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<DIV><FONT lang=0 face=Arial color=#000000 size=2 FAMILY="SANSSERIF" PTSIZE="10"><STRONG><EM> </EM></STRONG>Paper attached and embedded
below. Important new information. Laurie<BR></DIV>
<DIV>
<DIV> </DIV>
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<HR>
From: Gerald.Hayes@freshfromflorida.com<BR>To: Ladadams@aol.com<BR>Sent:
2/1/2011 10:21:06 A.M. Pacific Standard Time<BR>Subj: Paper<BR></DIV>
<DIV> </DIV><FONT style="BACKGROUND-COLOR: transparent" face=Arial color=#000000 size=2>
<DIV class=WordSection1>
<P class=MsoNormal>Take a look at the attached when you get a chance.
Thanks Jerry<o:p></o:p></P>
<P class=MsoNormal><o:p> </o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">G. W. Hayes,
Jr.</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">Chief,</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">Apiary Inspection
Section</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">Division of Plant
Industry</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">P O Bx
147100</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">Gainesville
FL 32614-7100</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">(352) 372-3505 ext
128</SPAN><o:p></o:p></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; COLOR: #92d050; FONT-FAMILY: 'Arial','sans-serif'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">email:gerald.hayes@freshfromflorida.com<o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 10pt; FONT-FAMILY: 'Arial','sans-serif'">Please note that
Florida has a broad public records law (Chapter 119, Florida Statutes). Most
written communications to or from state employees are public records
obtainable by the public upon request. emails sent to me at this email address
may be considered public and will only be withheld from disclosure if deeemed
confidential pursuant to the laws of the State of
Florida.</SPAN><o:p></o:p></P>
<P class=MsoNormal><o:p> </P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Cell
death localization <I style="mso-bidi-font-style: normal">in situ</I> in
laboratory reared honey bee (<I style="mso-bidi-font-style: normal">Apis
mellifera</I> L.) larvae treated with pesticides<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><st1:PersonName w:st="on"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Ales
Gregorc</SPAN></st1:PersonName><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">*,
James D. Ellis<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Honey
Bee Research and Extension Laboratory, Department of Entomology and
Nematology, University of Florida, P.O. Box 110620, Bldg 970 Natural Area
Drive, Gainesville, FL, USA 32611<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt 0pt 10pt; TEXT-ALIGN: justify; mso-pagination: widow-orphan lines-together; tab-stops: -72.0pt -36.0pt 0pt 21.4pt 42.8pt 64.25pt 85.65pt 108.0pt; mso-hyphenate: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 115%; FONT-FAMILY: 'Times New Roman'">*Corresponding
author email and present mail address: <A href="mailto:ales.gregorc@kis.si">ales.gregorc@kis.si</A>; <SPAN style="LETTER-SPACING: -0.1pt; mso-no-proof: yes">Agricultural Institute of
<st1:place w:st="on"><st1:country-region w:st="on">Slovenia</st1:country-region></st1:place>, Hacquetova 17, SI-1000
<st1:City w:st="on">Ljubljana</st1:City>, <st1:country-region w:st="on">Slovenia</st1:country-region>, tel: </SPAN><SPAN class=skypepnhtextspan><SPAN style="COLOR: black"> +386-1- 28 05
150</SPAN></SPAN></SPAN><SPAN class=skypepnhrightspan><SPAN style="FONT-SIZE: 10pt; COLOR: black; LINE-HEIGHT: 115%; FONT-FAMILY: Arial"> </SPAN></SPAN><SPAN style="FONT-FAMILY: Arial; LETTER-SPACING: -0.1pt; mso-bidi-font-family: 'Times New Roman'; mso-no-proof: yes"><o:p></o:p></SPAN></P>
<P class=MsoNormal><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always" clear=all></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; TEXT-ALIGN: justify"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Abstract<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">In
this study, cell death detected by DNA fragmentation labeling and
phosphatidylserine (PS) localization was investigated in the honey bee (<I style="mso-bidi-font-style: normal">Apis mellifera</I> L.) midgut, salivary
glands and ovaries after treating larvae with different pesticides offered via
an artificial diet. To do this, honey bee larvae reared in an incubator were
exposed to one of nine pesticides: chlorpyrifos, imidacloprid, amitraz,
fluvalinate, coumaphos, myclobutanil, chlorothalonil, glyphosate and simazine.
Following this, larvae were fixed and prepared for immunohistologically
detected cellular death using two TUNEL techniques for DNA fragmentation
labeling and <A name=OLE_LINK9></A><A name=OLE_LINK8><SPAN style="mso-bookmark: OLE_LINK9">Annexin V to detect the localization of
exposed PS specific <I>in situ </I>binding to apoptotic cells.</SPAN></A>
Untreated larvae experienced ~10% midgut apoptotic cell death under controlled
conditions. All applied pesticides triggered an increase in apoptosis in
treated compared to untreated larvae. The level of cell death in the midgut of
simazine-treated larvae was highest at 77% mortality and statistically similar
to the level of cell death for chlorpyrifos (65%), imidacloprid (61%),
myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to
fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any
pesticide treated larvae. Indications of elevated apoptotic cell death in
salivary glands and ovaries after pesticides applications were detected.
Annexin V localization, indicative of apoptotic cell deletion, had an
extensive distribution in the midgut, salivary glands and ovaries of
pesticide-treated larvae. The data suggest that the tested pesticides induced
apoptosis in tissues of honey bee larvae at the tested concentrations. Cell
death localization as a tool for a monitoring the subclinical and sub-lethal
effects of external influences on honey bee larval tissues is discussed.
