daf-7 and the development of strongyloides ratti and parastrongyloides trichosuri

Molecular & Biochemical Parasitology 139 (2005) 213–223

daf-7and the development ofStrongyloides rattiandParastrongyloides trichosuri�

Matt Crooka, Fiona J. Thompsona, Warwick N. Grantb, Mark E. Vineya,∗a School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UKb AgResearch, Wallaceville Animal Research Centre, Ward Street, Upper Hutt, New Zealand

Received 13 July 2004; received in revised form 15 November 2004; accepted 23 November 2004Available online 16 December 2004

Abstract

daf-7 is a key ligand in one of the three pathways that control dauer larva development inCaenorhabditis elegans. Given the similaritiesbetween dauer larvae of free-living nematodes and third stage infective larvae of animal parasitic nematodes, we hypothesised thatdaf-7may be involved in the development of these infective larvae. To investigate this, we cloneddaf-7orthologues fromStrongyloides rattiandPi edip tiono©

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arastrongyloides trichosuriand analysed their RNA level by semi-quantitative RT-PCR during theS. rattiandP. trichosuri life cycles andn a range of in vitro and in vivo conditions. We found that, in both species, the RNA level ofdaf-7was low in free-living stages but peakn the infective L3 (iL3) stage with little or no expression in the parasitic stages. This contrasts with thedaf-7RNA level inC. elegans, whicheaks in L1, decreases thereafter, and is absent in dauer larvae. The RNA level ofdaf-7 in infective larvae was reduced by larval penetraf host skin or development in the host, but not by a shift to the body temperature of the host.2004 Elsevier B.V. All rights reserved.

eywords: daf-7; C. elegans; Development; Parasitic nematode; Infection; RT-PCR

. Introduction

Strongyloides rattiandParastrongyloides trichosuriarearasitic nematodes of rats (Rattus norvegicus) and brush-

ailed possums (Trichosurus vulpecula), respectively. Thearasitic stages are female (S. ratti) and female and maleP. trichosuri). For both species, eggs and larvae are passed

Abbreviations: iL3, infective third stage larvae; p.i., post infection; RT-CR, reverse transcriptase PCR; P female/male, parasitic female/male; TGF-, transforming growth factor-beta; GMP, guanosine 5′-monophosphate;ST, expressed sequence tag; RACE, rapid amplification of cDNA ends;NOVA, analysis of variance; BCS, bovine calf serum; DMEM, Dulbecco’sodified Eagles medium; RNAi, RNA interference; BLAST, basic locallignment search tool� Note: DNA sequences reported in this paper have been deposited in theenBank database with accession numbersSr-daf-7(AY672707),Sr-act-(AY672708),Sr-plk-2 (AY672706),Pt-act-3 (AY672703) andPt-asp-1

AY672704).∗ Corresponding author. Tel.: +44 117 928 7469; fax: +44 117 925 7374.E-mail address:[email protected] (M.E. Viney).

in the faeces and develop into iL3s or free-living adult mand females. ForS. ratti, these free-living adults mate aproduce eggs, which develop into iL3s (Fig. 1(a)) [1]. ForP.trichosuri, these free-living adult males and females cango through a number of free-living adult cycles, after wha proportion of the progeny develop into iL3s (Fig. 1(b))[2]. In both species, iL3s persist in an arrested state, caof infection, for up to 2 months and only resume devement on entering a host, after which they migrate to the sintestine and develop via a fourth larval stage into paraadults. These iL3s are thought to be analogous to dauer lof free-living nematodes, includingCaenorhabditis elegan,as both are arrested, long lived, resistant stages that onsume development under appropriate conditions; enterhost for parasitic nematodes and low population densityabundant food forC. elegansdauer larvae[3–5].

The control of dauer formation has been extensivelyvestigated inC. elegans[6]. Dauer larvae form under condtions of high population density (sensed via a constituti

166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
oi:10.1016/j.molbiopara.2004.11.010

214 M. Crook et al. / Molecular & Biochemical Parasitology 139 (2005) 213–223

Fig. 1. (a) Life cycle ofS. rattiwith two discrete developmental switches:(1) a sex-ratio switch; (2) a female-only developmental switch[1] and (b)life cycle ofP. trichosuri. Larval stages are omitted for clarity.

