2021) 101690
Fig. 2. Synganglion (Sy), Haller’s organ (HO)
and olfactory lobes (OL) of Amblyomma sculp­
tum adult tick. A: Schematic drawing of adult
tick, the pink color indicates HO neuronal pro­
jections extending to reach the OL in the Sy. B:
Schematic drawing of HO (Drawing by Guil­
herme Campos Pereira). C: Fluorescence mi­
croscope (Axio Scope A1. Bar = 75 μm) on OL
in the Amblyomma sculptum male Sy. D:
Confocal laser microscopy (TCS SP8 confocal
laser microscope, with 63x/1.4 objective, using
immersion oil. Bar = 25 μm) on OL.
dark at room temperature. The synganglion was dehydrated in an
ethanol series (40 %, 60 %, 80 % and 100 %) for 15 min, in the same
shaker in the dark at room temperature. The preparations were mounted
on glycerol medium with Tris buffer (9:1). After that, the slides were
evaluated under a fluorescence microscope (Axio Scope A1, Carl Zeiss
AG, Jena, Germany) and those on which the olfactory lobes showed
fluorescence (n = 20; 13 males and seven females) were identified and
separated for analysis under a confocal microscope.
2.4. Microscopy and three-dimensional computational analysis
A general view of the olfactory projections was obtained using a
Leica TCS SP8 confocal laser microscope (Leica Biosystems Nussloch
GmbH, Nussloch, Germany), equipped with 488 and 552 nm diode la­
sers for excitation. Structures labelled with 1% TMR were observed
using a 63x/1.4 objective (with oil immersion). A series of images of the
same synganglion was obtained at intervals of 0.9 μm, with approxi­
mately 80–176 confocal images. The images obtained from the confocal
laser microscope were analyzed using the Reconstruct free software
(http://synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm).
2.5. Olfactory lobes, quantity, shape and volume of the glomeruli and
statistical analysis
To verify the olfactory lobes and glomeruli of A. sculptum adults, a
series of optical sections obtained from the confocal microscope was
imported to the Reconstruct software. Separate folders were created for
each synganglion, for observations on the olfactory lobes and glomeruli.
The olfactory lobes of different individuals were compared visually. The
K.M.F. Menezes et al. Ticks and Tick-borne Diseases 12 (2021) 101690

Fig. 3. Olfactory lobes (OL) of Amblyomma sculptum adult ticks. A and B: Confocal laser microscopy (TCS SP8 confocal laser microscope, with 63x/1.4 objective,
using immersion oil. Bar = 25 μm) on OL: A- male; and B- female. The images on the right side are the same as those on the left, where the glomeruli were outlined in
purple and identified. The glomerulus marked with a red asterisk (*) is at the intersection between the two OLs, and it was not possible to assign it to any OL. The
glomeruli indicated with a blue asterisk (*) were poorly marked. NA = neuronal axon; DA = dendritic arborizations; SDI = sparse and diffuse innervation; F = fibers;
G = glomeruli. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

same glomerulus on paired olfactory lobes was identified based on its
position, and this was outlined with the same color and received the
same number. After finishing the individual markings, the quantity of
glomeruli was recorded. Subsequent to this procedure, comparisons
were made between the olfactory lobes of the same tick and of different
individuals, as described by Ignell et al. (2005) and Barrozo et al.
(2009), to verify the percentage correspondence of the glomeruli.
After completion of the markings and numbering of all the glomeruli,
3D reconstruction of the olfactory lobe was performed, and the shape
and volume of each glomerulus was verified. The volume (μm3) of each
glomerulus, in a series of optical sections, was calculated using the
Reconstruct software, as an average from each mark that received the
same numbering, in all ticks of the same sex as mentioned above. The
glomerular volumes were tabulated and compared using Pearson’s
correlation with a significance level of P < 0.05. Pearson’s correlation
coefficients (r) vary between -1.0 and 1.0. When r = 1, this indicates a
perfect positive correlation between the two variables and when r = -1,
this indicates negative correlation between the two variables. Thus, the
closer to r = 1 that the coefficient is, the greater the correlation between
the variables is.
3. Results
3.1. Neuronal pathways between peripheral olfactory sensilla (Haller’s
organ) and the synganglion
In the current study, the 3D reconstruction was performed on four
ticks (three females and one male) out of the total of 20 ticks (13 males
and seven females) in which the olfactory lobes showed fluorescence.
Each tick received a number in ascending order from “1′′ to the total
number of ticks of each sex.
The olfactory lobes located in the tick synganglion received afferent
innervation from Haller’s organ and the backfilled neuronal axon
branched out and formed numerous dendritic arborizations for the
glomeruli (Fig. 2 and 3). In certain regions, fibers were more densely
packed than in others, which made it possible to delineate the limits of
the glomeruli through the dense innervation originating from the ol­
factory nerve. In most cases, it was possible to follow an innervation
branch in the lobe and to outline the associated glomerulus. However,
other glomeruli showed sparse and diffuse innervation from afferent
neurons and apparently exhibited connections with interneurons and/or
efferent projections. Due to poor labelling by the dye, it was not always
possible to delineate some glomeruli: see the blue asterisks in Fig. 3B.

