Histochemical Localization of Esterases in the Integument of the
Female Boophilus microplus (Acari: Ixodidae) Tick
M. A. VILLARINO,1S. D. WAGHELA, AND G. G. WAGNER
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University,
College Station, TX 77845Ð4467
J. Med. Entomol. 38(6): 780Ð782 (2001)
The cattle tick Boophilus microplus (Canestrini) is one of the most important ecto-
parasites affecting tropical cattle with worldwide distribution. Application of organophosphate
compounds (OP) is extensively used as a tick control method. However, the appearance of ticks
resistant to the OP decreases the therapeutic efÞcacy of such compounds. Esterases have been
implicated as potential biochemical mechanisms for detoxiÞcation in B. microplus larvae. We found
increased esterase activity in the inner layers of the integument of OP resistant adult female
B. microplus ticks as compared with the OP susceptible ticks. We discuss the potential role of these
enzymes during acaricide metabolism and propose future research.
Boophilus microplus, acaricide resistance, esterase activity, tick integument.
Boophilus microplus (CANESTRINI) ticks are economi-
cally important pests of cattle worldwide because of
the direct damaged caused and the diseases causing
organisms transmitted by them (Nun ˜ez et al. 1985).
Just as in other arthropods, ticks have a chitinous
exoskelton that serves as the primary protection
against water loss and physical damage (Sonenshine
Since the report of insecticide resistance in the
houseßy, Musca domestica (L.), in 1946, an increased
number of arthropods, including ticks, have been
shown to be resistant to insecticides such as organo-
phosphate (OP) compounds (Georghiou and Mellon
1983). Resistance to OP compounds frequently in-
volves detoxiÞcation of the insecticide through in-
creased esterase activity that can result from either
qualitative or quantitative changes in the esterases of
the resistant arthropods (Whyard et al. 1995). More
than 22 esterases have been implicated in OP resis-
tance in the Diptera (Healy et al. 1991). When an OP
acaricide is applied, the active agent must reach the
target molecule, acetylcholinesterase (Casida 1964).
The acaricide can penetrate the tick, by penetrating
ing. Previously, increased esterase activity was de-
and compared with other OP resistant insects (Vil-
larino et al. 2000). Such increases in the esterase ac-
tivity of OP resistant ticks implied that this group of
enzymes was important in OP detoxiÞcation. In this
article, we have determined that increased esterase
activity occurs in the integument of engorged female
B. microplus by histochemistry and light microscopy.
Materials and Methods
Tick Strains. Engorged females of an OP-resistant
strain (Mexico OP) (F14) and an OP susceptible
(Gonzalez) (F9) strain were used during this exper-
strain showed a LC90([AI], 95% CL) of 0.804 and the
Gonzalez strain showed a LC90([AI], 95% CL) of
0.100 (Davey and George 1999) using the method
collected before egg production began, frozen with
dry ice and transported to the College of Veterinary
Histochemical Analysis. Individual ticks were
thawed and washed with 100 ml 0.01 M phosphate
buffered saline (PBS) solution pH 7.2 that contained
2% Triton X-100 (Sigma, St. Louis, MO) for 15 min. A
transverse section 1 by 3 mm was cut from the middle
of the idiosoma of each tick. The sections obtained
were carefully washed in PBS, and the internal con-
tents removed. Each section was stored in PBS until
Histological Preparations. Sections of the integu-
ment of the ticks were Þxed in 4% formalin for 12 h,
washed in distilled water for 15 min and embedded in
warmed parafÞn. Serial sections of 3.5 ?m were cut
with a microtome (Leitz 1512, Stuttgart Germany)
Þtted with a metal knife. ParafÞn embedded sections
were placed on a glass microscope slide, stained with
hematoxylin and eosin (H&E) and Mallory stain (ba-
with a glass cover slip.
Cryostat Sections. Recently obtained integument
0022-2585/01/0780Ð0782$02.00/0 ? 2001 Entomological Society of America
O.C.T. compound (Sakura Laboratories, Torrence,
CA) and frozen into chunks by immersion in liquid
cryostat (Sakura Laboratories), mounted on a micro-
scope glass slide and air dried at 20?C for 15 min.