<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Keywords:</SPAN></B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"> <I style="mso-bidi-font-style: normal">Apis mellifera</I>, immunohistology, cell
death, TUNEL, insecticide, herbicide, fungicide<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Introduction<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Globally,
the environment around honey bee (<I style="mso-bidi-font-style: normal">Apis
mellifera</I>) colonies can be contaminated with toxic chemicals from
industrial, agricultural and domestic activities. In many cases, these
chemicals are pesticides which encompass an array of compounds designed to
repel or kill insects (insecticides), plants (herbicides), fungi (fungicides)
and other organisms considered pests. Though honey bees are non-target
organisms for most pesticide applications, they nevertheless can be exposed to
pesticides while collecting pollen and nectar from flowers, collecting resins
from various plants, drinking water from rivers/lakes/ponds/etc., breathing,
and during flight (if the pesticides are airborne). These pesticides may be
brought back inadvertently to the colony where their levels are concentrated
further in the waxy nest infrastructure. In surveys of North American honey
bee colonies conducted in 2007 and 2008, investigators found 121 different
pesticides and metabolites in wax, pollen, bees, and corresponding hive
samples </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[1],</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
thus illustrating the need to understand how pesticides may affect individual
honey bees and the social colonies in which they
reside.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Many
of the pesticides to which honey bees are exposed have insecticidal properties
and may be harmful to bees. For example, pesticides are known to lower the
developmental rate of queen honey bees, increase the occurrence of queen
rejection, and lower queen weight </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[2-4]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
affect honey bee cardiotoxicity </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[5]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
and affect forager bee mobility and communicative capacity </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[6]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
all among other effects documented in the literature. In our effort, we
broaden the study of pesticide effects on honey bees by investigating
pesticide effects on cell death and localization in pesticide-treated, honey
bee larvae.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: AdvGulliv-R">There
are many reasons to look at pesticide effects in bee larvae tissues. First,
toxic effects of pesticides have been shown to manifest in mammalian tissue
and alter enzymatic levels, blood biochemistry and tissue histology
</SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[7]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: AdvGulliv-R">,
thus providing evidence that toxins can affect tissues in pesticide-exposed
organisms. Second, </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">histological
changes in treated individuals provide a rapid detection method for the
effects of toxicants, especially chronic irritants, in various tissues and
organs </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[8]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Third, many of the studies where the effects of pesticides on honey bees are
discussed focus on toxin effects on adult bees rather than immature ones,
resulting in a lack of information concerning the latter. Fourth, previous
immunocytochemical studies of cell death and the localization of heat-shock
proteins in larval honey bee tissues after acaricide application have fostered
a better-understanding the adverse effects acaricides may have on bees
</SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[9-11]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Finally, there is an overall lack of histopathological studies on pesticide
treated animal tissues. For all of these reasons, we studied the effects of
pesticides on larval honey bees at the cellular level.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">To
determine pesticide effects on the cellular tissues of larval honey bees, we
looked specifically at unintentional cell death (necrosis) and programmed cell
death (apoptosis)<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[12]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Necrotic cell death is induced by external influences with evident
morphological changes: i.e. the chromatin condenses and clumps are formed at
the nuclear periphery </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[12]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Necrosis refers to the <I>post mortem </I>changes that occur following the
death of the cell </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[13]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Apoptosis on the other hand presents a range of morphological symptoms
including cell shrinkage and chromatin margination, the latter of which is
followed by DNA fragmentation and the formation of apoptotic bodies
</SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[14]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Apoptosis originally was defined as the physiological death of cells and
tissues associated with developmental remodeling </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[15]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
and can be induced by genetic </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[16] </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">and
non-genetic </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[17]
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">means.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">We
used multiple cell death assays to determine the effects of various pesticides
on honey bee larvae. The first method we used to determine the progression of
cell death <I style="mso-bidi-font-style: normal">in situ</I> was the TUNEL
(terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling) method
which assesses DNA breakdown preceding the nuclear collapse of apoptotic
nuclei </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[18]
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">and
consists of the visualization of fragmented DNA in the nucleus </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[19]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Cell death previously has been characterized using the terminal TUNEL
technique method in the honey bee midgut </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[10, 20] </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">and
larval salivary glands </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[1</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">1]
where the death of salivary gland tissues in honey bee larvae was detected
[21]. We decided to use two TUNEL methods in our experiment because others
have provided data which show that different TUNEL kits can indicate different
levels of cell death in target tissues [10]</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">.
For example, the <I style="mso-bidi-font-style: normal">in situ</I> cell death
detection kit AP was unable to differentiate between apoptosis and necrosis in
different human tissues and detected both </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">[22]</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">.
Furthermore, DNA fragmentation and a TUNEL-positive reaction can occur after
different kinds of cell death using various kits. Regardless, </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">immunocytochemical
methods assaying DNA fragmentation [24] are useful techniques for detecting
impending apoptosis due to larval exposure to pesticides while feeding
</SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[25]</SPAN><SPAN lang=AF style="mso-bidi-font-family: Arial; mso-ansi-language: AF; mso-fareast-language: SL"><FONT face=Calibri>. </FONT></SPAN><SPAN lang=AF style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><SPAN style="mso-spacerun: yes"> </SPAN></SPAN></FONT><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB"><o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
second method we employed to monitor cell death was via our use of Annexin V
to detect the localization of exposed phosphatidylserine (PS) specific <I>in
vivo </I>binding to apoptotic cells. In dying cells, PS is externalized
actively to the plasma membrane’s outer leaflet parallel to the extracellular
environment </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[26]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Most forms of cell death share the phenomenon of cell surface expression of PS
</SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[27]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Externalization of PS is an early event in the sequence of steps leading to
cell death which starts well before changes in the cell nuclei and plasma
membrane integrity are compromised </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[28]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
PS on the cell surface can be detected using Annexin V, a member of the
annexin protein family that binds in a calcium-dependent way to PS-containing
membranes </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[29]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
The Annexin V affinity assay discriminates among living cells, cells in the
early phase of cell death and (secondary) necrotic cells that have a
compromised cell membrane </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[30]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">In
our study, induced cell death and PS localization was investigated in honey
bee midguts after treating larvae with one of nine different pesticides
offered via an artificial diet. The tested pesticides (with insecticide class
in parentheses) included 2 fungicides [myclobutanil (azole), chlorothalonil
(substituted benzene)], 2 herbicides [simazine (triazine), glyphosate
(phosphonoglycine)], and 5 insecticides/miticides [fluvalinate (pyrethroid),
imidacloprid (nicotinoid), coumaphos (organophosphate), chlorpyrifos
(organophosphate), amitraz (amidine)] and represent a range of
modes-of-actions and pesticide families. With the exception of glyphosate, all
have been found as residues in honey bee colonies </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[1]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Immunohistological methods using both TUNEL assays and Annexin 5 were employed
in order to reduce the probability of extraneous artifacts </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[25]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
in an attempt to define the specific modes of cell death, and for the broad
quantification of cell death observed in larval midguts. We hypothesized that
increased apoptotic cell death (determined using the TUNEL technique) occurs
in pesticide treated larvae in comparison to untreated larvae and that PS
exposure on the plasma membrane of apoptotic cells (determined using Annexin
V) would be present in pesticide treated larvae.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">2.
Materials and Methods<o:p></o:p></SPAN></B></P>
<P class=05BodyText style="MARGIN: 0pt; TEXT-INDENT: 0pt"><I style="mso-bidi-font-style: normal"><FONT size=3><FONT face="Times New Roman">2.1. Larval rearing, treatment and
sampling<o:p></o:p></FONT></FONT></I></P>
<P class=05BodyText style="MARGIN: 0pt"><FONT face="Times New Roman"><FONT size=3>Experiments were conducted at the University of Florida Honey Bee
Research and Extension Laboratory, Department of Entomology and Nematology,
Gainesville, FL. Queens in three production honey bee colonies housed in
10-frame Langstroth-style equipment were confined to a section of newly-drawn
comb using a metal queen excluder cage (~10 × 10 × <st1:metricconverter w:st="on" ProductID="3 cm">3 cm</st1:metricconverter>) at time t = -12 h. The
caged queen and frame were returned to the center of the brood nest where
worker bees could access and tend the queen. After 24 h of queen confinement,
t = 12 h </FONT><SPAN style="FONT-SIZE: 11pt; LINE-HEIGHT: 200%">[ 31,
32]</SPAN><FONT size=3>, we removed the queen from the cage and replaced the
cage on the comb as before but this time for 108 h (from t = 0) to allow the
eggs to hatch and larvae to reach an appropriate age for grafting. During this
time, worker bees were able to access the comb to feed the developing larvae.
