Research Article The Role of AChE in Swimming Behavior of Daphnia magna: Correlation Analysis ...

12 Pages·2017·1.62 MB·English

Hindawi
Journal of Toxicology
Volume 2017, Article ID 3265727, 11 pages
https://doi.org/10.1155/2017/3265727
Research Article
The Role of AChE in Swimming Behavior of
Daphnia magna: Correlation Analysis of Both Parameters
Affected by Deltamethrin and Methomyl Exposure
QingRen,1RuibinZhao,1ChengWang,2ShanggeLi,1TingtingZhang,1ZongmingRen,1
MeiyiYang,1HongweiPan,1ShiguoXu,1JianpingZhu,1andXunWang3
1InstituteofEnvironmentandEcology,ShandongNormalUniversity,Jinan250014,China
2ManagementCollege,OceanUniversityofChina,Qingdao266100,China
3DepartmentofElectricalandComputerEngineering,TandonSchoolofEngineering,NewYorkUniversity,Brooklyn,NY11201,USA
CorrespondenceshouldbeaddressedtoZongmingRen;[email protected];[email protected]
Received 23 February 2017; Revised 25 May 2017; Accepted 13 September 2017; Published 19 October 2017
AcademicEditor:RobertTanguay
Copyright©2017QingRenetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
The unpredictable toxicity of insecticides may cause behavior disorder of biological organisms. In order to assess the role of
acetylcholinesterase(AChE)inswimmingbehaviorofDaphniamagna,acorrelationanalysisofbothparametersin24hexposureof
deltamethrin(DM)andmethomyl(MT)wasinvestigated.ThebehaviorresponsesofD.magnainDM(13.36𝜇g/Land33.40𝜇g/L)
andMT(19.66𝜇g/Land49.15𝜇g/L)suggestedthatrecoverybehaviorintheadjustmentphasewascrucial,andbehaviorhomeostasis
providedthemwithanoptimalwaytoachieveawidertoleranceagainstenvironmentalstress.Duringtheexperiment,positive
effectsonAChEactivityoccurredinthebeginningoftheexposure.EventhoughthedenovosynthesisofAChEinD.magnamight
helpitrecover,theAChEinhibitionindifferenttreatmentscouldbeobserved.SomeinductioneffectsonAChEactivityatthe
beginningofexposureoccurred,anda50%decreasemaycausetoxiceffectsonbehavior.Inmosttreatments,theresultsshowed
thatbothbehaviorstrengthandAChEactivitystayedinthesamefieldwithinacorrelationcircle.Theseresultsillustratedthat
theenvironmentalstresscausedbybothDMandMTcouldinhibitAChEactivityandsubsequentlyinduceastepwisebehavior
response,thoughbothpesticidesaffectitasdirectandindirectinhibitors,respectively.
1.Introduction evenoneenvironmentalfactorchangesandlimitsorganisms,
this can drive others to compensate and strengthen the
Numerous pesticides pose incidental threats to nontarget accommodation in the fluctuating habitat [7]. For instance,
organisms by creating environmental pollution and dam- recognition of burrow’s olfactory signature was an efficient
aging people’s health. Pyrethroids and carbamate pesticides discrimination mechanism when light becomes a limiting
have been used throughout the world to control pests in factorinthedark[8].
agricultural crops, forests, and wetlands [1–3]. However, Theadaptationofindividualsinapollutedaquaticenvi-
the extensive utilization of these insecticides may impair ronmentisimperativetotheirownsurvival.Homeostasisisa
biological communities, inducing an unbalance in aquatic set of processes to achieve and maintain a state of dynamic
ecosystems,andcauseunpredictabletoxicitytohumansand equilibrium. It is a way to maintain internal stability and
numerousotherbiologicalorganisms.Accordingtothelaw appropriate responses to both internal and external stimuli
oftolerance[4–6],oncethequantityorqualityofonefactor [9]. Behavior homeostasis is proposed as a neutral term
exceedsthetoxicitythreshold,thegrowthandreproduction that can be applied across phylogeny from aneural single-
of organisms will be limited. Therefore, these insecticides celledprotozoatocomplexmammalswhendiscussingissues
can exert their toxicity to organisms as limiting factors. If related to stimulus detection and assessment. Behavior is
2 JournalofToxicology
being used in its broadest sense to include any measurable D. magna to environmental stress have been reported [39–
and observable response to an iterative stimulus across 41], and there have been a multitude of researches on the
phylogeny[10].Abehaviorhomeostasismechanismprovides toxic endpoint at different levels, for example, individual
aperfectwayforaquaticindividualstodevelopanabilityto growthandreproductivity[42],embryonicdevelopmentand
tolerateawidervarietyofenvironmentallimitingfactors[11], sex differentiation [42], acetylcholinesterase activity [43],
and this behavior response to environment pollution is an and cellular and molecular level [44]. Lovern et al. [45]
adaptiveprocess:aquaticorganismcanswimfrompolluted have investigated the effects of different chemicals on both
environmenttocleanonebasedontheirinstinct:Avoidance behavior and physiological changes of D. magna, Jensen et
Behavior [12]. Previous research has suggested that aquatic al.[46]haveestablishedacause-effectrelationshipbetween
organisms have the ability to adapt to the aquatic environ- acetylcholinesteraseinhibitionandalteredlocomotorbehav-
mental stress by adjusting the tolerance of their limiting ior in the carabid beetle, Sismeiro-Vivas et al. [47] have
factors, which might induce a stepwise behavior response investigatedtheshort-termeffectsofquirlanonthebehavior
includingacclimation,adjustment,andsoon[13].Behavior andacetylcholinesteraseactivityofGambusiaholbrooki,and
responseofdifferentaquaticorganismsunderenvironmental Tilton et al. [48] have made relationship analysis between
stresshasbeenreportedtobesensitivetosublethalchemical AChEinhibitionandbehaviorinzebrafishexposedtocopper
concentrations,suchascrustaceans[14,15],snails[16],insects orchlorpyrifosseparatelyorasmixtures.However,therehas
[17],andfish[18].Meanwhile,movementchangesaresuitable been hardly any study on the correlation analysis between
indicatorsintheecologicalriskassessment[19]andbehavior the online behavior responses and the continuous AChE
monitoringisreportedasausefulmeansfortoxicitychecking inhibitionlevel.