Histochemistry. Mounted sections were Þxed in a
methanol, acetic acid, water solution (3:1:6) for 1 h,
for 12 h at room temperature in 100 ml of PBS that
contained 10 mM ?-naphthyl acetate (diluted in 100
?l of acetone), and 10 mM fast garnet (Sigma). The
PBS then air dried and mounted in a water base so-
lution and covered with a glass cover slip. ParafÞn
mounted and cryostat sections were stained at the
same time under the same conditions. Integument
samples were incubated without substrate and in-
cluded for detection of nonspeciÞc reactions.
Image analysis. All the samples were observed at
400? magniÞcations using an Eclipse E-400 light mi-
croscope (Nikon, Tokyo, Japan). Images were re-
corded with a HV-C20 M digital camera (Hitachi,
Tokyo, Japan) mounted on the microscope. The im-
ages were captured using Pax-it Version 3.0.4f (MIS,
Houston, TX) for Windows (Microsoft, Roselle, IL),
The Mallory stained parafÞn embedded tissues
showed a red layer superimposed onto a blue layer
(Fig. 1, top left). The sections stained with H&E
showed a protein stratum and connective tissue layer
beneath it. Also, a muscle stratum and a basophilic
stratum under the lowest integument base formed by
adipocite cells were observed (Fig. 1, top right). Be-
cause the chemicals used during Mallory and H&E
staining process can affect esterase activity, frozen
cryostat tissues using ?-naphthyl acetate, a dark red
and outer endocuticle. The tissue sections from Mex-
ico OP engorged females processed under frozen cir-
cumstances showed darker brown color in the en-
docuticle part of the integument as a consequence of
the Gonzalez females (Fig. 1, bottom left). This ob-
servation was found in 12 of the 15 OP-resistant ticks
analyzed. No dark brown coloration was detected in
any of the formalin Þxed tissues.
Most of the chemical agents used as pesticides for
arthropod control are esters of substituted phospho-
parafÞn embedded engorged female integument of Mexico OP strain. OC, outer cuticle; IC, inner cuticle. Top right: Mexico
OP engorged female integument. H&E stain. 400?. Differences in tissue are evident: Muscles and connective tissue. PS,
protein strata; BS, basophilic strata; M, muscle. Bottom right: Esterase B histochemistry, Mexico OP strain engorged female
integument. Bottom left: Esterase B histochemistry, Gonzalez strain engorged female integument. Esterase activity is higher
in Mexico OP strain, causing a darker dye precipitation in the IC strata of the integument.
Top left. Mexico OP engorged female integument. Mallory stained. 400?. The stain differentiates strata found in
November 2001VILLARINO ET AL.: LOCALIZATION OF ESTERASES IN FEMALE B. microplus781
ric, carbamic or cyclopropane carboxylic acids, there- Download full-text
fore subject to degradation by esterases (Devonshire
1991). Several authors have discussed the esterases
as factors in the pesticide resistance phenomenon
thol produced gives a strong color reaction with dia-
zonium salts, and the insoluble resulting dye makes it
ideal for histological studies (Devonshire and Moores
1982). Thus, the increased intensity of deposition of
the dye in the Mexico OP strain of the resistant
B. microplus is due to the increases esterase activity.
These increased esterase activity suggests that in the
a putative mechanism of resistance. Potentially, ester-
ase detoxiÞcation of the OP compounds in the integ-
ument may reduce the amount of active OP com-
pound reaching the nervous tissues as previously
highly lipid soluble. Organic solvents and ionic deter-
gents are often used as vehicles to facilitate their
miscibility with water. This chemical characteristic
anism is present in integument tissue. Previous met-
abolic analysis using a radio labeled OP compound
showed signiÞcant greater concentration of hydro-
lytic products and a lower concentration of couma-
located in the integument strongly suggests that this
may be the initial detoxifying mechanism in ticks that
are resistant to OP. Strong conclusions about the role
of the integument based just on the results as pre-
sented are incomplete; however, we believe our Þnd-
ings are important and further research on esterase
characterization, synergy studies, and enzyme kinetic
studies using commercial acaricides and their metab-
olites will be conducted and reported.
TX) for providing tick material and to the technical person-
nel of the Veterinary Histology Laboratory, College of Vet-
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