At 108 h, we removed the test frames (now containing 36 </FONT></FONT><FONT size=3><SPAN style="FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN style="mso-char-type: symbol; mso-symbol-font-family: Symbol">±</SPAN></SPAN><FONT face="Times New Roman"> 12 h old larvae) from the colonies and took them to
the laboratory. <o:p></o:p></FONT></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">At
the laboratory, the larvae were grafted to sterile, 96-well tissue culture
plates (well volume = 0.32 mL, Fisher Scientific, <st1:place w:st="on"><st1:City w:st="on">Pittsburgh</st1:City>, <st1:State w:st="on">PA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). Prior to grafting the larvae
into plates, we pipetted 20 µL of larval diet into the bottom of each cell.
The diet had a pH that ranged from 4.0-4.5 and consisted of 50% royal jelly
(Glory Bee Foods, <st1:City w:st="on">Eugene</st1:City>, <st1:State w:st="on">OR</st1:State>, <st1:country-region w:st="on">USA</st1:country-region>), 6% D-glucose (Fischer Chemical, <st1:City w:st="on">Fair Lawn</st1:City>, <st1:State w:st="on">NJ</st1:State>,
<st1:country-region w:st="on">USA</st1:country-region>), 6% D-fructose
(Fischer Chemical, <st1:City w:st="on">Fair Lawn</st1:City>, <st1:State w:st="on">NJ</st1:State>), 37% double distilled water, and 1% yeast extract
(Bacto™, <st1:place w:st="on"><st1:City w:st="on">Sparks</st1:City>,
<st1:State w:st="on">MD</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>) by volume </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'"><FONT size=3>[32]</FONT></SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Prior to adding the diet to each cell, we pre-warmed it to 35<SUP>o</SUP>C in
an incubator (Percival Scientific Inc, <st1:place w:st="on"><st1:City w:st="on">Perry</st1:City>, <st1:State w:st="on">IA</st1:State>,
<st1:country-region w:st="on">USA</st1:country-region></st1:place>).<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Each
subsequent day, we transferred larvae to a clean culture plate provisioned
with fresh diet. The amount of artificial diet provided to each larva depended
on the larva’s age. We fed larvae 20 µL of diet at hours 108 and 132, 30 µL on
hour 156, 40 µL on hour 180, and 50 µL on hour 204 </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[33, 34]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
At 204 h post oviposition (larvae are 132 </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol"><SPAN style="mso-char-type: symbol; mso-symbol-font-family: Symbol">±</SPAN></SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"> 12
h old), we transferred the larvae to a 48-well plate (Becton Dickinson
Labware, <st1:PlaceName w:st="on">Franklin</st1:PlaceName> <st1:PlaceName w:st="on">Lakes</st1:PlaceName>, NJ, <st1:place w:st="on"><st1:country-region w:st="on">USA</st1:country-region></st1:place>, wells were 13 ×
<st1:metricconverter w:st="on" ProductID="17 mm">17 mm</st1:metricconverter>)
because the growing larvae were too large to handle delicately in a 96-well
plate. Throughout the study, trays containing larvae were incubated in the
dark at 35<SUP>o</SUP>C and ~96% RH </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[31]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">To
test the effects of pesticides on developing larvae, specific pesticide
concentrations were mixed with the larval diet daily for 4 days beginning the
second day larvae were in the laboratory (132 h = 60 h old larvae). Nine
treatment groups of larvae were established in all, each group being composed
of 12 treated larvae. Each group of test larvae was treated with 1 of the
following pesticide doses: 1.6 ppm chlorpyrifos, 400 ppm imidacloprid, 400 ppm
amitraz, 200 ppm fluvalinate, 100 ppm coumaphos, 400 ppm myclobutanil, 400 ppm
chlorothalonil, 400 ppm glyphosate, and 400 ppm simazine. The respective
pesticide doses are at or below LC<SUB>50</SUB> values known for honey bee
larvae (unpublished data). Originally, we wanted to standardize the dose
delivered across all pesticides at 400 ppm to bracket the upper residue limit
that any of these pesticides have been found in honey bee colonies [1].
However, chlorpyrifos has a low LC<SUB>50</SUB> value and
fluvalinate/coumaphos LC<SUB>50</SUB> values do not fit standard toxicity
curves (unpublished data). As such, these three pesticides were administered
at different doses than were the other pesticides. All applied pesticides were
obtained from Chem Service, <st1:place w:st="on"><st1:City w:st="on">West
Chester</st1:City>, <st1:State w:st="on">PA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>. <o:p></o:p></SPAN></P>
<P class=MsoPlainText style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Prior
to administration to the larval diet, each pesticide was diluted individually
in an acetone solvent. The diet/pesticide combinations were prepared and
stored in 1.5 ml snap-top plastic vials (Fisher Scientific, <st1:place w:st="on"><st1:City w:st="on">Pittsburgh</st1:City>, <st1:State w:st="on">PA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). We included two control
groups in the study: larvae feeding on diet containing acetone and larvae
feeding on an untreated diet. All larvae were sampled on day 6 (h = 228), 24
hours after the application of the last pesticide treatment. Sampled larvae
were fixed in 10% formalin for 24 h, dehydrated in a series of alcohols and
xylene, and finally embedded in paraffin wax as described by Gregorc and Bowen
</SPAN><SPAN style="FONT-SIZE: 11pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">[9]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Sections of 5 μm were cut on a 2030 Rechert/Young Microtome (<SPAN>Cambridge
Instrument GmbH., <SPAN style="FONT-WEIGHT: normal; mso-bidi-font-weight: bold">Germany</SPAN></SPAN>),
floated on distilled water at <st1:metricconverter w:st="on" ProductID="40ÿ°C">40°C</st1:metricconverter>, collected on cleaned slides, and
kept in an drying oven at <st1:metricconverter w:st="on" ProductID="60ÿ°C">60°C</st1:metricconverter> for ~4 h. Slides then were stored
at room temperature until later analyses.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-style: italic"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">2.2.
Immunohistology<o:p></o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
paraffin wax was removed from the tissue sections in three washes of xylene
and three washes of absolute alcohol. Sections then were rinsed in Phosphate
Buffer Solution </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(PBS,
<st1:metricconverter w:st="on" ProductID="0.01 M">0.01
M</st1:metricconverter>, pH 7.1) </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">and
prepared for staining.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-style: italic"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">2.3.