secreted product)[7,8], low food abundance and high tem-perature[3], i.e. conditions that are not conducive to repro-ductive development. Extensive genetic analyses of the con-trol of dauer larva formation has shown that three pathways,containing TGF-�-like [9], insulin-like[10] and guanylyl cy-clase genes[11] together coordinate this developmental de-cision. There is some evidence that these pathways inter-act, since members of the guanylyl cyclase pathway can actboth upstream, and in parallel to, the TGF-�-like pathway[12]. Given the similarities between iL3s of parasitic nema-todes and dauer larvae we sought to determine if the mecha-nisms that control their development are the same. Membersof these pathways have been found in parasitic nematodes[13]. These includeBm-tgh-2, a tgf-β-like gene fromBrugiamalayi[14]; putativegpa-2andgpa-3homologues, possible

members of the guanylyl cyclase pathway[15] andfktf-1, aproposed homologue ofdaf-16, a fork-head transcription fac-tor in the insulin-like pathway fromStrongyloides stercoralis[16]. Activation ofAncylostoma caninumiL3s by 8-bromo-cyclic GMP also demonstrates the involvement of the guany-lyl cyclase pathway in resumption of iL3 development[17].Comparison of the control of development of larvae of para-sitic and free-living nematodes will begin to address the ques-tion of whether the same pathways have been repeatedly usedin the evolution of parasitism, a life style that has evolved onat least four occasions among nematodes[18]. This compar-ison will also answer the question of whether mechanismsinvolved in the control of development are conserved be-tween distantly related genera;S. rattiandP. trichosuribe-ing members of Clade IV andC. elegansa member of CladeV [18,19].

To investigate whether the molecular control of the initia-tion ofC. elegansdauer development is also conserved in thecontrol of the initiation of development of iL3s of parasiticnematodes, we cloneddaf-7homologues fromS. rattiandP.trichosuri. DAF-7 is a TGF-�-like ligand, which suppressesthe development ofC. elegansdauer larvae at low populationdensity and high food abundance via the TGF-� pathway[9]. daf-7 is an ideal candidate gene to test the relationshipbetween developmental pathways inC. elegansand parasiticn fromo ep lig-aa sd ringi L2sp ltsw .

lst tt re,t mtt steds

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ematodes for two reasons. First, it can be distinguishedther members of the TGF-� superfamily by its distinctivattern of nine cysteine residues within the C-terminalnd domain, shared only withC.briggsaeDAF-7,Bm-TGH-2nd vertebrate TGF-� homologues[14]. Secondly,C.eleganaf-7mRNA shows an expression pattern that varies du

ts life cycle; high in L1s, decreasing thereafter and off inreparing to enter the dauer stage, dauer larvae and adu[9],hich facilitates testable comparisons between generaHere we describedaf-7homologues and their RNA leve

hrough theS. rattiandP. trichosurilife cycles. We find thahe RNA level ofdaf-7 peaks in iL3s and that, therefohe pattern ofdaf-7RNA levels in these species differs frohat inC. elegans. The possibility thatdaf-7of S. rattiandP.richosurimay be involved in the maintenance of the arretate of iL3s is discussed.

. Materials and methods

.1. Parasites and maintenance

S. ratti: Line ED321 Heterogonic[1] was used throughut and infections were maintained and larvae isolatereviously described[20].P. trichosuri: Kahurangi isolate, originally isolated fro

ossums in the Kahurangi National Park, South Island,ealand was maintained by serial passage in vitro on af Escherichia coliHB101 on LB agar at 20◦C. Possumere infected percutaneously by the application of iL3

reshly wetted flank fur. Faeces were collected from infe

M. Crook et al. / Molecular & Biochemical Parasitology 139 (2005) 213–223 215

animals overnight and cultured by placing 4–6 pellets ontowater saturated filter paper draped over an inverted 50 mmdiameter petri dish, within a 90 mm diameter petri dish con-taining distilled water, and incubated at 20◦C.

2.2. Cloning and sequence analysis

TheP. trichosuri expressed sequence tag (EST) project(http://www.nematode.net) identified an EST (BI743385)with similarity to C. elegans daf-7[9]. This sequence wasused as the basis for the discovery of adaf-7 homologuefromS. ratti. To do this, primers (Pt-daf-7F and R,Table 1)designed to nucleotide positions 1–22 and 226–249 of thisEST were used to amplify (94◦C, 3 min; 94◦C, 1 min; 50◦C,1 min; 72◦C, 1 min—for 35 cycles; 7 min, 72◦C final exten-sion, with a final concentration of 2.5 mM Mg2+) a fragmentfrom S. ratti genomic DNA. To obtain further sequence,S.ratti iL3 total RNA was used as the template for 5′ and 3′RACE (Invitrogen, USA) using the RACE primers detailedin Table 1(Roche, UK) and gene specific primers designedto the genomic fragment (not shown). Sequence fragmentsresulting from these RACE reactions were assembled into acontiguous sequence. To determine whether an intron waspresent, primers (Sr-daf-7F2 and R,Table 1) designed tothe 5′ and 3′ ends, were used to amplify this contiguous se-q eaw gt atso any)M con-c siont ict with

1000 iterations, were carried out in DNAMAN (Lynnon Cor-poration, USA).

