patterns of covert infection by invertebrate pathogens

Molecular Ecology (1995) 4, 447-457

Patterns of covert infection by invertebrate pathogens:iridescent viruses of blackflies

T. WILLIAMSNERC Institute of Virology & Environmental Microbiology, Oxford OXl 3SR, UK and ECOSUR, Tapachula, Chiapas, Mexico

Abstract

RecentIy, it has been recognized that blackfly populations mar host two forms of infec-tion by iridescent viruses (IVs)¡ a covert (inapparent, nonlethal) form which was commonin springtime populations in the River Ystwyth, Wales, and a patent (obvious, lethal)form which was rafe. This study aimed to investigate the changes in frequency of the twotypes of infection in blackfly populations ayer the reproductive period of the flies,April-September 1992. Blackfly larvae sampled from three different sites along the riverwere bioassayed for the presence of covert IV infection. Of 870 larvae assayed, 17 werefound to be infected. AII the infected larvae appeared to be Simulium variegatum, thedominant species during the sampling periodo IV infections were common in the spring(17-37% depending on site) but appeared absent in the S. variegatum population for mostof the summer months, reappearing again in the autumn (0-20% infected). These fluctu-ations were concurrent with biotic and abiotic factors: elevated levels of covert infectionoccurred at low population densities, high water flow rates, low temperatures (and pre-sumably slower growth rates), although it is not clear if any cause-and-effect relationshipexists. Patent infections occurred immediately alter the peak of covert infection in thespring, and again in the autumn. Virus characterization of isolates from covertly infectedlarvae showed that three distinct groups of isolates were present in the blackfly popula-tion. Isolates from the springtime populations were mostIy variants of an isolate found inpatentIy infected blackfly larvae in the 1970s (Aberystwyth IV). Isolates from the autumnpopulations were mostIy variants of an isolate from a patentIy infected larva found inSeptember the previous real. A third group comprised a single novel isolate which wásdetected in a covertly infected larva. The mechanisms by which IVs persist in blackflypopulations remain unknown, although the role of altemative hosts is a possibility whichneeds to be studied.

Keyworás: blackfly; fluctuations in incidence; inapparent infection; iridescent viruses;persistence; Simulium variegatum

Received 19 December 1994; accepted 27 March 1995

Introduction

In many animal populations there exist extended periodswherein conditions are not suitable for pathogen transmis-sion, perhaps due to fue host population failing to attainsome critical threshold density or due to host dormancy,for example. During such periods pathogens must adoptstrategies by which they can persist until conditions fortransmission become favourable. The mechanisms by

Present address: El Colegio de la Frontera Sur (ECOSUR), AptdoPostal 36, Tapachula 30700, C~apas, Mexico. Tel.: (52) 962 81077.Fax: (52) 96281015.

which pathogens persist in host populations are still poor-ly understood in many systems (Grenfell & Dobson, inpress). One possible mechanism involves fue pathogenpersisting within fue host by adopting nonlethal, e.g.latent, forms of infection. As the probability of transmis-sion falls e.g. in low-density host populations,host-pathogen population models predict that pathogenswhich adopt strategies of host-exploitation which are notlethal to fue host and which exploit vertical routes of trans-rnission will be favoured ayer fue lethal forms of disease(Anderson & May 1981; Nowak 1991; Sasaki & Iwasa

1991).Such predictions are equally applicable to virulent

T. WILLIAMS448

insect pathogens which have been considered as biocon-trol agents for insect pests and vectors. With a few excep-tions (e.g. sex ratio distorting diseases of parasiticHymenoptera) nonlethal infections mar not produce overtsymptoms in fue insect host and where signs of disease doexist they mar be subtle or difficult to detect (Sait et al.1994b; Goulson & Cory, in press). Consequently, fue ecolo-gy of invertebrate pathogens usually focuses strongly onfue behaviour and dynamics of fue overt, lethal forro of fuedisease.1ñerefore, due to difficulties in detection or due toa lack of interest in pathogens which do not appear to havebiocontrol potential, covert (inapparent) infections havelargely been neglected (Kelly 1985), despite their wide-spread occurrence in certain systems, e.g. many smallRNA viruses of Drosophila and other insects (e.g. Dobos etal. 1991; Moore 1991). Invertebrate iridescent viruses (IVs)are also one such group.