DeadEnd colorimetric TUNEL system<o:p></o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><SPAN style="mso-tab-count: 1">
</SPAN>The DeadEnd system (Promega, <st1:place w:st="on"><st1:City w:st="on">Madison</st1:City>, <st1:State w:st="on">WI</st1:State>,
<st1:country-region w:st="on">USA</st1:country-region></st1:place>) labels
fragmented DNA of apoptotic cells <I style="mso-bidi-font-style: normal">in
situ</I> using the TUNEL assay. After applying proteinase K, the larval
sections were incubated with the TdT reaction mixture and then with a
horseradish peroxidase-labeled streptavidin solution. Diaminobenzidine (DAB)
substrate was applied onto the tissue sections to develop a brown reaction
product. The sections were counterstained with </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">Mayer’s
hematoxylin</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Negative control labeling was achieved by substituting the deoxynucleotidyl
transferase (TdT) enzyme with PBS.</SPAN><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'"><o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></I></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">2.4.
</SPAN></I><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">In
situ<I style="mso-bidi-font-style: normal"> cell death detection kit, AP
(ISCDDK)<o:p></o:p></I></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">Dewaxed
and rehydrated tissue sections were incubated with proteinase K (20 μg/mL in
<st1:metricconverter w:st="on" ProductID="10 mM">10 mM</st1:metricconverter>
Tris/HCl, pH 7.4). Labeling was conducted by covering the tissue section with
a TUNEL reaction mixture composed of terminal deoxynucleotidyl transferase
(TdT) from calf thymus. TdT enzymes with fluorescein were detected using
“converter-AP” consisting of anti-fluorescein antibodies from sheep,
conjugated with alkaline phosphatase. The substrate solution was obtained
using a Vector® Red Alkaline Phosphatase Substrate Kit (Vector Laboratories,
<st1:place w:st="on"><st1:City w:st="on">Burlingame</st1:City>, <st1:State w:st="on">CA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). Sections were incubated with
the substrate (AP) and washed in tap water for 5 min. Counterstaining was
accomplished by transferring the sections into Mayer’s hematoxylin and then
rinsing the sections under running tap water. As a negative control, we
labeled a subgroup with terminal transferase, rather than TUNEL reaction
mixture.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">2.5.
Quantification of cell type and apoptosis<o:p></o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">TUNEL
labeled tissue slides were used for quantification of cell type and apoptosis
as determined using Dead End and ISDDK kits. For each treated group of larvae,
approximately 300 total cells from at least three larvae on different slides
were counted in random fields within the tissue. The results were expressed as
the proportion of cells counted that gave positive staining. To confirm
reproducibility, 25% of the slides were chosen randomly and scored twice. The
proportion of cells that gave positive staining was analyzed by treatment (9
pesticides and 2 controls) with a one way ANOVA for both staining techniques
(Dead End and ISDDK). Furthermore, we used a two way ANOVA to test the effects
of technique, overall treatment and the interaction of treatment × technique
on the proportion of cells with positive staining. Prior to all analyses, the
proportion data were transformed with an asin √x transformation. The
untransformed means are reported in the manuscript. Where necessary, we used
Student’s T-tests to compare means, accepting differences at <I style="mso-bidi-font-style: normal">P</I></SPAN><FONT size=3><FONT face=Calibri> </FONT><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">≤
0.05.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><A name=OLE_LINK11></A><A name=OLE_LINK10><SPAN style="mso-bookmark: OLE_LINK11"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-weight: bold">2.6.
Immunohistochemical localization of
PS<o:p></o:p></SPAN></I></SPAN></A></P><SPAN style="mso-bookmark: OLE_LINK11"></SPAN><SPAN style="mso-bookmark: OLE_LINK10"></SPAN>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">Dewaxed
and rehydrated tissue sections were placed in PBS (<st1:metricconverter w:st="on" ProductID="0.01 M">0.01 M</st1:metricconverter>, pH 7.1) and
incubated with a primary antibody solution. Rabbit antibodies polyclonal to
Annexin V were obtained from<I style="mso-bidi-font-style: normal"> </I>Abcam
(Abcam Inc., <st1:place w:st="on"><st1:City w:st="on">Cambridge</st1:City>,
<st1:State w:st="on">MA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). Antibodies were used at a
concentration of 2 µg/ml in PBS with 1% bovine serum albumin.<I style="mso-bidi-font-style: normal"> </I>After incubating the primary
antibodies overnight<I style="mso-bidi-font-style: normal"> </I>at
<st1:metricconverter w:st="on" ProductID="4ÿ°C">4<SPAN style="mso-fareast-font-family: Calibri">°C</SPAN></st1:metricconverter>, the
sections were covered with biotinylated universal secondary antibodies for 30
min. Alkaline phosphatase reagent also was applied for 30 min. Both reagents
were obtained in the Vecastain Universal ABC-AP kit (Vector Laboratories,
<st1:place w:st="on"><st1:City w:st="on">Burlingame</st1:City>, <st1:State w:st="on">CA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). The substrate solution was
obtained using the Vector® Red Alkaline Phosphatase Substrate Kit (Vector
Laboratories, <st1:place w:st="on"><st1:City w:st="on">Burlingame</st1:City>,
<st1:State w:st="on">CA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></st1:place>). Sections were incubated with
the substrate (AP) and counterstaining was accomplished by transferring
sections into Mayer’s hematoxylin. As a control, </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">no
primary antibody was applied to the tissue sections. </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">Sections
were mounted in Faramount aqueous mounting medium (Dako, </SPAN><st1:place w:st="on"><st1:City w:st="on"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Carpinteria</SPAN></st1:City><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
<st1:State w:st="on">CA</st1:State>, <st1:country-region w:st="on">USA</st1:country-region></SPAN></st1:place><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">).
All slides were examined with a Leica light microscope (</SPAN><SPAN lang=EN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN">Leica
Microsystems, <st1:place w:st="on"><st1:country-region w:st="on">Germany</st1:country-region></st1:place></SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">)
at 400× magnification.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt 0pt 0pt 18pt; TEXT-INDENT: -18pt; LINE-HEIGHT: 200%; mso-list: l4 level1 lfo10"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'"><SPAN style="mso-list: Ignore">3.<SPAN style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN></SPAN></B><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Results<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">3.1.