[20]. Furthermore, there have been many investigations on We have focused our study on the behavior response
the intrinsic mechanisms of the behavior response under ofaquaticorganismsafteracuteexposure,becausebehavior
environmentalstress,forexample,thehormonallevels[21], homeostasis can provide them with a perfect way to have
theacetylcholinesteraseactivityinbrain[22],dysregulation wide tolerance ability against environmental limiting fac-
of the right brain [23], and the modulating covariation in tors. However, there is no direct evidence to clearly show
physiologicaltraits[24]. the intrinsic mechanisms of behavior homeostasis yet. We
Previousresearchsuggestedthatdeltamethrin(DM)and hypothesize that AChE acts as a dominant factor in nerve
methomyl (MT) are two kinds of pesticides with different conduction for swimming behavior within different chem-
toxicmechanisms.DMisasynthetictypeIIpyrethroid[25]. icals that may directly or indirectly inhibit AChE activity
It could inhibit ATPase in synaptosomal membrane, which continuously;thentheaimofthisstudyisto(i)investigate
willcausetheaccumulationofneurotransmitterAChE,and the swimming behavior and AChE inhibition of D. magna
theninducethetoxiceffect.Itisknowntobetoxictodiverse under the stress of two kinds of pesticides with different
aquaticorganismsbecauseoftheeffectsithasonthenervous toxicmechanismsthatactasindirect(DM)anddirect(MT)
system, which is involved in signal transduction and the inhibitorsofAChE[34],(ii)revealtherelationshipbetween
proteomeregulationasregistered[26,27].MTisacommonly AChEinhibitionandbehaviorresponsesbasedoncorrelation
used monomethyl carbamate insecticide to control a wide analysis,and(iii)discusstherolethatAChEplaysinbehavior
range of insects and spider mites through direct contact homeostasis.
and ingestion [28]. MT may also exert its toxic effect on
nontarget organisms, by inducing an oxidative stress that 2.MaterialsandMethods
alters in enzymatic and nonenzymatic antioxidant systems
[29,30]. 2.1.Materials. TheexperimentalD.magna(24hyoung)were
Acetylcholinesterase (AChE) is a key enzyme that culturedinourlaboratoryformorethanthreegenerations.
hydrolyzestheneurotransmitteracetylcholineincholinergic Theculturewasmaintainedat20±2∘Cundera16hlight:8h
synapses of both vertebrates and invertebrates; this may dark photoperiod (illumination ranged between 3000 and
affect a nerve’s ability to conduct due to the accumulation 4500lx). Culture medium was prepared according to the
of acetylcholine in the body once AChE is inhibited [31– componentsoftheStandardReferenceWater(SRW)[49]and
33]. Behavior movement of organisms is related directly D. magna were fed Scenedesmus obliquus two times a day.
to nerve conduction [34]. Swimming behavior of aquatic Before feeding D. magna, the culture medium of the algae
organismswouldbenefitpredation,antipredation,andavoid- wasfilteredandthendilutedbySRWuntiltheconcentration
ance abilities, which could increase the survival chance of reached1×105cells/ml.Thequantityofthealgaewasapprox-
theseorganismsintheiraquaticenvironment.Therefore,the imately1%beakervolume.Beforetheexposureexperiments,
inhibition of AChE would decrease the ability of behavior the gravid female D. magna already carrying eggs were
homeostasis[35],potentiallyinducingahighersurvivalrisk. removed and cultured individually in 50ml glass beakers
Daphnia magna, a small planktonic invertebrate crus- of SRW until they oviposited. The healthy and uninjured
tacean(0.5–5.0mm)withashortlifecycle,isverysensitive neonateswerethentakenandusedforthisexperiment.