IVs are nonoccluded icosahedral particles containing adsDNA genome of 140-200 kbp in size. 1ñe patent forro ofIV infection appears as a stunning opalescent colorationthroughout fue body of fue host, usually an insect or crus-tacean. Paracrystalline arrays of virus particles in infectedhost tissues interfere with fue passage of light and result infue characteristic iridescence, fue colour of which is depen-dent on particle size and spacing (Klug et al. 1959;Hemsley et al. 1994). 1ñe patent disease is invariablylethal, but in most host populations, fue incidence of fuesedramatic infections is minuscule. Consequently, IVs arenot currently considered as useful agents for pest control.In an isopod-IV system, where transmission occurs viacannibalism of infected conspecifics, fue patent lethal dis-ease is the only recognized forro of infection (Grosholz1992). However, in another system, nonlethal covert IVinfections of blackfly larvae (Diptera: Simuliidae) weredemonstrated at levels of up to c. 30%, many orders ofmagnitude greater than fue patent disease (Williams 1993).Covert IV infections are not believed to be latent in fuegeneral sense used for viruses: latent viruses mar integrateinto fue host genome or exist as naked nucleic acid in hostcells which show few or no signs of infection until fuevirus is activated (see Hughes et al. 1993 for an insect virusexample). Rather, fue fact that covert IV infection can betransmitted to altemative hosts suggests that they exist asparticles within host cells but at a very low density.Precisely which host tissues harbour covert IV infectionsare unknown although cells lining fue midgut have beensuspected (Glare 1992). IV isolates from both fue covert(nonlethal) and the patent (lethal) infections exhibited amarked degree of genetic variability; no two isolatesappeared identical and, with one exception, all appearedas variants of an original patent isolate discovered inblackflies at fue same location in fue 1970s (Batson et al.1976; Williams 1993; Wi~liams & Cory 1993). Reference willbe made to fuese isolates during fue course of this study as

marked similarities among the various isolates fromcovert and patent infections of blackfly larvae becameapparent. The route of transmission of fuese viruses inblackfly populations is not known (Kelly 1985; Ward &Kalmakoff 1991).

Recently, changes have been proposed in the classifica-tion and nomenclature of IVs (Williams & Cory 1994). Theprevious system of naming isolates according to the hostand fue sequence of discovery was shown to be no longeruseful and an altemative nomenclature based on geo-graphical origin of the isolate was proposed.Consequently, IV type 22 from Simulium sr. (Batson et al.1976) was renamed Aberystwyth IV in accord with its dis-covery in fue River Ystwyth near Aberystwyth, Wales, UK.The new system of nomenclature is used here.

Blackflies are haematophagous Diptera with aquaticjuvenile stages. Eggs are laid on stones, trailing vegetationor directly in fue water. First-instar larvae disperse down-stream on strands of silk and attach to substrates (stones oraquatic plants) in fast flowing sections of fue river. Larvaeundergo ~ 6 instars before pupating in a silk tent. Adultflies emerge, mate and the female seeks a blood meal froma vertebrate host before returning to fue river to lar eggs.In fue River Y stwyth, simuliids over-winter mainly as lar-vae. Pupation occurs in the early spring and the first gen-eration of larvae arrear in early April. S. variagetumappears to have a number of generations annually proba-bly three, but fuese generations are not discreteo

Because fue original report of covert IV infection waslimited to a single sample in the springtime Simulium pop-ulations, a complete study was undertaken to determinechanges in the abundance of covert iniéétion for fue wholereproductive period of fue River Y stwyth blackfly popula-tions. In particular, three questions were addressed:1 Is this a single-host-single-virus interaction?2 How does fue incidence of covert IV infection changeover the spring and summer months?3 Can these changes be correlated with biotic factors suchas host density, age structure, community structure or abi-otic factors such as water chemistry or temperature?

Materials and methods

Field sampling

Simulium larvae were sampled at monthly intervalsbetween April 1992 and September 1992 from fue threesites previously used along fue River Ystwyth, Wales, UK(map references given in Wi1liams 1993). These were anupstream mountainous fast-flowing site near Blaenycwm,an intermediate site near Abermagwr and a tranquildownstream site near Pentre-Ilyn. These sites were-= 32 km, 13 km and 7 km from fue river estuary, respec-tively. Water chemistry data for fue study period were

COVERT IV INFECTIONS OF BLACKFLIES 449

Oxford were treated in fue same way. No evidence hasbeen found for IV infection in Simulium populationsaround Oxford, despite extensive studies (Unpublished

data).