DeadEnd colorimetric TUNEL system<o:p></o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
brown reaction product obtained from the Promega DeadEnd kit indicated
DAB-positive, impending apoptotic cell death in all test larvae. Pesticide
specific levels of apoptosis detected in the midgut tissue are shown in Table
1. The DAB reaction product was detected in the midguts of all
pesticide-treated larvae in larger percentages than in control larvae fed
either a diet containing acetone or pure diet (Table 1). In all DAB-positive
cells, the brown reaction product was localized to the nuclei. The largest
percentages of DAB-positive cells in the midgut epithelium were observed in
larvae exposed to simazine, glyphosate, myclobutanil and amitraz (>60%,
Table 1). There were some incongruities between the two TUNEL techniques used
to estimate cell mortality, but these usually were orders of magnitude
differences in the data because the trends detected by both TUNEL techniques
were similar (Table 1). In general, pesticides that resulted in high levels of
apoptosis as detected by the ISDDK technique resulted in the same as detected
by the DeadEnd technique (Table 1). Notably, fluvalinate, on average, resulted
in the lowest level of apoptosis of any tested pesticide (Table 1).
<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
DAB reaction product was observed in columnar midgut epithelial cells in
simazine treated larvae (Fig. 1A) and in nearly all of the regenerative cells
in chlorpyrifos treated larvae (Fig. 1B). Furthermore, there were midgut
regions in amitraz treated larvae with both DAB-positive columnar and
regenerative epithelial cells (Fig. <st1:metricconverter w:st="on" ProductID="1C">1C</st1:metricconverter>), and regions in imidacloprid treated
larvae with columnar DAB-positive and regenerative negative cells (Fig. 1D).
In larvae with high proportions of DAB-positive cells, the positive cells were
localized in compartmental areas of the midgut, but tissues also were observed
containing only solitary DAB-positive cells (chlorothalonil treated larvae,
Fig. 1E). In untreated larvae, ~10% of the midgut epithelial cells were
DAB-positive (Fig. <st1:metricconverter w:st="on" ProductID="1F">1F</st1:metricconverter>). <o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
DAB reaction product also was localized in the salivary glands and the ovaries
of treated larvae. Salivary gland tissue expressed high levels of DAB-positive
cells in larvae exposed to amitraz (Fig. 1G). Similar levels also were found
in salivary glands in simazine, imidacloprid, glyphosate, myclobutanil or
fluvalinate treated larvae. In the ovarian tissue, high levels of DAB-positive
nurse cells were found in imidacloprid-treated larvae (Fig. 1H). In ovaries of
larvae treated with the remaining pesticides, the DAB reaction product was
found in similar amounts as in ovaries of untreated larvae. At normal tissue
turnover, up to 20% of nurse cells were DAB-positive (Fig. 1I). Negative
control sections showed no presence of the DAB reaction product, and
endogenous peroxidase also was quenched successfully (Fig. 1J).
<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">3.2.
</SPAN></I><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">In
situ<I style="mso-bidi-font-style: normal"> cell death detection kit, AP
(ISCDDK)<o:p></o:p></I></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Twenty-four
hours after honey bee larvae were exposed to the last of four pesticide
treatments, the red azo-dye reaction product was found in increased levels of
the midgut columnar-cell nuclei and also in the midgut regenerative-epithelial
cells. In chlorpyrifos-treated larvae, the level of positive-reaction product
in the columnar midgut cells (Fig. 2A) had risen to ~74% (Table 1). In
simazine, myclobutanil, imidacloprid, chlorpyrifos, chlorothalonil and
glyphosate-treated larvae, the level of positive columnar epithelial cells
with red azo-dye reaction product was ≥65% (Table 1). Simazine induced
localization of red azo-dye reaction product to the columnar and regenerative
cells (Fig. 2B). In coumaphos-treated larvae, the reaction product was found
in ~48% of all columnar and regenerative epithelial cells (Fig.
<st1:metricconverter w:st="on" ProductID="2C">2C</st1:metricconverter>). The
reaction product in the salivary glands was found in myclobutanil-treated
larvae, where a majority of cells were positive (Fig. 2D). In untreated
larvae, low amounts of reaction products were observed, though sporadic cells
were positive (Fig. 2E). The red azo-dye product in the ovarian tissue of all
treated and untreated control larvae ranged from 5 to 10 % (Fig.
<st1:metricconverter w:st="on" ProductID="2F">2F</st1:metricconverter>).<SPAN style="mso-spacerun: yes"> </SPAN><o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-bidi-font-weight: bold">3.3.
Immunohistochemical localization of PS<o:p></o:p></SPAN></I></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">In
the pesticide-treated larvae, the red azo-dye reaction product detected by
Annexin V, which characteristically localizes PS, was found to be present
abundantly in the midgut epithelium, salivary glands and ovaries. Thus, it was
possible to delineate the PS boundary at the apical columnar cell membrane in
the brush border and at the basal cell cytoplasm bound to basal membrane by
immunostaining of Annexin V. The Red azo-dye reaction product was localized
and bound to the apical brush border in chlorpyrifos- treated larvae (Fig.
3A). Annexin V staining spread throughout the midgut epithelium cells
noticeably, where immunostaining was diffuse and the entire cell cytoplasm of
glyphosate-treated tissue was stained (Fig. 3B). In the glyphosate-treated
larvae, Annexin V was abundant and PS was localized in the basal and apical
cell cytoplasm (Fig. <st1:metricconverter w:st="on" ProductID="3C">3C</st1:metricconverter>). Staining of the cytoplasm in a group
of columnar cells at the basal area was uneven and spotty and bound to the
basal membrane in simazine-treated larvae (Fig. 3D). Red azo-dye was present
abundantly in salivary glands of mycobutanil (Fig. 3E) and ovaries of
glyphosate-treated larvae (Fig. <st1:metricconverter w:st="on" ProductID="3F">3F</st1:metricconverter>). Staining was less intensive in
salivary gland cells in untreated larvae (Fig. 3G). In untreated larvae,
Annexin V was present in some sections of the midgut epithelium and
immunostainning was bound to the apical and basal cell membrane (Fig. 3H)
while the cytoplasm of the midgut cells was not stained. Results indicate that
Annexin V binds to cells of the midgut, salivary glands and ovaries of all
pesticide treated larvae abundantly while in untreated larvae Annexin V
binding was not as evident. In both groups of control larvae, the general
morphology of the epithelium was unchanged.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt 0pt 0pt 18pt; TEXT-INDENT: -18pt; LINE-HEIGHT: 200%; mso-list: l4 level1 lfo10"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'"><SPAN style="mso-list: Ignore">4.<SPAN style="FONT: 7pt 'Times New Roman'">
</SPAN></SPAN></SPAN></B><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Discussion<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Honey
bee larvae reared in an incubator and treated with one of nine pesticides
undergo subclinical, cellular changes that can be detected using
immunohistochemical methods. ISCDDK showed comparable levels of apoptosis with
that shown using the DeadEnd kit. Both TUNEL kits indicated induction of DNA
strand breaks after pesticide treatments and differences in apoptosis levels
in the tissue sections. There were variations in the distribution of
apoptosis, which was uneven and inconsistent. ISCDDK was found to demonstrate
DNA-fragmentation after both apoptotic and necrotic cell death </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[22, 23]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">There
were differences in apoptosis appearance in the midgut and apoptotic cells
were <SPAN style="mso-spacerun: yes"> </SPAN>observed randomly in the
epithelium of pesticide-treated larvae. Normal apoptotic cell death level in
the epithelium observed in both groups of control larvae (untreated diet and
acetone treated diet) was ~10%. Observed elevated death rates in the midgut
columnar cells and in ovarian or salivary gland cells of pesticide treated
larvae may be triggered by an apoptotic pathway after pesticide application.