to environmental changes [36, 37]. D. magna is a stan- MT and DM were purchased from J&K Chemical Ltd
dard organism for toxicity tests, and the species has often (Beijing). All compounds were of technical grade (95%
∘
been used in bioassays and environmental monitoring of purity).Stocksolutions(storedat4 Cuntiluse),eachhaving
aquatic systems due to the ease and the low economical aproperconcentration,werepreparedindimethylsulfoxide
cost of maintaining cultures [38]. Behavior responses of to make each test solution. The concentration of dimethyl
JournalofToxicology 3
sulfoxideinwaterwaslessthan0.5%inalltheexperiments. Oncetherewerelessthan10livetestindividuals,theAChE
Studies showed that dimethyl sulfoxide of such concentra- experiment stopped. The death of D. magna was defined as
tion would neither lead to acute toxicity to D. magna nor theinabilitytoswimformorethanafewstrokesfor15safter
affect the mobility of D. magna [50, 51]. Acetylthiocholine gentleagitationofthetestvessel[57].
iodide (ATCh), 5,5-dithio-2,2-nitrobenzoic acid (DTNB), 81ml 0.1M disodium hydrogen phosphate and 19ml
SephadexG-25,bovineserumalbumin(BSA),andTritonX- 0.1M sodium dihydrogen phosphate were mixed and then
100werepurchasedfromSigma(Sigma-AldrichCorporation, diluted with deionized water to 100ml to get phosphate
St.Louis,MO,USA).Allofthesechemicalswereofanalytical
buffer (0.1M, pH 7.4). Homogenates were prepared in an
grade(95%purity).
ice-cold phosphate buffer using mechanically driven Teflon
fittedPotter-Elvehjemhomogenizerfor2minsat3000r.p.m.
2.2.ExperimentalSetup. Wesetthelogarithmspacingcon-
in ice till total disruption of water fleas. The homogenates
centration of the insecticide in the exposure experiment to were then centrifuged at 12,000×g for 20min at 4∘C [58].
measure LC50-24h (50% lethal concentration) of juveniles Thesupernatantwasusedasanenzymesourceformeasuring
(48hyoung).Accordingtothepreviousreport,LC50-24hof
DMonD.magnarangedfrom0.113𝜇g/Lto9.4𝜇g/L[42,52– theactivityofAChE.AChEactivityinthehomogenateswas
54], and MT on D. magna was from 7.3𝜇g/L to 20.0𝜇g/L detected as follows: 50𝜇L enzyme and 50𝜇L ATCh (5mM
∘
[55], so the concentrations of DM (0.113𝜇g/L, 0.341𝜇g/L, final concentration) were incubated at 30 C for 15min in a
1.031𝜇g/L, 3.113𝜇g/L, and 9.400𝜇g/L) and MT (7.30𝜇g/L, final volume of 0.1mL, and then the reaction was stopped
9.39𝜇g/L,12.08𝜇g/L,15.55𝜇g/L,and20.0𝜇g/L)wereusedto by0.125mMDTNB-phosphate-ethanolreagentinside0.9ml
conduct the acute toxicity tests. During the experiment, D. (12.4mg of DTNB dissolved in 125mL 95% ethanol, 75mL
magnawerefednothingandwereplacedinthreereplicates distilled water, and 50mL 0.1M phosphate buffer, pH 7.5)
per concentration with 20 young fleas per treatment. This as the thiol indicator. The color was detected immediately
experimentwasmadeinthesameconditionofculture. at 412nm using an ELIASA (Infinite M200) [59]. Based on
The swimming behavior of D. magna was monitored the Bradford Protein Assay of the protein concentration of
by an online system built in the Research Center for Eco- enzymatic extracts [60], the AChE activity was detected,
Environmental Science, Chinese Academy of Sciences [56]. whichwasinunitofnmol/min⋅mg.Thewhole-bodyAChE
Six test D. magna were placed in each flow-through test activity (% of control) was used to analyze the effects of
chamber (3cm long, 2cm in diameter), which was closed differentchemicalsontheAChEactivity.