obtained from fue National Rivers Authority (NRA) forthree sites along fue Y stwyth which corresponded to eachof fue sampling sites. In addition fue NRA provided dailymean flow data for fue river at Pont Llolwyn near to fuedownstream Bite (Pentre-llyn). At each sampling occasion50 stones were selected at random at each Bite. The num-ber of Simulium larvae on each stone was estimated andfue afea of fue uppermost surface of the stone (over whichfue larvae attached) was measured and noted. Stoneswithout larvae were not considered. This gave an estimateof fue density of larvae per m2 of suitable substrate. Thedensity of pupae was not recorded as simuliids tend toselect sites with different flow characteristics for pupationcompared to larval feeding sites (Crosskey 1990). Larvaeof all sizes were collected en masse from stones using softforceps and placed in an antibiotic solution (10 mg/mLstreptomycin and 10 000 IV / mL penicillin) and transport-ed back to the laboratory in insulated boxes. All sampleswere frozen and stored at -20 °C. In May, June andSeptember, c. 500 larvae of all instars were taken fromAbermagwr and reared in fue laboratory to check for fueoccurrence of patent IV infections. Patently infected larvaefound during searches of each Bite during this period havealready been reported by Williams & Cory (1993).

Characterization of virus from covert infections

Galleria mellonella larvae with patent IV infections werehomogenized in sterile distilled water and debris pelletedby centrifugation at 1500 g for 5 mino Virus in fue super-natant was pelleted by centrifugation at 10 000 g for 5 minoONA was extracted from fue sernipure virus sample using50S and Proteinase K followed by phenol-chIoroformtreatment and dialysis (Williams & Cory 1993). IV ONAwas treated with EcoRI or HindIII following manufacturersprotocols (Boeringer) and subject to electrophoresisthrough 0.6% agarose in TBE buffer. Gels were pho-tographed and then 50uthern blotted onto Hybond(0.45 .um) membrane (Amersham) (Sambrook et al. 1989).

A probe was made comprising a pUC19 plasmid con-taining a 1.4-kb Sal! fragment of fue Aberystwyth IV majorcapsid profein (MCP) gene described by Cameron (1990).This plasrnid plus 100 ng of phage lambda ONA werenick-translated with a32P-dATP to high specific activityand purified on a Sephadex G50 column. Initially; blotswere prehybridized for at least 4 h at 37 °C in hybridiza-tion buffer (50-rnM HEPES, 0.02% Ficoll 400, 0.02% bovineserum alburnin, 0.02% polyvinylpyrrolidone, 0.1% SOS)with 50% formarnide and 100 .ug/mL of salmon spermONA followed by hybridization overnight under fue sameconditions. Blots were subject to stringent washes at 63 °Ctwice for 1 h each, followed by aut6radiography.Following this, blots were stripped in 0.4-M NaOH at45 °C, neutralized in 200-rnM Tris-HCI (pH 7.0), 0.1 x SSC,0.10;0 50S and rinsed in 2 x SSC. Blots were then prehy-bridized and hybridized as before but at a lower strin-

Bioassay to detect covert IV infections

Bioassay methods generally followed those given previ-ously (Williams 1993). Covert IV infections in Simuliumwere detected following injection of individual blackflylarval homogenates into a permissive lepidopteran whichsubsequently developed a patent infection. Briefly, 50Simulium larvae from each sample were thawed and iden-tified (Davies 1968; Crosskey 1991). The length of eachlarva was measured to an accuracy of 0.2 mm using abinocular dissecting microscope at x 40 magnification.These larvae were chosen unsystematically in an attemptto select a subsample representative of the size and speciesdistribution of the sample as a whole. First- and second-instar larvae were not used due to difficulties in speciesidentification of these stages. Each larva was individuallyhomogenized in 130 .uL of antibiotic solution (above) andspun at 1500 g to pellet insect debris. Groups of 8-10 third-instar Galleria mellonella (Lepidoptera: Pyralidae) larvaewere individually injected with ~ 8 .uL of supematant fromeach Simulium larva. This species is highly permissive to alarge number of invertebrate IVs and has become the stan-dard host used for producing large quantities of theseviruses (Ward & Kalmakoff 1991). G. mellonella larvae werereared on artificial diet at room temperature and periodi-cally assessed for patent IV infections. If no overt diseasehad appeared by 21 days postinjection, the result was con-sidered to be negative. Cont¡rollarvae (S. equinum and S.erythrocephalum) taken fromSeacourt Stream at Wytham,

i 4.i 3

30

u 25~~~ 20Se 15 ~J.9. 10 ~Lo.

5

oJan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Month

Fig.l Flow late of fue River Ystwyth during each month of 1992:daily mean [bars] with daily minimum and daily maximum foreach month [lines].

T. WI.LLIAMS450

gency: 20% formamide, 37 °C overnight, followed bywashes in 2 x SSC at 41°C.