4
K.M.F. Menezes et al.
Fig. 4. Numbering of glomeruli in male (A and B) and female (C and D), found in the olfactory lobes of Amblyomma sculptum adults, obtained through the
Reconstruct software. TCS SP8 confocal laser microscope with 63x/1.4 objective, using immersion oil. Bar = 25 μm.
Also in Fig. 3B, a glomerulus marked with a red asterisk can be seen: this
is at the intersection between the two olfactory lobes and, for this
reason, it was not assigned to any side.
3.2. Quantitative analysis, shape and percentage correspondence of
glomeruli of the olfactory lobe of A. sculptum adults
Male 1 and female 2 were selected as references for marking the
glomeruli of the other ticks because they had the most complete olfac­
tory lobes. Among the olfactory lobes evaluated, the number of
glomeruli in each lobe of the male was estimated to range from 26 to 30,
whereas in females, this number ranged from 24–29. The olfactory lobes
presented glomeruli with a rounded shape (Figs. 4 and 5).
The percentage correspondence of the numbers of glomeruli found in
both olfactory lobes (i.e. the glomeruli in olfactory lobe 1 that corre­
sponded to glomeruli in olfactory lobe 2) of each tick evaluated ranged
from 86 to 100 %. Specifically, the glomerulus correspondences were 87
% (26/30), 90 % (26/29), 100 % (27/27) and 86 % (24/28) for male 1,
female 1, female 2 and female 3, respectively (Table 1).
5
Ticks and Tick-borne Diseases 12 (2021) 101690
3.3. Volumes and statistical analysis between the volumes of
corresponding glomeruli
The glomerular volumes of the male and females ranged from 46.819
μm3 to 402.041 μm3. Glomerulus 3 showed the largest range, from
74.631 μm3 to 402.041 μm3 (184.679 μm3 ± 44.688 μm3); while
glomerulus 30 presented the smallest range, from 64.505 μm3 to 77.433
μm3 (70.686 μm3 ± 2.116 μm3). Regarding the male alone, the average
volume of the glomeruli was 144.870 μm3, while among the females, the
averages were: female 1, 228.031 μm3; female 2, 131.681 μm3; and fe­
male 3, 146.323 μm3. Pearson’s correlation coefficient was significantly
different from zero (P < 0.05). There were positive correlations between
the volumes of corresponding glomeruli in the females (r = 0.8547) and
between the volumes of corresponding glomeruli in the females and the
male (r = 0.8061) (Fig. 6).
4. Discussion
In the present study, a neuronal projection of Haller’s organ to the
synganglion of A. sculptum adults was done for the first time. In addition,
the first 3D reconstruction of the olfactory lobes of a tick species was
made. The olfactory sensilla of Haller’s organ were projected into the
paired olfactory lobes in the synganglion, which were composed of
K.M.F. Menezes et al. Ticks and Tick-borne Diseases 12 (2021) 101690

Fig. 5. Three-dimensional reconstruction of olfactory lobes of male (A-D) and female 2 (E-H) of Amblyomma sculptum ticks, sectioned at different ventral-dorsal
levels, obtained through the Reconstruct software. L1 = olfactory lobe 1; L2 = olfactory lobe 2.