2.3. Sample preparation and RT-PCR

Worm preparations (detailed below) were pelleted by cen-trifugation, the supernatant removed and the pellet resus-pended in a small volume of TRI-reagent (Sigma, UK) andstored at−80◦C until use. ForS. ratti, samples were groundin liquid nitrogen using a microgrinder[22]; forP. trichosuri,samples were homogenised using an Omni pH homogeniser(Omni International, Warrenton, USA). RNA was extractedin TRI-reagent, treated with DNAse (Promega, UK) andcDNA synthesised from equal quantities, measured spec-trophotometrically, of total RNA using Superscript II reversetranscriptase (Invitrogen, UK), all according to the manufac-turers instructions.

The RNA level ofdaf-7was compared with that of two,putative constitutively expressed genes. To select such con-trol genes, commonly used reference genes (actin and tubu-lin) and the 20 most abundantly expressed ESTs in theS.ratti EST project (http://www.nematode.net) were amplifiedfrom S. ratti cDNA and genomic DNA. The criteria for theuse of such a gene was that the amplified region must spanan intron and expression levels must be approximately thesE pers.c -ma withP nta1c g-

TO

G

R

V

uence fromS. ratti genomic DNA and cDNA, using thbove PCR conditions. Primers (Pt-daf-7F and R,Table 1)ere used withP. trichosurigenomic DNA and cDNA, usin

he same conditions as forS. ratti. Three independent repef these PCRs were sequenced (Operon, Qiagen, Germotif sequence searches were carried out, using the

eptual translation of transcripts of interest, by submiso the ProfileScan server[21]. Alignments and phylogenetree construction, using the Neighbour Joining method

able 1ligonucleotide primers (5′–3′) used in this study

S. ratti

ene specific primersdaf-7 F2: ATA TTT ATT GGT AAT TGG TTC G

F: TTA AAG AAA CAA GAA TTC CAA C a

R: TTA TAA AAA TCA CAT TTG AGA C a

act-3 F: GCT GAA CGT GAA ATT GTT CGa

R: GAA GCC AAG ATA GAT CCT CCa

plk-2 F: GAT GAA CTA GCT CGT TTA CCT Ga

R: TTA TAT GCC CTT ACA TCA GGA Ga

asp-1

ACE primersOligo dT anchor GAC CAC GCG TAT CGA TGT CGA C (T)16VAnchor primer GAC CAC GCG TAT CGA TGT CGA C

= A, C or G.a Primers used in analysis ofdaf-7RNA levels.

.

ame across all life cycle stages. By these criteria,S. rattiST clusters SR00015 and SR00113 (Thompson FJ,ommun.,http://www.nematode.net/), with significant alignent by BLASTX[23] to C. elegans plk-2(E= 5e−25) andct-3 (5e−76), were chosen. This process was repeated. trichosuri, which resulted in two genes with significalignment by BLASTX toC. elegans act-3(1e−132) andasp-(3e−59), respectively. ForC. elegans, act-3 (X16798) washosen, as previously used[9]. Primers to these gene fra

P. trichosuri C. elegans

F: GAT GGA ATT TTA TTG TAGCAC CAa

F: AAT TTC TCC GAT CCAAGG TAT CTT Ca

R: TTA ATG GAA TAT ACTCAG TTG GAa

R: TTT CTT GGC GAT CAT TCCGTT GACa

F: GCT GAG CGT GAA ATCGTC CGT Ga

F: ATT ACC TCA TGA AGATCC TTA CCa

R: GTT TTC TCT CTG GTGGAG CAT Ga

R: TAT TCT TGC TTG GAG ATCCAC ATCa

F: CAA CTA TTG CTG AAGTTT TCG Ga

R: TTG TTG TTT TCT GGT TTCACT Ga

http://www.nematode.net/




ments are listed inTable 1. To determine the exponentialphase of amplification of each of these gene fragments, theywere amplified (using the above conditions) from total RNAof their respective species. PCRs were sampled from 14 to35 cycles in three cycle increments, resolved on 2.5% (w/v)agarose gels containing ethidium bromide and the PCR prod-uct quantified by comparing the number of pixels represent-ing the PCR product of interest with that of a DNA marker(Genetools, Syngene, UK). PCR product quantity was plot-ted against cycle number (data not shown) and the highestcycle number from the exponential phase of each gene wasused.