offonbothsideswithnylonnets(250𝜇m),and3replicates
perconcentrationwereused.Apairofelectrodeslocatedon 2.3. Data Analysis. The 50% lethal concentration (LC50)
thewallsofthetestchamberssentahigh-frequencysignalof values were calculated by probit analysis in the MATLAB
alternatingcurrent,whichwasthenreceivedbyasecondpair 2009 (© 1984–2009 The MathWorks, Inc.). In order to
ofnoncurrent-carryingelectrodes.Thebehaviorstrengthof assess the role of AChE in swimming behavior of Daphnia
allD.magnaineachtestchamber,whichwasusedtoshow magna in different treatments, the average values of three
swimmingintensity,wastransformedbyanA/Dtransformer replicates of both the behavior data and whole-body AChE
and the signal changes formed by the A/D transformer activity (% of control) were calculated and then analyzed
were analyzed automatically by software installed on the based on the linear fitting in MATLAB. The Kalman fil-
equipment.Thebehaviorstrengthwassampledautomatically tering with linear regression analysis was used in the con-
bythesystemeverysecond,andtheaveragebehaviorstrength tinuous changes over the 24-hour period of both whole-
data were taken twice an hour in the first two hours and body AChE activity (% of control) and behavior strength
after that once every hour to analyze the trends found in of D. magna in different treatments with 95% confidence
differenttreatments.Behaviorstrengththatchangedfrom0 bounds to eliminate the influence of the environmental
(losingtheabilityofmovement)to1(fullbehaviorexpress) noise (Fileds et al., 1991). Linear regression equations of
wasintroducedtoillustratethebehaviorresponsedifferences AChE activity (% of control) and behavior strength could
ofD.magna[13].Exposureconcentrationsweremadebased becalculatedwithdifferentconstantsindifferenttreatments,
ontheresultsofacutetoxicity(LC50-24h),and2×LC50and which might be applied in the toxic effect analysis. After
5×LC50treatmentswerechosen. Kalman filtering with linear regression with 𝑝 < 0.05,
Invivo,AChEinhibitiontestswereperformedbyexpos- PrincipalComponentAnalysis(PCA)wassubsequentlyused
ingjuvenilestoseveraltreatments(0,2×LC50and5×LC50) toillustratethecorrelationbetweentheinhibitiondegreeof
in 5000ml beaker. In the analysis of AChE activity, a 16h AChEandthebehaviorresponsesofD.magna.Thelearning
light:8h dark photoperiod was applied for 24h exposure process of PCA was conducted using the Self-Organizing
∘
at 21–23 C. We applied three replicates in this experiment Map toolbox developed by the Laboratory of Information
with500individualsin2×LC50 and5×LC50 exposureand andComputerScience,HelsinkiUniversityofTechnologyin
270 individuals in control. No food was added during the MATLAB environments [61]. One-way analysis of variance
experiments. Samples (10 from each treatment) were taken (ANOVA)wasperformedtocomparethewhole-bodyAChE
once an hour in the first 4h and after that once every 2h activity (% of control) and behavior strength in different
in treatments. Once the individual number of D. magna behavior stages with different significance (𝑝 < 0.01 and
that sunk to the bottom of the beaker (not dead) is higher 𝑝 < 0.05)basedontheStepwiseBehaviorResponseModel
than10,AChEactivityoftheseindividualswasalsodetected. [13].
4 JournalofToxicology
Table1:The24hacutetoxiceffectsofbothDMandMTonD.magna.
Chemicals LC50(𝜇g/L) 95%confidenceinterval(𝜇g/L) Equationoflinearregression 𝑟
DM 6.68 6.441–7.026 𝑌=2.026𝑋+15.485 0.992
MT 9.83 9.148–10.184 𝑌=3.411𝑋+22.081 0.984
3.ResultsandDiscussion from8thto16thh,behaviorstrengthfurtherdecreasedto0
(redshadows).Under19.66𝜇g/LMTexposure(Figure1(c)),
3.1.AcuteToxicity. Accordingtothechemicaltoxicitytothe behaviorstrengthshowedaslightdecline,andthebehavior
aquaticorganism[55],DMandMThadhightoxicityto D. adjustmentafter3hwasnotasstrongasinthe13.36𝜇g/LDM
magna.TheacutetoxiceffectsofDMandMTonD.magna exposure (yellow shadows). Under 49.15𝜇g/L MT exposure
are shown in Table 1 after probit analysis in MATLAB with (Figure1(d)),thebehaviorresponsetrendsweresimilartothe
95% confidence interval. The measured LC50-24h values of exposureof19.66𝜇g/LMT,butontheotherhandbehavior
DM and MT on D. magna were 6.68𝜇g/L and 9.83𝜇g/L. strengthwasmuchlower.
Accordingtothepreviousreport,24hacuteecotoxicitydata Some difference could be observed in different treat-
(asLC50)rangedfrom0.113𝜇g/Lto9.4𝜇g/L[42,52–54],and ments:First,thenumberofadjustmentsinalowerconcentra-
MTonD.magnawasfrom7.3𝜇g/Lto20.0𝜇g/L[55].These tionexposurewasmorethaninahigherconcentrationexpo-
resultssuggestedthatthemeasuredLC50-24hvaluesinthis sure,andsometimestherecoverybehaviorstrengthinfollow-
studywereacceptable. ingadjustmentsmightbemuchgreaterthanpreviousones,
for example, 21h later in 13.36𝜇g/L DM exposure. Second,
3.2. The Toxic Effects of DM and MT on Both Swimming the behavior adjustment in DM was more evident than in
Behavior and AChE Activity. The swimming behavior and MTexposure,specificallyinalowerconcentrationexposure;
the AChE activity of D. magna under the exposure to thismaybepartlyduetothedifferenceintoxicmechanisms.