All applied pesticides induced significant apoptotic cell death in the larvae
midgut as demonstrated through the use of both TUNEL kits. Necrosis, which
usually is caused by a lethal accident or disease opposed to a programmed
process, can be detected by TUNEL as found in Orita et al. </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[33]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
and in previous experiments where larvae were water-treated </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[10]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Interestingly,
fluvalinate resulted in the lowest levels of observed cell death of any
pesticide treated larvae. Fluvalinate has been used in the <st1:place w:st="on"><st1:country-region w:st="on">U.S.</st1:country-region></st1:place>
for over two decades to control <I style="mso-bidi-font-style: normal">Varroa
destructor </I>Anderson and Trueman, the varroa mite. Our data suggest that
honey bee larvae may have developed some level of resistance to fluvalinate
exposure. Equally interesting is that the herbicides glyphosate and simazine
and fungicides myclobutanil and chlorothalonil induced elevated apoptotic cell
death in an insect. Though unclear how this may affect honey bees at the
individual organism or colony level, the data suggest that herbicides and
fungicides cannot be presumed innocuous to bees. Regardless, the level of
stress-induced apoptosis related to pesticide treatment in bee larvae in our
experiment was comparable to that experienced by two invasive bivalves exposed
to a molluscicide </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[35]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">In
our experiment, the tested insecticides, fungicides and herbicides induced
elevated level of apoptosis in the larval midgut. In previous experiments,
lower concentrations of coumaphos applied to adult worker bees did not trigger
increased levels of apoptosis in hypopharyngeal glands compared to that in
untreated bees </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[20]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
In contrast, honey bee larvae treated with acaricides experienced apoptosis
and stress-induced, necrotic cell deletion </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[10]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">,
indicating that these different types of cell death can occur simultaneously
after exposure to pesticides </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[32]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Follicular
maturation during oogenesis involves necrosis along with apoptosis
</SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[36]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
and investigators have shown that necrosis potentially can accompany apoptosis
during normal development as shown in experiments with mouse cell embryos
</SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[37]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Thus, necrotic and apoptotic cell death often occur simultaneously during many
pathological processes, as seen in the present study, and during normal
processes such as tissue renewal, embryogenesis, and immune response.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">In
our study, we confirmed elevated levels of apoptosis in larvae treated with
pesticides. The epithelial cell nuclei remained morphologically unchanged but
became TUNEL-positive, indicating that the DNA was fragmented but not
different from neighboring cell nuclei otherwise. It is possible that the
induced larval cell apoptosis trigged by pesticide treatment in our study may
have been a reversible process in the midgut tissue, one from which the
affected larvae could recover. On the other hand, the appearance of apoptosis
may precede further tissue deletion, the development of necrosis in the midgut
cells, or cell death altogether. The TUNEL method thus is a useful diagnostic
tool to monitor subclinical changes in honey bee larvae induced by external
influences.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">The
apoptosis of regenerative cells observed in the basal area of the epithelium
of pesticide treated larvae may function to maintain the proper ratio of cells
in the midgut, i.e. large numbers of regenerative cells may die to compensate
for the inadequate number of epithelial cells. This apoptotic mechanism has
been suggested for <I style="mso-bidi-font-style: normal">Drosophila</I> cell
mechanisms which cause dying germline and follicle cells in <I style="mso-bidi-font-style: normal">Drosophila</I> ovaries </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[38]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Other investigators observed that the percentage of epithelial cells labeled
with digoxigenin using the ISCDDK increased to 70% in 3-day-old larvae when to
the larvae were treated with formic acid </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[39]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
The high cell death levels detected using ISCDDK likely indicated accidental
cell death leading to necrosis, triggered by necrotic injury </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[39]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Further studies should be performed to establish whether higher pesticide
concentrations can decrease apoptosis and increase necrosis in honey bee
larvae and how this may affect clinical symptoms or larvae mortality.
<o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><SPAN style="mso-tab-count: 1">
</SPAN>Our data indicate that Annexin V has a widespread distribution in
pesticide treated larvae being found in the midgut epithelium, salivary glands
and ovaries. Microscopic analyses on cellular localization of Annexin V would
help to obtain information on its function. Intracellular and extracellular
localizations of Annexin V have been reported in human cardiac muscle and vary
based on changes in disease states </SPAN><FONT size=3><SPAN style="FONT-FAMILY: 'Times New Roman'">[40]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
It has also been reported that in the ischemic rat heart, Annexin V leaked
from cardiac cells into the extracellular space and that the cardiac cell
membrane was stained intensely by the anti-Annexin V antibody </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[41]</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
In our study, the varied localization of Annexin V suggests that this protein
is related closely to apoptotic cell death in tissues of bee larvae exposed to
pesticides. These findings may contribute to a better understanding of
potential cell injury during and after pesticide exposure, especially due to
the possibility of false-negatives produced using TUNEL kits </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[28</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">].
</SPAN><SPAN lang=SL style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'; mso-ansi-language: SL; mso-fareast-language: SL">Moreover,
cell death detection and quantification can be more accurate and potential
artifacts can be reduced by using more than one assay </SPAN><SPAN style="FONT-FAMILY: 'Times New Roman'">[25]. </SPAN><SPAN lang=SL style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: SL"><o:p></o:p></SPAN></FONT></P>
<P class=MsoNormal style="MARGIN: 0pt; TEXT-INDENT: 36pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Collectively,
our data indicate that the nine test pesticides can induce apoptosis in
tissues of honey bee larvae reared in an incubator. The data also suggest that
the pesticide doses we tested were tolerable to larvae because apoptosis
likely was initiated as a protective mechanism in the midgut, salivary glands
or ovaries, though further expansion into necrosis, tissue deletion and larval
death is a potential development of these events. Future studies will be
necessary to explore the effects and modes of action of different doses of
these pesticides on larvae at the cellular and tissue levels. The
quantification of cell death could be used to monitor the subclinical and
sub-lethal effects of applied pesticides on larval tissue. Honey bee larvae
reared <I style="mso-bidi-font-style: normal">in vitro</I> could be used in
the future as models for studying the effects of chemicals on living tissues
at the cellular level. <B style="mso-bidi-font-weight: normal"><BR style="PAGE-BREAK-BEFORE: always" clear=all>Acknowledgements
</B><o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt 0pt 10pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">We
would like to thank Jeanette Klopchin and Michelle Kelley (<st1:place w:st="on"><st1:PlaceType w:st="on">University</st1:PlaceType> of
<st1:PlaceName w:st="on">Florida Honey Bee
Research</st1:PlaceName></st1:place> and Extension Laboratory) for their
technical assistance with this project. We also thank Catherine Zettel Nalen
(UF HBREL) for editing an earlier draft of the manuscript and Michael Scharf
(UF Department of Entomology and Nematology) for assistance with pesticide
dosing. We would like to thank <SPAN style="mso-no-proof: yes">Prof. Dr.