To analyse the RNA level ofdaf-7, species specific primerswere used (Table 1). For each cDNA preparation two compar-isons, unless otherwise stated, were carried out:daf-7: act-3(all species, 29 cycles) anddaf-7: plk-2(S. ratti, 32 cycles) ordaf-7: asp-1(P. trichosuri, 29 cycles), using the above condi-tions. Each PCR was repeated three times and products werequantified, as above. Ratios of RNA levels were calculated asthe quantity ofdaf-7amplicon/quantity of reference gene am-plicon and the log of these ratios taken to normalise the data,which were then analysed by one-way ANOVA (SPSS, SPSSInc., USA) where the log ratios were the response variableand life cycle stage or experimental treatment, in respectiveexperiments, the explanatory variable. Post hoc analysis ofd wasc et

2t

so-l ain-t resw st el, asa -t /w)st time

points, a small number of worms from each collection pointwere fixed in 10% (v/v) steaming formal saline and subse-quently examined microscopically. At 6, 24, 48 and 72 h thepreparations of different life cycle stages ofS. ratti showthat these sample times essentially correspond to L1, L2, L3and free-living adults, respectively (Table 2(a)). Parasitic fe-males were collected as previously described[26]. A singlecDNA synthesis was carried out for each of two biologicalreplicates.P. trichosuri: To prepare different life cycle stages, eggs

were extracted from fresh faeces by flotation on a saturatedsalt solution. L1s were hatched from these eggs by incubationfor 6 h at 20◦C in distilled water. For other stages, 100�l ofa mixture of 104 eggs/106 Escherichia coliHB101 cells permillilitre was incubated on LB agar plates in the presence ofa small piece of sterile possum faeces for 12, 24, 36 and 48 h,after which worms were washed off these plates with distilledwater and collected by centrifugation. iL3s were recoveredfrom 14-day-old, 20◦C plates. At 12, 24, 36 and 48 h thepreparations of different life cycle stages ofP. trichosurishowthat these sample times essentially correspond to L1, L2, L3,L4 and free-living adults, respectively (Table 2(b)). Parasiticmales and females were collected manually from mucosalscrapings of the small intestine. Duplicate cDNA syntheseswere carried out for each of two biological replicates.

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ifferences in ratios of RNA levels between treatmentsarried out by the Tukey HSD test.P values of <0.05 weraken as significant.

.4. daf-7 RNA levels in the life cycle of S. ratti, P.richosuri and C. elegans

S. ratti: To prepare different life cycle stages, we first iated L1s from fresh faeces with a Baermann funnel mained at 19◦C for 6 h[24]. For other stages, faecal cultuere maintained at 19◦C for 24, 48 and 72 h, and the worm

hat had developed were isolated with a Baermann funnbove. iL3s were recovered from 6-day-old, 19◦C faecal cul

ures. All stages were recovered by flotation on a 60% (wucrose solution and washed in M9 buffer[25]. To determinehe percentage of each life cycle stage present at these

able 2ercentages of each life cycle morph at different sample times

ime (h) L1 L2 L3 iL3s

a)S. ratti6 83 174 30 708 8 42 46 3.52 8 10 3 23

b)P. trichosuri6 1002 11 894 2.5 7 886 68 1 1

C. elegans: daf-7RNA levels inC. eleganswere measurey semi-quantitative RT-PCR to determine the pattern ofdaf-RNA levels inC. elegansand to compare it to publisheorthern analyses[9]. L1, L2, L3, L4 and hermaphroditeere collected from well-fed synchronous populations grt 19◦C using standard methods[25]. Arrested L1s (L1 arrere grown from freshly hatched eggs on NGM plates[25]ithout food for 24 h at 19◦C. Dauer larvae were grownauer assay agar plates containing pheromone[3] with lim-

ted food for 72 h at 25◦C. Duplicate cDNA syntheses wearried out for each of two biological replicates.