both DM and MT is shown in Figure 1. Different color ThetoxiceffectsofDMonorganismsbycombiningwiththe
+
shadows mean the different stages based on the Stepwise Na channelonthecellmembranemadethenumberofopen
+
BehaviorResponse Model: no effectsandstimulationstand Na channelsincrease[63].ForMT,inhibitingtheactivityof
for the exposure period before the first significant decrease AChEmadetheAChElosesitsnormalphysiologicalactivity
ofbehaviorstrength(SD-BS)occurred(grayshadows)(20%, which leads to the disorder of ACh metabolism and blocks
[13]), acclimation is the period after the first SD-BS until the normal conduction of the nervous system [64]. These
the first adjustment (20% behavior strength increase) (blue results suggest that behavior homeostasis did exist for D.
shadows),(re)adjustmentisfromthefirstadjustmenttotoxic magnaunderenvironmentalstress.Meanwhile,theseresults
effects(yellowshadows),andtoxiceffectstartsfromthetime are consistent with previous results, which advised evident
when behavior strength of D. magna is lower than 0.2 and stepwisebehaviorresponsesincludingnoeffect,stimulation,
thereisnorecovery(redshadows). acclimation,adjustment(readjustment),andtoxiceffect[13].
The average behavior strength in control changed from For example, in 33.40𝜇g/L DM treatment, the acclimation
0.87to0.72andtheaverageAChEactivitywasaround100% phaseoccurredin3h,adjustmentstartedin8h,andthetoxic
(from 88% to 105%) (Figure 1(e)). As it is reported by Ren effect phase occurred in 16h. In 49.15𝜇g/L MT treatment,
etal.[62],thebehaviorresponsesofD.magna,whichhavea acclimationbeginsat1h,adjustmentphasebeginsat18h,and
fluctuationof36%,aremoreintensivethantheAChEactivity at21hthetoxiceffectoccurred.
(lessthan20%fluctuation)undertheDDVPexposure.These Similartotheresultsfoundinswimmingbehavior,AChE
suggest that both the swimming behavior and the AChE activity (% of control) of D. magna in both DM and MT
activity of D. magna exposed to DM and MT are relatively exposureshowedevidentinhibition.InthetreatmentsofDM
stable in control group based on the data from each time (Figures 1(a) and 1(b)), the AChE inhibition trends in both
point. 13.36𝜇g/L and 33.40𝜇g/L were similar. At the beginning of
However, the swimming behavior in other treatments the exposure, AChE activity increased to more than 105%;
wasinhibited.Under13.36𝜇g/LDMexposure(Figure1(a)), then the inhibition occurred with some recovery. AChE
behavior strength showed fluctuating changes. In the first activity was almost as its lowest type at the end of each
6h, it maintained approximately 0.8, and from the 6th to treatment;obviouslyindeadindividuals,itwasmuchlower
the 9th hour, a drastic decrease occurred from 0.8 to less than live ones under the same exposure time. However,
than 0.3. There were more than 3 behavior adjustments there were some differences: (i) 50% inhibition of AChE
9h later, which happened from 11th to 15th, 16th to 19th, activity occurred about 4h earlier in 33.40𝜇g/L than in
19th to 24th hour, separately according to the adjustment 13.36𝜇g/L DM; (ii) the ability of AChE activity recovery
criteriainourpreviousresearch(yellowshadows)[13].Under was much higher in lower concentration treatments. In the
33.40𝜇g/L DM exposure (Figure 1(b)), behavior strength treatments of MT (Figures 1(c) and 1(d)), AChE inhibition
showedasuddendecreaseafter2h,anditreachedlessthan differed in 19.66𝜇g/L compared with that in 49.15𝜇g/L. In
0.4inthe7thh(blueshadows).After2behavioradjustments lower concentration treatments, AChE activity was slowly
JournalofToxicology 5
1 150 1 150
0.8 120 0.8 120
%) %)
0.6 90 vity ( 0.6 90 vity (
BS 0.4 60 E acti BS 0.4 60 E acti
h h
C C
0.2 30 A 0.2 30 A
0 0 0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
BS BS
AChE activity (%) AChE activity (%)
(a) (b)
1 150 1 150
0.8 120 0.8 120
%) %)
0.6 90 vity ( 0.6 90 vity (
BS 0.4 60 E acti BS 0.4 60 E acti
h h
C C
0.2 30 A 0.2 30 A
0 0 0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
BS BS
AChE activity (%) AChE activity (%)
(c) (d)
1 150
0.8 120
%)
0.6 90 y (
BS 0.4 60 activit
E
h
0.2 30 C
A
0 0
0 4 8 12 16 20 24
BS
AChE activity (%)
(e)
Figure1:TheswimmingbehaviorandtheAChEactivity(%ofcontrol)inhibitionofD.magnaafterinvivoexposureinbothDMandMT.