Elaine C. M. Silva-Zacarin (</SPAN>Universidade Federal de Sa˜o Carlos
(UFSCar), Campus Sorocaba, Brazil) <SPAN style="mso-no-proof: yes">for reading
the manuscript and useful<SPAN style="mso-spacerun: yes">
</SPAN>suggestions. </SPAN>This work was supported by the National Honey Board
and the Florida Department of Agriculture and Consumer Services through the
work of the Honey Bee Technical Council. <o:p></o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always" clear=all></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">References<o:p></o:p></SPAN></B></P>
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</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Phospholipid
degradation in energy-deprived cardiac myocytes: does annexin V play a role?,
J. Mol. Cell Cardiol. 29 (</SPAN><SPAN lang=NL style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: NL">1997)
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">1401–1410.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; TEXT-ALIGN: justify"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; TEXT-ALIGN: justify; mso-layout-grid-align: none"><FONT size=3><SPAN lang=EN-GB style="FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">[</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">41</SPAN><SPAN lang=EN-GB style="FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">]</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">
N. Kaneko, R. Matsuda, F. Chiwaki, S. Hosoda, </SPAN></FONT><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Purification
of cardiac annexin V from the beagle dog heart and changes in its localization
in the ischemic rat heart, Heart Vessels 9 (</SPAN><SPAN lang=EN-GB style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-ansi-language: EN-GB">1994)
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">148–154.<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; TEXT-ALIGN: justify; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always; mso-special-character: line-break" clear=all></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal>
<TABLE class=MsoNormalTable style="BORDER-RIGHT: medium none; BORDER-TOP: medium none; BORDER-LEFT: medium none; BORDER-BOTTOM: medium none; BORDER-COLLAPSE: collapse; mso-table-layout-alt: fixed; mso-border-alt: solid black .5pt; mso-yfti-tbllook: 480; mso-padding-alt: 0pt 5.4pt 0pt 5.4pt; mso-border-insideh: .5pt solid black; mso-border-insidev: .5pt solid black" cellSpacing=0 cellPadding=0 border=1>
<TBODY>
<TR style="mso-yfti-irow: 0; mso-yfti-firstrow: yes">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 473.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=789 colSpan=5>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><B style="mso-bidi-font-weight: normal"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Table 1.
M</SPAN></B><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">ortality in the
midgut columnar cells determined using two TUNEL kits, DeadEnd and
ISCDDK. Data are mean ± s.e. proportion cells positively stained, N out
of at least 300 cells counted from a minimum of 3 larvae. When both
treatments were analyzed together, neither technique (DeadEnd or ISCDDK
- <I style="mso-bidi-font-style: normal">F</I> = 1.1; df = 1,123; <I style="mso-bidi-font-style: normal">P</I> = 0.29) nor the interaction
between technique and treatment (<I style="mso-bidi-font-style: normal">F</I> = 1.6; df = 10,123; <I style="mso-bidi-font-style: normal">P</I> = 0.13) affected the
proportion of cells positively stained. Data in columns followed by the
same letter are not different at <I>P </I>< 0.05. Students T tests
were used to compare means. <o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 1">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt; mso-border-top-alt: solid black .5pt" vAlign=top width=114>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Type of
pesticide<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt; mso-border-top-alt: solid black .5pt" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Treatment
↓<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Dead End
technique<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">ISDDK
technique<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Both techniques
analyzed together<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 2">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=114 rowSpan=5>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Insecticide<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Chlorpyrifos
<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.56 ± 0.08,
6abc<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.74 ± 0.04,
6a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.65 ± 0.05,
12abc<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 3">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Imidacloprid<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.51 ± 0.12,
6bcd<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.76 ± 0.02,
4a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.61 ± 0.08,
10abc<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 4">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Amitraz*<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.64 ± 0.07,
5abc<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.37 ± 0.03,
4c<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.52 ± 0.06,
9c<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 5">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Fluvalinate*<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.29 ± 0.04,
7d<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.32 ± 0.02,
3c<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.30 ± 0.03,
10d<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 6; mso-prop-change: czettel 20100721T0918">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Coumaphos*<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.58 ± 0.12,
8abc<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.48 ± 0.03,
5bc<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.54 ± 0.07,
13c<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 7">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=114 rowSpan=2>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Fungicide<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Myclobutanil<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.62 ± 0.11,
5abc<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.78 ± 0.06,
4a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.69 ± 0.07,
9ab<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 8">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Chlorothalonil<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.49 ± 0.08,
7cd<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.66 ± 0.06,
4ab<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.55 ± 0.06,
11bc<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 9">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=114 rowSpan=2>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Herbicide<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Glyphosate<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.74 ± 0.05,
5ab<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.65 ± 0.15,
5a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.69 ± 0.08,
10ab<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 10">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Simazine<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.76 ± 0.06,
7a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.77 ± 0.06,
7a<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.77 ± 0.04,
14a<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 11">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=114 rowSpan=2>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Control<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Diet with
acetone<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.10 ± 0.01,
8e<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.11 ± 0.03,
5d<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.11 ± 0.01,
13e<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 12">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Diet
only<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.10 ± 0.02,
8e<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.09 ± 0.02,
5d<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">0.10 ± 0.02,
13e<o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 13">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 68.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=114>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 81pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=135>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">ANOVA→<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">F</SPAN></I><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"> = 10.6; df =
10,71; <I style="mso-bidi-font-style: normal">P</I> <
0.01<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><I style="mso-bidi-font-style: normal"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">F</SPAN></I><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'"> = 16.3; df =
10,51; <I style="mso-bidi-font-style: normal">P</I> <
0.01<o:p></o:p></SPAN></P></TD>
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 108pt; PADDING-TOP: 0pt; BORDER-BOTTOM: black 1pt solid; BACKGROUND-COLOR: transparent; mso-border-bottom-alt: solid black .5pt" vAlign=top width=180>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">Treatment
effect: <I style="mso-bidi-font-style: normal">F</I> = 22.2; df =
10,123; <I style="mso-bidi-font-style: normal">P</I> <
0.01<o:p></o:p></SPAN></P></TD></TR>
<TR style="mso-yfti-irow: 14; mso-yfti-lastrow: yes">
<TD style="BORDER-RIGHT: #d4d0c8; PADDING-RIGHT: 5.4pt; BORDER-TOP: #d4d0c8; PADDING-LEFT: 5.4pt; PADDING-BOTTOM: 0pt; BORDER-LEFT: #d4d0c8; WIDTH: 473.4pt; PADDING-TOP: 0pt; BORDER-BOTTOM: #d4d0c8; BACKGROUND-COLOR: transparent; mso-border-top-alt: solid black .5pt" vAlign=top width=789 colSpan=5>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: normal; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; FONT-FAMILY: 'Times New Roman'">*Used in honey
bee colonies to control varroa
mites<o:p></o:p></SPAN></P></TD></TR></TBODY></TABLE><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always" clear=all></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Figure
1. </SPAN></B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Staining
of formalin-fixed, paraffin-embedded larvae on which the DeadEnd colorimetric
apoptosis detection system (Promega, <st1:place w:st="on"><st1:City w:st="on">Madison</st1:City>, <st1:State w:st="on">WI</st1:State>,
<st1:country-region w:st="on">USA</st1:country-region></st1:place>) was used.