.5. Temperature and daf-7 RNA levels

To prepare iL3s grown at a range of temperatures, faultures were incubated in water at 13, 19, 25 or 30◦C for

L4 Free-living adult male Free-living adult fem

0.59 23 24

2.576 7 11

4.5 47.5 46


8, 6, 4 and 2 days, respectively (S. ratti) and 15, 20, 25 and30◦C for 14, 12, 10 and 8 days, respectively (P. trichosuri).For both species, iL3s were collected from each tempera-ture treatment and divided into two equal samples. For eachtemperature treatment, one cohort was placed at the originalincubation temperature and the other was placed at host bodytemperature (37◦C forS. rattiand 35◦C forP. trichosuri) for3 h, after which all iL3s were collected by centrifugation. ForS. ratti, two cDNA syntheses were carried out for the first bi-ological replicate and one for the second; forP. trichosuri,there was a single cDNA synthesis from one biological repli-cate. The RNA level ofdaf-7in iL3s from each treatment wasdetermined as described above, with the exception that forS.ratti, daf-7RNA levels were compared withplk-2only.

2.6. Host infection and daf-7 RNA levels

S. ratti: To investigatedaf-7RNA levels in the within hoststages of the life cycle, 16 female Wistar rats were infectedsubcutaneously with 10,000 iL3s and eight rats sacrificed at1 and 2 days post infection (p.i.). The fronto-nasal region ofeach rat was removed, macerated and incubated in a Baer-mann funnel containing low glucose DMEM (Sigma, UK)at 37◦C for 7 h and larvae collected by centrifugation. Du-plicate cDNA syntheses were carried out for one biologicalr

h ctedw ol-l , andL malli andv riedoi ribeda

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iumo dfcr /v)B inew ctedt lys n-d to thel me-t wasi ), inw n-vP ove,f laced

onto the upper surface of the skin. Larvae that had penetratedinto the media after 5 h or overnight were then collected.All media were supplemented with 50 ng ml−1 ampicillinand streptomycin and 10 ng ml−1 tetracyclin (Sigma, UK).For each of the aboveS. rattiexperiments, duplicate cDNAsyntheses were carried out for one biological replicate. ForP. trichosuri, a single cDNA synthesis was carried out foreach of two biological replicates. The RNA level ofdaf-7in iL3s from each treatment was determined, as describedabove.

3. Results

3.1. Cloning and sequence analysis

S. ratti: 1143 bp of sequence was cloned from cDNA.Amplification of this sequence from genomic DNA failedto demonstrate the presence of an intron. Analysis showedsignificant alignment by BLASTX withC. elegans daf-7(2e−13) andB. malayi tgh-2(3e−13). The conceptual trans-lation of the entire sequence was 16.5 and 15.8% identicaltoC. elegansDAF-7 andB. malayiTGH-2, respectively, butwas 28.3 and 26.3% identical to the C-terminal ligand do-main (from protease cleavage site to stop codon). The ninec er oft o-m wasf

85)s C-t f-7 edt n oft e C-t2 iner

s en-ca nFq rt andt ,w illra iticn

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eplicate.P. trichosuri: To investigatedaf-7RNA levels in the within

ost stage of the life cycle one female possum was infeith 5000 iL3s and sacrificed 5 days p.i. iL3s were c

ected from the fronto-nasal region and lungs, as above4s were collected from mucosal scrapings of the s

ntestine. iL3s and L4s were differentiated by lengthulval development. A single cDNA synthesis was carut for one biological replicate. The RNA level ofdaf-7

n larvae from each treatment was determined, as descbove.

.7. Host-like conditions and daf-7 RNA levels

To investigate the effect of temperature or culture medn the RNA level ofdaf-7 in iL3s, 40,000 iL3s were place

or 5 h in either: (a) water at 19◦C (S. ratti) or 20◦C (P. tri-hosuri); (b) water at host body temperature (37 and 35◦C,espectively) or (c) DMEM supplemented with 10% (vCS (Sigma, UK) at host body temperature. To determhether skin components diffusing into the medium affe

he RNA level ofdaf-7 in S. ratti, shaved skin from freshacrificed rats was cut into 5 cm× 5 cm squares and the uerside lightly lacerated. These pieces were placed on

ids of 90 mm diameter petri dishes over a 50 mm diaer hole in the lid such that the underside of the skinn contact with the media (as for treatment c, abovehich 40,000S. ratti iL3s were incubated for 5 h. To iestigate the effect of penetration of skin onSr-daf-7andt-daf-7RNA level, skin cultures were prepared, as ab

rom rats and possums, respectively, and 100,000 iL3s p

onserved cysteine residues, characteristic of a membhe DAF-7 TGF-� subfamily, were present in the ligand dain (Fig. 2(a))[9]. No signal or spliced leader sequence

ound.P. trichosuri: The 292 bp EST sequence (BI7433

howed significant alignment by BLASTX with theerminal domain ofB.malayi tgh-2(2e−5) andC.elegansda(8e−5). The sequence of this from genomic DNA show

hat no intron was present. The conceptual translatiohis sequence was 27.6, 25.9 and 71.6% identical to therminal ligand domain ofC.elegansDAF-7,B.malayiTGH-

and theS. ratti sequence, respectively, with all cysteesidues present (Fig. 2(a)) [9].