M±SDwasapplied.(a,b,c,d,ande)ShowingthebehaviorresponsesandAChEactivityofD.magnain13.36𝜇g/LDM,33.40𝜇g/LDM,
19.66𝜇g/LMT,49.15𝜇g/LMT,andcontrol,separately.AChEactivityofD.magnaincontrolatthebeginningoftheexperimentsisregarded
as100%.ThesolidlinewithrounddotsrepresentedthebehaviorresponsesofD.magna.ThesolidlinewithtrianglesrepresentedtheAChE
activityofliveD.magnaandthedottedlinewithtrianglerepresentedtheAChEactivityofdeadD.magnaoncethereweresomeindividuals
thatdiedduringtheexposureperiods.Grayshadowsmeannoeffectsandstimulation,blueshadowsmeantheacclimation,yellowshadows
meanthe(re)adjustment,andredshadowsmeanthetoxiceffect.
decreasingwitharecoveryatabout18h,andthelevelswere deviationinFigure1showedthevibrationamplitudeofboth
constant at above 50% during the 24-hour period. AChE behaviorstrengthandtheAChEactivity,anditsuggestedthat
activity was comparatively lower than 50% after 6h in a the vibration amplitude was different in different behavior
higher concentration with several recoveries. The standard responsesaccordingtothecriteriafordifferentstagesbased
6 JournalofToxicology
on the Stepwise Behavior Response Model (Table 3): it is 1
biggerinbothacclimationandadjustmentthaninnoeffects-
0.8
stimulationandtoxiceffects.
After the first period (approximately hour 2 to hour 6), 0.6
there were effects on AChE activity in different treatments; 49.15-MT-AChE
significantinhibitionwith𝑝 < 0.05occurredinallthetreat- 0.4 19.66-MT-AChE
49.15-MT-BS
mentswithseveralrecoveries.Meanwhile,AChEactivityin
0.2 19.66-MT-BS
DMwaseasilyinhibited,whichsuggeststhatsomedifference
in the toxic mechanisms might induce these results. It is
0
clear that pyrethroid insecticides possess inhibitory effects
onAChEactivityofD.magnaandcarbamates,whichcould −0.2 33.40-DM-AChE 33.40-DM-BS
exert their toxic effect by inducing oxidative stress with
−0.4
an alteration in enzymatic and nonenzymatic antioxidant
13.36-DM-AChE
systems[27,29].
−0.6
TheseresultssuggestthatAChEactivitymightincreaseat 13.36-DM-BS
thebeginningoftheexposure,anditwasobviouslylowerin −0.8
deadindividualsthanintheliveindividuals.AChEactivity
inhibitionshowedthathigherrecoverydegreehappenedin −1
−1 −0.8 −0.6 −0.4 −0.2 0 0.2 0.4 0.6 0.8 1
higherconcentrationtreatments.Thoughcontinuousdetec-
tionofAChEactivityoftestorganismsindifferenttreatments
Figure2:ThecorrelationanalysisofswimmingbehaviorandAChE
did not attract much attention in previous research, AChE activityofD.magnabasedonPrincipalComponentAnalysis.
activityofD.magnawassimilartothepreviousresults[35].It
showedpositiveeffectsonAChEactivityatthebeginningof
theDMexposureandsomeevidentrecoveryofAChEactivity
occurred during the exposure in different treatments. The
reasonsforthepositiveeffectsandtheactivityrecoverymight
analysis of variance for the modeling of response data, and
bethattheseinsecticidesstimulatedthedenovosynthesisof
theresultsoftheanalysisdependonthescalingofthematrix
AChEinD.magna[65];theseeffectsweresimilarlyobserved
[68].Basedontheanalysisoftheobservedresultsasshown
inthreeridgemussels(A.plicata)[66].
inFigure1,PrincipalComponentAnalysisinSelf-Organizing
Map was applied to construct a circle correlation analysis
3.3. Correlation Analysis of Both Swimming Behavior and
of the relationship between behavior responses and AChE
AChE Activity. The swimming behavior of D. magna in
activity(Figure2).
differentinsecticidestreatmentsshowedthatthefluctuating
environmental stress would indeed induce evident behav- Thecorrelationcircleresultsindicatethatthechangesin
ior responses, including no effect, stimulation, acclimation, behavior strength show a positive relationship with AChE
adjustment (readjustment), and toxic effect. The behavior activityineachtreatment.Inmosttreatments,bothbehavior
homeostasisdependsontheadjustmentsmadeunderenvi- strength and corresponding AChE activity inhibition of D.
ronmentalstress;thisprovidesaperfectmethodforindividu- magnastayedwithinthesamefieldofthecorrelationcircle.
alstodevelopwidertoleranceabilitytoenvironmentalstress. For example, the results of both parameters in 19.66𝜇g/L
Itisshownthatphysiologicalresponsesmightbeimportant and 49.15𝜇g/L MT and 33.40𝜇g/L DM exposure were in
forindividualstoovercomethesestresses[43].Someresults the upper-right, upper-left, and lower-left corner, respec-
of the relationship between behavior responses and AChE tively.Thoughbehaviorstrengthin13.36𝜇g/LDMexposure
activityindifferenttreatmentscouldbeobservedinFigure1: stayedinthelower-rightcornerofthecircle,AChEactivity
First, the tendency of both behavior strength and AChE inhibition was in lower-left area. The reason for this might
activity decreased, but there were some differences in each be due to the fact that behavior homeostasis (behavior
treatment.Second,thevaluesofbothbehaviorstrengthand adjustment) in this treatment was stronger than in others.