Larvae were 6 d old, 24 h after the last of four consecutive daily pesticide
treatments and prepared for immunohistology. Peroxidase conjugated
anti-digoxigenin secondary antibody and DAB as a substrate were used to obtain
a specific brown reaction product. </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">The
DAB reaction product localized to the nuclei of the columnar midgut epithelial
cells is indicated by black arrows and the DAB reaction product localized to
the regenerative cells is indicated by a black arrow head. When the DAB
reaction product is absent, either the arrow (columnar midgut epithelial
cells) or arrow head (regenerative cells) is white. Panel </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">1A
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">shows
the midgut, of a simazine-treated larva. The DAB reaction product is localized
to the nuclei of the </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">most
of the epithelial columnar</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
and regenerative cells. Panel B shows chlorpyrifos treated larva with the DAB
reaction product localized to the regenerative epithelial cells but not the
columnar ones. Panel C shows an amitraz treated larva where the DAB reaction
product was localized in columnar and regenerative epithelial cells.
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Panel
D shows sections of the midgut of an imidacloprid treated larva with the DAB
reaction product localized in the nuclei of columnar cells</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
but not in</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
regenerative cells. Panel E shows a chlorothalonil treated larva. The DAB
reaction product was found sporadically in the migut columnar epithelial
cells. Panel F shows an untreated larva where only ~10 % DAB positive midgut
epithelial cells were found. Panel G shows an amitraz treated larva where DAB
positive and negative salivary glands cells</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
were seen</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Panel H shows an imidacloprid treated larva with indicative DAB staining in
ovariole nurse cells. Panel I shows a control larva where the DAB reaction was
distributed sporadically in ovariole nurse cells. Panel J shows a control
section of an imidacloprid treated larva where endogenous peroxidase was
quenched successfully and enzyme incubation was omitted. No DAB reaction
product was found. Magnification of all panels: 400</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: txsy">×.</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'"><o:p></o:p></SPAN></P>
<P class=MsoNormal><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always" clear=all></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Figure
2. </SPAN></B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Sections
of formalin-fixed, paraffin-embedded, 6 d old larvae, 24 h after the last of
four consecutive daily pesticide treatments. Cell death was detected using the
TUNEL technique ISCDDK (Roche). TdT-mediated dUTP for DNA labeling was
employed, followed by the application of anti-fluorescein alkaline phosphatase
conjugated antibody, using fast red for visualization, and counterstaining
with haematoxylin. Dense red azo dye staining localized to the nuclei of the
midgut epithelial cells, in the salivary gland cells, or in ovary nurse cells
indicative of impending cell death is </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">indicated
by a black arrow. Reaction product localization to the regenerative cells is
indicated by a black arrow head. Where the reaction product is absent, either
the arrow (midgut epithelial cells) or arrow head (regenerative cells) is
white. </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Panel
A shows a chlorpyrifos-treated larva with red azo dye staining localized to
the midgut columnar epithelial-cell nuclei</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
but not the regenerative epithelial cells. </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Panel
B shows midgut epithelium in a simazine-treated larva. The Red azo-dye
reaction product localized to the columnar and regenerative cells. Panel C
shows a midgut epithelium section of a coumaphos-treated larva. The Red
azo-dye reaction product localized to the columnar</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">and
regenerative</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">epithelial
cells. Panel D shows salivary gland tissue of a myclobutanil-treated larva
where the majority of cells were alkaline phosphatase positive</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">.
Panel E shows </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">untreated,
control larvae with red azo-dye reaction product to sporadic salivary gland
cells. Panel F shows the red azo-dye product sporadically in nurse cells in
ovaries of a simazine-treated larva. Magnification of all panels:
400</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: txsy">×.</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p></o:p></SPAN></P>
<P class=MsoNormal><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA"><BR style="PAGE-BREAK-BEFORE: always" clear=all></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Figure
3. </SPAN></B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-bidi-font-weight: bold">Immunohistochemical
localization of AnnexinV cells of </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">formalin-fixed,
paraffin-embedded 6 d old larvae, 24 h after the last of four consecutive
daily pesticide treatments. <o:p></o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Panel
A shows the red azo-dye reaction product bound to the apical brush border
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow)</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"> in
a chlorpyrifos-treated larva. The reaction product was not localized in the
midgut cells </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(white
arrow head).</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">
Panel B shows cells from a glyphosate-treated larva and indicates Annexin V
staining throughout the remaining midgut epithelium cytoplasm </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow). </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">Panel
C shows a midgut section of a glyphosate-treated larva with red azo-dye
staining indicating PS localized to the basal and apical cell cytoplasm
</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow head)</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Panel D indicates that an alkaline phosphatase reaction product in a
simazine-treated larva was localized to the cytoplasm of the columnar cells at
the basal area </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow head)</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Panel E shows red azo-dye localized in the salivary gland tissue of a
myclobutanil-treated larva (black arrow head). Panel F shows a
glyphosate-treated larva in which red azo-dye was localized to ovarian nurse
cells </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow head)</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
Panel G shows no intensive staining in the salivary gland tissue of a larva
consuming untreated food. Panel H shows cells from an untreated, control larva
with red azo-dye reaction product to some sections of the midgut epithelium
bound to the apical and basal cell membrane </SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: 'Times New Roman'">(black
arrow head)</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.
The cell cytoplasm of the midgut cells was not stained. Magnification of all
panels: 400</SPAN><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'; mso-fareast-font-family: txsy">×</SPAN><B><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'">.<o:p></o:p></SPAN></B></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN style="FONT-SIZE: 12pt; LINE-HEIGHT: 200%; FONT-FAMILY: 'Times New Roman'"><o:p> </o:p></SPAN></P>
<P class=MsoNormal style="MARGIN: 0pt; LINE-HEIGHT: 200%; mso-layout-grid-align: none"><SPAN lang=PT-BR style="mso-ansi-language: PT-BR"><o:p><FONT face=Calibri size=3> </FONT></o:p></SPAN></P>
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