The phylogenetic relationship between the sequenceoding the ligand domains fromS. ratti andP. trichosurind other members of the TGF-� superfamily is shown iig. 2(b). This shows that theS. ratti andP. trichosuri se-uences cluster together with a 100% bootstrap suppo

hat these are related to the nematodedaf-7-like gene groupith 89% support (Fig. 2(b)) [27]. For these reasons, we w

efer to theS. ratti andP. trichosurisequences asSr-daf-7ndPt-daf-7, respectively, following the standard parasematode nomenclature system[28].

.2. daf-7 RNA levels in the life cycle of S. ratti, P.richosuri and C. elegans

For S. ratti, the mean RNA level ofdaf-7 was greatst in iL3s, low in the other free-living stages and not

ectable in parasitic females (Fig. 3(a)). Thedaf-7 RNAevel in iL3s was significantly different, compared with


Fig. 2. (a) Alignment of the ligand domain amino acid sequences, from cleavage site to stop codon, of the conceptual translation ofdaf-7 from S. rattiandP. trichosuriand six other members of the TGF-� superfamily. The start of thePt-DAF-7 sequence was unavailable. Residues with 100,≥75 and≥50%identity between the eight sequences are represented by black, dark grey and grey shaded boxes, respectively. Dots represent gaps introduced to allow optimalalignment; amino acid residue number within the ligand domain is shown.� and� represents the limits of the sequence used in the phylogenetic analysis.The two cysteine residues found only in the DAF-7 TGF-� sub-family are shown by�. (b) Phylogenetic tree of the nucleotide sequences encoding the liganddomains of members of the TGF-� superfamily. Numbers represent bootstrap values and scale bar represents branch length as a fraction of the total treelength. The accession numbers are as follows:C. elegans daf-7, U72883;C. elegans tig-2, (cosmid F39G3);C. elegans dbl-1, AF004395;C. briggsae daf-7,CBG24910;B. malayi tgh-2, AF104016;B. malayi tgh-1, AF010495;H. sapiens tgf-β-1, P01137;H. sapiens activin, X82540;H. sapiens inhibin, X72498;H.sapiens myostatin, AF019627;H. sapiens bmp-2, P12643;D. melanogaster dpp, P07713;D. melanogaster 60A, P27091.


meandaf-7RNA level for all other life cycle stages com-bined (F1,11= 18.01,P= 0.002;F1,6= 8.98,P= 0.03 forplk-2 andact-3, respectively). A very similar expression patternwas seen inP. trichosuri (Fig. 3(b)), with a significantlygreater meandaf-7RNA level in iL3s, compared with allother life cycle stages (F1,15= 47.2,P< 0.0001 forasp-1),though this difference was not significant when RNA levelwas compared toact-3 (F1,15= 3.58,P= 0.08).daf-7RNAwas only detected in parasitic males (0.016± 0.007, mean± 1 S.E.M.) and females (0.07± 0.026 foract-3) after 32PCR cycles, at a similar level to that seen in L1s to free-living adults (overall mean 0.048± 0.021) at this higher cyclenumber.

ForC. elegans, the mean RNA level ofdaf-7was greatestin arrested L1s and growing L2s, lower in L1s, L3s, L4s andadults, and only occasionally detected in dauer larvae (Fig. 4).There was a significant effect of life cycle stage ondaf-7RNA level (F1,68= 3.86,P= 0.002), with a significantly lowermean RNA level in dauer larvae, compared with all other lifecycle stages combined (F1,68= 4.74,P= 0.033). These resultsare consistent with the RNA level ofdaf-7 determined byNorthern analysis[9].

Fsa

Fig. 4. The mean RNA level (±1 S.E.M.) ofdaf-7 to act-3 in different lifecycle stages ofC. elegans.