AChEactivitywerehigherinlowerconcentrations(Figures The results of both behavior strength and AChE activity in
1(a) and 1(c)) than in higher concentrations (Figures 1(b) different treatments (Figure 1) indicated that the behavior
and 1(d)) at the end of the exposure. All of these results responses were similar to each other, which included no
suggest that the behavior response of D. magna is similar effect, stimulation, acclimation, adjustment (readjustment),
totheinhibitionofAChEactivityinthosetreatments.50% andtoxiceffect[13].AsreportedbyXuerebetal.[34],theloss
inhibitionofAChEactivitywouldinduceanevidentdecrease ofthenerveconductionabilitycorrelatedwithAChEactivity
in behavior strength due toxic effects, especially in higher inhibitionplayedasignificantroleintheprocess.
concentrationtreatments. Table 2 shows the Kalman filtering linear regression
Principal Component Analysis is a statistical procedure analysis of both whole-body AChE activity (% of control)
thatusesorthogonaltransformationtoconvertasetofobser- and behavior strength of D. magna in different treatments
vationsofpossiblycorrelatedvariablesintoasetofvaluesof with 95% confidence bounds. The results showed higher
linearly uncorrelated variables called principal components concentration treatments (33.40𝜇g/L DM and 49.15𝜇g/L
[67]. Principal Component Analysis is more suitable than MT)couldinduceahigherslope,whichwasmorethan0.03
JournalofToxicology 7
Table2:Linearregressionanalysisofbothwhole-bodyAChEactivity(%ofcontrol)andbehaviorstrengthofD.magnaindifferenttreatments
with95%confidencebounds.
BS AChE
Treatments Linearregression Slope 𝑦-intercept Linearregression slope 𝑦-intercept
equation equation
13.36𝜇g/LDM 𝑌=−0.016𝑋+0.722 (−0.027,−0.006) (0.581,0.863) 𝑌=−0.034𝑋+1.082 (−0.044,−0.023) (0.934,1.229)
33.40𝜇g/LDM 𝑌=−0.030𝑋+0.732 (−0.035,−0.026) (0.665,0.799) 𝑌=−0.031𝑋+0.862 (−0.040,−0.023) (0.748,0.976)
19.66𝜇g/LMT 𝑌=−0.018𝑋+0.766 (−0.021,−0.014) (0.718,0.813) 𝑌=−0.011𝑋+0.915 (−0.017,−0.005) (0.833,0.997)
49.15𝜇g/LMT 𝑌=−0.039𝑋+0.776 (−0.043,−0.034) (0.715,0.838) 𝑌=−0.049𝑋+1.155 (−0.058,−0.040) (1.032,1.277)
Theslopevaluesandthe𝑦-interceptvaluesareshownwith95%confidencebounds.
intheequationsforbothparameters.Inlowerconcentration resultsclearlyshowedthattheenvironmentalstresscausedby
treatments, the slope in the equations was less than 0.02, bothDMandMTinhibitsAChEactivityandtheninducesa
except for the 13.36𝜇g/L DM treatment, in which the slope stepwisebehaviorresponse,thoughthetoxiceffectsofthese
in the equation was more than 0.03; this might induce the insecticides act as indirect and direct inhibitors of AChE
correlationanalysisresultsinFigure2.The𝑦-interceptinthe separately [34]. Therefore, it can be ascertained that AChE
linearregressionequationsofbothparametersdidnotshow activityinhibitionispartoftheintrinsicresponsemechanism
evidentdifferenceamongdifferenttreatments.Theseresults ofbehaviorhomeostasisbasedonbehaviorstrength.
suggested that both circle correlation and linear regression
analysis are not accurate enough to support the positive 4.Conclusion
relationshipbetweenbothparameters.
Toillustratetheeffectsmoreclearly,bothparameterswere This study demonstrates the role of AChE in the behavior
compared after one-way analysis of variance as shown in homeostasis of D. magna under both DM and MT stress.
Table 3. The criteria of different swimming behavior were Differenceswereregisteredindifferenttreatmentsbasedon
based on the stepwise behavior responses [13]. The results behaviorstrengthandAChEactivity.Suchresultsconfirmed
advisedthatallswimmingbehaviorwasregardedasnormal that AChE could be used as one of the biomarkers for
(no effects) in control and AChE activity data of D. magna both indirect and direct inhibitors, and a clear stepwise
keptapproximately97%with0.79behaviorstrength.During behavior response could be induced by these inhibitors.