6
K.M.F. Menezes et al. Ticks and Tick-borne Diseases 12 (2021) 101690

Table 1
Quantity and volumes of glomeruli identified in the olfactory lobes of male and females Amblyomma sculptum ticks that were selected for the three-dimensional
reconstruction using the Reconstruct software.
M1 F1 F2 F3
Glomeruli Minimum – maximum volume (μm3) Average ± standard error (μm3)
L1 L2 L1 L2 L1 L2 L1 L2

1 1 1 1 1 1 1 1 1 100.329 – 309.427 168.806 ± 23.450

2 1 1 1 0 1 1 1 0 77.370 – 325.733 139.755 ± 37.864
3 1 1 1 1 1 1 1 1 74.631 – 402.041 184.679 ± 47.773
4 1 0 1 1 0 0 1 0 46.819 - 89.758 117.428 ± 34.807
5 1 1 1 1 1 1 1 1 63.781 – 156.442 97.188 ± 12.981
6 1 1 1 1 1 1 1 1 73.387 – 288.243 133.628 ± 25.330
7 1 1 1 1 1 1 1 1 88.073 – 134.575 100.555 ± 6.296
8 1 1 1 1 0 0 1 1 84.785 – 301.557 151.008 ± 38.591
9 1 1 1 1 1 1 1 1 75.609 – 102.12 88.655 ± 3.668
10 1 1 1 1 1 1 1 1 69.003 – 103.242 89.048 ± 3.660
11 1 1 1 0 1 1 1 1 79.645 – 120.345 96.352 ± 5.641
12 1 1 1 1 1 1 1 1 106.397 – 185.249 137.056 ± 8.743
13 1 1 1 1 1 1 1 1 81.935 – 129.865 110.792 ± 6.363
14 1 1 1 1 1 1 1 1 65.060 – 105.917 87.673 ± 4.698
15 1 1 1 1 1 1 1 1 98.431 – 136.057 116.866 ± 5.127
16 1 1 1 1 1 1 1 1 165.971 – 288.497 217.708 ± 12.876
17 1 1 1 1 1 1 1 1 54.021 – 120.309 96.728 ± 8.899
18 1 1 1 1 1 1 1 1 81.699 – 128.657 102.640 ± 5.430
19 1 1 1 1 1 1 1 1 78.999 – 135.429 113.886 ± 6.424
20 1 1 1 1 1 1 1 1 77.058 – 116.896 93.100 ± 5.702
21 1 1 1 1 1 1 1 1 91.242 – 113.645 101.692 ± 3.025
22 1 1 1 1 1 1 1 0 62.370 – 195.309 106.922 ± 18.593
23 1 1 1 1 1 1 1 1 116.626 – 182.869 140.272 ± 7.961
24 1 0 1 1 1 1 1 0 96.302 – 117.632 107.551 ± 3183
25 1 1 0 0 1 1 1 1 141.129 – 202.293 182.267 ± 9.103
26 1 1 1 1 1 1 1 1 91.648 – 123.378 106.102 ± 3.353
27 1 1 1 1 1 1 1 1 82.741 – 143.273 108.881 ± 7.119
28 1 0 1 1 1 1 1 1 110.501 – 182.767 133.803 ± 11.659
29 1 1 1 0 0 0 0 0 160.952 – 211.424 183.002 ± 14.914
30 1 0 1 1 1 1 0 0 64.505 – 77.433 70.686 ± 2.366
Total 30 26 29 26 27 27 28 24

M1, male 1; F1, female 1; F2, female 2; F3, female 3; L1, olfactory lobe 1; L2, olfactory lobe 2.

Fig. 6. Pearson’s correlation between the volumes of male and female glomeruli (P < 0.05). A: Pearson’s correlation between females 1, 2 and 3 (r = 0.8547). B:
Pearson’s correlation between the male and females 1, 2 and 3 (r = 0.8061).