3.3. Temperature and daf-7 RNA levels

ForS. rattiiL3s, there was a significant effect of the origi-nal incubation temperature ondaf-7expression (F1,24= 4.05,P= 0.033) (Fig. 5(a)), but there was no significant difference

ig. 3. The mean RNA level (±1 S.E.M.) ofdaf-7 in different life cycletages of (a)S. ratti to plk-2 (white columns) and toact-3 (grey columns)nd (b)P. trichosurito act-3(grey columns) and toasp-1(white columns).

Fiaf

ig. 5. The mean RNA level (±1 S.E.M.) ofdaf-7at different temperaturesn iL3s of: (a)S. ratti to plk-2and (b)P. trichosurito act-3(grey columns)nd toasp-1(white columns). The “shift” suffix represents samples shifted

rom that temperature to host body temperature.


between temperatures by Tukey HSD post hoc analysis. How-ever, there was a significant difference indaf-7RNA lev-els at 19 and 25◦C combined, when compared with thoseat 13 and 30◦C combined (F1,24= 4.63,P= 0.043). ForP.trichosuri iL3s, there was no significant effect of the origi-nal incubation temperature ondaf-7RNA level (F1,11= 1.35,P= 0.34;F1,11= 0.06,P= 0.98 foract-3andasp-1, respec-tively) (Fig. 5(b)). For iL3s of both species, there was nosignificant difference indaf-7RNA level between the origi-nal incubation temperatures, combined, and the shift to hosttemperature, combined (S. ratti, F1,51= 0.09,P= 0.77;P. tri-chosuri,F1,22= 2.43,P= 0.13;F1,22= 2.15,P= 0.16 foract-3andasp-1, respectively). However, inS. rattiiL3s, a decreasein daf-7RNA level appears to occur on the shift to host tem-perature from 25◦C, although this difference is not significant(F1,17= 0.85,P= 0.37) (Fig. 5(a)).

3.4. Host infection and daf-7 RNA levels

ForS. ratti, there was a significant effect of stage ondaf-7RNA level (F1,18= 42.8,P< 0.0001;F1,18= 32.9,P< 0.0001for plk-2 andact-3, respectively) (Fig. 6(a) and (b)), witha significant difference in mean RNA level between pre-infective iL3s and post-infective iL3s recovered 1 day p.i.

(P< 0.0001;P< 0.0001 forplk-2andact-3, respectively), butnot between post-infective iL3s recovered 1 and 2 days p.i.(P= 0.086;P= 0.99 forplk-2 andact-3, respectively). Thisshows that RNA level has decreased by 24 h p.i. in the hostand stays low thereafter. ForP. trichosuri, there was a signif-icant effect of stage on mean RNA level ofdaf-7(F1,6= 22.8,P= 0.002 foract-3), with a significant difference betweenpre-infective iL3s and post-infective L4s (P= 0.01) but notbetween post-infective iL3s and post-infective L4s. Therewas no effect of stage on mean RNA level ofdaf-7, com-pared withasp-1(F1,6= 4.99,P= 0.089) (Fig. 6(c)). Thesedata indicate thatdaf-7RNA level decreases gradually withtime in the host.

3.5. Host-like conditions and daf-7 RNA levels

ForS. ratti, there was a significant effect of treatment onmean RNA level ofdaf-7, compared withact-3 (F1,27= 5.2,P= 0.02), but not when compared withplk-2 (F1,27= 1.6,P= 0.35). (Fig. 7(a) and (b)). Meandaf-7 RNA level wassignificantly different between iL3 that had penetrated skinovernight and treatments that did not involve skin penetra-tion, but was not significantly different between iL3 that hadpenetrated skin for 5 h and overnight (data not shown). For

F(s

ig. 6. The mean RNA level (±1 S.E.M.) ofdaf-7 in S. ratti iL3s to (a)plk-2, (b)white columns). The +1 and +2 days p.i. representS. ratti iL3s recovered from thtages recovered from the host.

act-3, andP. trichosuri iL3s and L4 to (c)act-3 (grey columns) andasp-1e host at these time points. iL3s+ and L4+ representP. trichosuriiL3 and L4


Fig. 7. The mean RNA level (±1 S.E.M.) ofdaf-7following treatment ofS. ratti iL3s to (a)plk-2, (b)act-3, andP. trichosuriiL3s to (c)act-3(grey columns)andasp-1(white columns). Treatments were: water, DMEM, DMEM and skin, and skin penetration. Incubation temperatures (◦C) and penetration times (for5 h or overnight (o/n)) are shown.