no effects and stimulation, the behavior strength in all of Meanwhile,50%AChEinhibitionmaycausethetoxiceffect
the treatments kept constant, which was 0.79. There was of swimming behavior in a fluctuating environment, which
somedifferenceinAChEactivity,whichshowedasignificant clarifiesthatAChEactivityinhibitionispartoftheintrinsic
difference with 𝑝 < 0.05 in both higher concentration response mechanism of behavior homeostasis based on
treatments(33.40𝜇g/LDMand49.15𝜇g/LMT);furthermore, behaviorstrength.Nevertheless,currentdataofbothswim-
it was higher in 33.40𝜇g/L DM than in control but lower mingbehaviorandAChEactivityofonekindofindividuals
in 49.15𝜇g/L MT than in control. The increase of AChE cannot serve as direct and thorough evidence for the key
activityin33.40𝜇g/LDMsuggestsaninductioneffectinthe roleofAChEinthebehaviorhomeostasis.Thelinkbetween
first exposure period, which was also observed by Xuereb AChEinhibitionandacutebehaviorhomeostasisofdifferent
et al. [34, 43]. Comparing these to the control results, both chemicalstodifferentorganismshasyettobeanalyzedand
thetwoparametersintheotherbehaviorresponsesshowed willbeestablishedinfuturestudies.
significantdifference(𝑝 < 0.01),whichrevealedanextreme
behaviordecreaseandAChEactivityinhibition.Theresultsin ConflictsofInterest
Table3alsoillustratethatthebehaviorhomeostasisinlower
concentration treatments (13.36𝜇g/L DM and 19.66𝜇g/L Allauthorsdeclaredthattheyhavenoproprietary,financial,
MT)dependedmainlyonadjustment,whichstartedfromthe professional,orotherpersonalinterestofanynatureorkind
11thhourofexposurewherenotoxiceffectsoccurred. inanyproductscitedinthepaper.
Previous research on the relationship between AChE
activity levels and behavior homeostasis has illustrated that Authors’Contributions
AChEactivityinhibitionresultedinunregulatednerveend-
ing activation and paralysis in organisms and could induce QingRen,RuibinZhao,andChengWangcontributedequally
abnormalbehaviorresponses[31].Ourobservationsshowed tothismanuscript.
that a 50% activity decrease of AChE may cause the toxic
effectofswimmingbehavior,andfluctuatingenvironmental Acknowledgments
stress may cause an induction effect of AChE activity at
times. Though the de novo synthesis of AChE in D. magna ThisstudywasfinanciallysupportedbytheNationalNatural
might help AChE activity recover, the trends during the ScienceFoundationofChina(21107135)andtheJinanHigh-
24hexposureindifferenttreatmentsweredownward.These LevelTalentPlan(2013041).Theauthorswouldliketothank
8 JournalofToxicology
∗∗ ∗∗ BS)wer
Table3:Statisticalanalysisofwhole-bodyAChEactivity(%ofcontrol)andbehaviorstrengthindifferentbehaviorresponses. #1#2#3#4Acclimation(Re)adjustmentToxiceffectNoeffectsandstimulationTreatmentsStarttimeAChEBehaviorStarttimeAChEBehaviorStarttimeAChEBehaviorStarttimeAChEBehavior(h)activitystrength(h)activitystrength(h)activitystrength(h)activitystrength∗∗∗∗∗∗∗∗𝜇95±120.79±0.0382±90.46±0.2211.049±280.42±0.1913.3g/L-DM0.06.0///∗∗∗∗∗∗∗∗∗𝜇102±20.79±0.0295±50.58±0.148.063±270.31±0.1418±170.03±0.0333.40g/L-DM0.03.016.0∗∗∗∗∗∗∗∗𝜇94±40.78±0.0378±60.57±0.0911.071±70.46±0.0719.66g/L-MT0.05.0///∗∗∗∗∗∗∗∗∗∗∗𝜇92±110.79±0.0158±160.44±0.1918.034±170.25±0.1620±30.27±0.1849.15g/L-MT0.01.021.097±30.79±0.04Control0.0/////////∗∗∗#1𝑝𝑝criteriaexposure<0.05<0.01ComparedwithcontrolfordifferentstagesbasedontheStepwiseBehaviorResponseModel:periodbeforethefirstsignificantdecreaseofbehaviorstrength(SD-and;234%start%afterthefirstSD-BSuntilthefirstadjustment(20behaviorstrengthincrease);fromthefirstadjustmenttotoxiceffects;ingfromthetimewhenbehaviorstrengthofD.magnaislooccurred(20[13]);than0.2andthereisnorecovery.AChEactivitydataofimmovableindividualsindifferenttreatmentswerenotshowninthetable.
JournalofToxicology 9
Ryan C. Wallis from Whitman College and Tae-soo Chon [16] M.R.S.Coffin,D.Drolet,D.J.Hamilton,andM.A.Barbeau,
fromPusanNationalUniversityforpolishingtheEnglishof “Effectofimmersionatlowtideondistributionandmovement
themanuscript. ofthemudsnail,Ilyanassaobsoleta(Say),intheupperBayof
Fundy,easternCanada,”JournalofExperimentalMarineBiology
andEcology,vol.364,no.2,pp.110–115,2008.
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