glomeruli. The numbers of glomeruli found in both sexes were quite
similar. The volumes and shapes of the glomeruli between the two ol­
factory lobes showed correspondence in different individuals, regardless
of gender.
The olfactory sensilla of Haller’s organ in A. sculptum had neuronal
projections confined to the olfactory lobes located in the synganglion, as
seen in other ixodid tick species (Borges et al., 2016). The number of
glomeruli found in adults of A. sculptum was slightly higher than what
was observed in the predatory mite Phytoseiulus persimilis (between 14
and 21) (van Wijk et al., 2006) and in the tick A. americanum (between
16 and 22) (Borges et al., 2016). However, it was lower than what was
found in the hematophagous insects Aedes aegypti, with 49 (Ignell et al.,
2005); Anopheles gambiae, with 61 (Ghaninia et al., 2007); and Culex
quinquefasciatus, with 62 in the female and 44 in the male (Ye et al.,
2020). Moreover, it was much lower than in social insects (quantities
greater than 200) (Kleineidam et al., 2005; Kuebler et al., 2010; Zube
et al., 2008). As previously noted, the number of glomeruli is associated
with the repertoire of chemical signals perceived by the arthropod
(Sachse et al., 1999).
In the present study, the mean glomerular volumes of the male and
the females were similar and rather smaller than those of other hema­
tophagous arthropods (Ghaninia et al., 2007; Ignell et al., 2005; Ye et al.,
2020). Females and males of the mosquitoes A. gambiae and A. aegypti
present sexually dimorphic glomeruli: this condition is associated with
different food preferences, since only the females are hematophagous
(Ghaninia et al., 2007; Ignell et al., 2005). Both males and females of
A. sculptum are blood feeders, so there are no differences in food pref­
erences (Randolph, 2014).
The number of glomeruli was apparently lower in the females than in
the males in the current study, but the number of individuals evaluated
did not allow inferences regarding whether this difference was signifi­
cant. Recently, the existence of morphological sexual dimorphism of

7
K.M.F. Menezes et al.
Haller’s organ in Dermacentor variabilis was reported, but this was not
seen in Ixodes scapularis and A. americanum. These findings suggest that
males and females of D. variabilis have more sex-specific sensory re­
quirements (Josek et al., 2018). Therefore, further studies regarding
morphological sexual dimorphism of Haller’s organ in A. sculptum are
desirable.
In the current study, 10 % (n = 20) of the prepared synganglia
showed fluorescence and were then subjected to confocal microscopy.
However, some areas of the olfactory lobes showed intense labelling,
probably due to extravasation of TMR, and it was only possible to
perform 3D reconstruction on 2% of the ticks. Previous studies on
P. persimilis and A. americanum used this same labelling and presented
similar results (Borges et al., 2016; van Wijk et al., 2006). TMR is
perhaps not the best labelling for anterograde backfills. It would be
advisable to accomplish labelling through using an anti-synapsid anti­
body that might provide better resolution of the glomeruli, as has been
reported in a centipede species (Sombke et al., 2012) and in crustaceans
(Krieger et al., 2010; Schachtner et al., 2005).
3D mapping of antennal lobes in arthropods (insects and crusta­
ceans) is important for understanding the fundamental principles of
integration and discrimination of olfactory information that occurs in
the olfactory neuropiles and in higher cerebral centers (Sombke et al.,
2012, 2011). There are some important data in the literature regarding
the chemical ecology of A. sculptum (Ferreira et al., 2020, 2019; Gachoka
et al., 2012, 2011; Louly et al., 2008; Soares and Borges, 2012) and the
present study adds some new information on neuronal olfactory pro­
jection and the morphology of the olfactory lobes. However, new studies
to obtain better 3D maps of the olfactory lobes in male and females ticks
are desirable for subsequent comparison, given that neuroethology
might improve the strategies for controlling harmful arthropods like
ticks.
5. Conclusion
The olfactory sensilla of Haller’s organ in A. sculptum adults project
to the olfactory lobes, which are organized into glomeruli located in the
synganglion. 3D reconstruction showed that males and females have
glomeruli that are similar in quantity, shape and volume.
CRediT authorship contribution statement
Karolina Martins Ferreira Menezes: Methodology, Investigation,
Data curation, Writing - original draft, Writing - review & editing. Jaires
Gomes de Oliveira Filho: Formal analysis, Writing - review & editing.
Lorena Lopes Ferreira: Writing - original draft, Methodology, Writing -
review & editing. Lígia Miranda Ferreira Borges: Conceptualization,
Methodology, Writing - review & editing, Supervision.
Acknowledgements
This study was financed in part by the Coordination Office for
Improvement of Higher-Education Personnel (Coordenação de Aperfei­
çoamento de Pessoal de Nível Superior, Brazil; CAPES), under Finance
Code 001. We thank the Microscopy Platform of the Gonçalo Moniz
Institute (Fiocruz Bahia) for providing confocal microscopy services;
and Pia Olafson, at USDA, for granting the material for labelling the
synganglion.
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