P. trichosuri, there was no significant effect of treatment onmeandaf-7RNA level (F1,24= 1.45,P= 0.24;F1,28= 1.38,P= 0.25 foract-3andasp-1, respectively) (Fig. 7(c)). Theseresults indicate that inS. ratti, but notP. trichosuri, pene-tration through skin may be the cue for a reduction indaf-7RNA level upon entering the host.

4. Discussion

We hypothesised that parasitic nematodes use pathwaysto control the initiation of iL3 development that are simi-lar to those used byC. elegansto control the initiation ofdauer development. To investigate this hypothesis we clonedputativedaf-7 homologues fromS. ratti andP. trichosuri.Overall, amino acid sequence identity was low, but substan-tial within the ligand domain, with the same characteristicpattern of cysteine residues between the three species. Thisimplies a conservation of ligand domain folding and there-fore, possibly, function. This and the clustering ofSr-daf-7andPt-daf-7to the nematodedaf-7cluster (Fig. 2(b)), justi-fies calling these genesdaf-7. However, further work, such ascomplementation studies ofC. elegans daf-7mutants, will beneeded to confirm the functional conservation of these genes.

daf-7RNA level in the life cycle ofS. rattiandP. trichosuriwas at its greatest in iL3s, low in other free-living stages andessentially absent in the parasitic stages. Very similar resultshave been found with the expression oftgfb-1 in S. sterco-ralis (Massey HC, Castelletto M, Bhopale VM, Schad GA,Lok JB, Personal Communication).daf-7RNA levels in theseparasitic nematodes are different toC. elegans, where RNAlevel peaks in arrested L1 and L2 (the stages during whichthe dauer development decision is taken), declines thereafterand is absent in dauer larvae (the analogue of iL3s of parasiticnematodes)[9,29,30]. Therefore, the expression ofSr-daf-7andPt-daf-7does not support the hypothesis that these genesand the pathways within which they act, play a role in theinitiation of iL3 development.

The peakSr-daf-7andPt-daf-7RNA levels in iL3s leadsus to propose an alternative hypothesis: that, inS. ratti andP. trichosuri, daf-7maintains the developmentally arrestedstate of iL3s until appropriate conditions for continued de-velopment arise, that is infection of a host. This is consistentwith the peak RNA level oftgh-2 in B. malayimicrofilar-iae, the arrested L1 stage of filarial nematodes[14], and maybe consistent with the highdaf-7RNA level in arrestedC.elegansL1s, compared with non-arrested L1s. This hypothe-sis is also consistent with the significantly lowerdaf-7RNA


level seen in bothS. rattiandP. trichosuri larvae recoveredfrom the host and which have therefore resumed parasiticdevelopment. These decreases are similar to the decrease intgh-2RNA level in B. malayimicrofilariae recovered fromthe mosquito intermediate host, where development has alsoresumed[14]. These comparisons suggest that the mecha-nism of maintaining an arrested larval state may be conservedamong parasitic nematodes, irrespective of the stage that isarrested.

Entering a host could be sensed via a range of cues;shift to host temperature, an increase in nutrient availabil-ity and, or cues associated with skin penetration. Ifdaf-7is involved in the maintenance of developmental arrest iniL3s, one, or a combination, of these cues would be expectedto result in a reduction indaf-7RNA levels. Shift to hosttemperature is an important cue in the resumption of devel-opment of infective stages ofS. venezuelensis[31] andB.pahangi[32]. Shift to host temperature also induces a lipid-phase change in the cuticle ofS. ratti iL3s [33], a processthought to occur on infection of a host[34]. The temper-ature at which iL3s are grown and maintained affects theRNA level of Sr-daf-7, though specific shift to the temper-ature of their host did not significantly affect the expres-sion of eitherSr-daf-7or Pt-daf-7. Incubation of iL3s inDMEM and 10% BCS, intended to mimic host conditions,s -d tiont dd iftt andpR geste

a ana ningo ic re-l ingm oneo haso d that a-t hf enica n oft thish

r-tt fort F-7w NAik in-a nt ap ases,a

Acknowledgements

We thank the staff at AgResearch, Wallaceville, withoutwhose help the work onP. trichosuriwould not have beenpossible, Mike Gardner for statistical advice, Simon Harveyfor C. eleganshelp and Stephanie McInnis for the RACEprimers. MC was supported by a University of Bristol Schol-arship. This work was supported by the MRC.

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daf-7 and the development of strongyloides ratti and parastrongyloides trichosuri

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