Relationships among isolates were further investigatedby calculating fue coefficient of similarity (Dice coefficient)(e.g. Grothues & Tümmler 1991) for pairwise comparisonsof isolate restriction profiles using fue program Molmatch(University of Glasgow) which had been used for similarcomparisons (Williams & Cory 1994). Molmatch calculatesF, fue proportion of fragments of common size which twoprofiles share, using fue formula F = 2 m/(a + b) (Nei & Li

1979) expressed as a percentage. Where m is the number ofrestriction fragments in common and a, b are fue totalnumber of restriction fragments for each isolate. For theEcoRl comparisons, variation in fragment size across fuegel was limited to 5%. Due to fue abundance of fragmentsof HindIll profiles, variation was limited to 2.5% exceptwhen compared to profiles published previously, forwhich fue fragment size variation was limited to 5% fol-lowing fue Molmatch recommendations.

Results

(BaO) levels were low, reflecting fue clean, fast-flowingnature of fue river (Table 1). Levels of nitrogen consistent-ly increased towards fue downstream site. The concentra-tion of metals (mostly zinc) increased sharply (x 10)between fue upstream and central blackfly sampling sites(Esgair Wen bridge and Llanafar bridge) where fue riverflows through a disused mining afea. Oaily mean flowrates fell steadily through fue spring and dropped to as lit-tle as 0.12 m3/ s in July before increasing again in fueautumn (Fig. 1). It became evident during sequential visitsto the river that changes in the river flow rate had markedeffects on the total area available for larval blackfly devel-opment at each site. By July, river flow was restricted tochannels in the river bed at the central and downstreamsite which mar be responsible for fue sharp decline in fuedensity of larvae recorded in fue August sample.

Larval population densities increased by approximate-Iy two orders of magnitude at all sites between March andJune before falling by an order of magnitude in late sum-roer. The highest larval density was observed at the down-stream site (Pentre-llyn) but blackfly distribution waspatchy here. The central site (Abermagwr) actually sup-ported Iarger populations of the larvae and for most of thesampling period had fue highest larvaI densities.

In March, fue dominant species was S. variegatum (62%)followed by S. reptans (270;0). Larvae of S. agyreatum, S.

Blackjly sampling and river data

For all the water samples, leve¡s of dissolved oxygen werehigh (90-106% saturation) and biological oxygen demand

Table 1 Details ofwater chemistry of fue

RivJ;r Ystwyth duringth~ sampling penad in1992 at three sitescorresponding to fuethree blackflysampling sites

TotalBOD(mg/L)

Totaloxidized

nitrogen(mg/L)

Water

temp(OC)

Totaldissolvedmetals(Cd + Zn + Pb)

(mg/L)DissolvedO2 (rng/L)

11.810.89.39.2

10.510.2

0.6

1.5

0.9

0.5

0.6

1.2

0.480.280.170.070.120.16

0.0170.0150.0340.0300.0300.006

99

ND151110

1.11.00.81.60.70.5

0.860.860.271.000.370.64

0.2620.4260.3250.3550.3690.449

810

NO151312

11.7

11.6

ND

9.8

9.9

9.7

Upstream (Esgair Wen Bridge)March

ApriIEarIy JuneLate June

SeptemberOctober

Central (Llanafan Bridge)March


SeptemberOctober

Downstream (LlaniIar Bridge)March


SeptemberOctober

1.632.120.682.191.221.84

0.2430.3680.3190.3630.3830.449

99

NO161412

11.8

11.5

9.1

10.4

9.511.4

0.91.61.10.60.50.5

ND = No data

IV INFECTIONS OF BLACKFLIESCOVER 451

ing (c. 70% pupation). None of these larvae developedpatent infections indicating that fue covert form of infec-tion is not an early stage of fue patent disease which wi1ldevelop later in fue life of fue insecto The same result wasreported previously for larvae taken in April (Williams

1993).

Bioassays for covert infection

Of fue 870 larvae bioassayed in this study, covert infec-tions were detected in 17, all of which appeared to be S.variegatum. The striking result was that covert infections ofblackfly larvae were not detected for most of fue surnmerperiod, but appeared in appreciable levels (8-37%) inspring and autumn samples (Table 2). For fue April sam-pIe, bioassay results from fue central site (Abermagwr)were combined with previous data: 2/20 additionallarvaewere positive for covert infection (Table 2) and 8/30 hadbeen demonstrated positive by bioassay previously (total= 50 larvae) (Williams 1993). These two larvae were 6.2and 6.8 rnm in length, compared to a mean length of

intermedium and S. aureum were algo presento In April,

Prosimulium sp. (probably P. tomosvaryi judging from fue

postgenal cleft) was observed at fue central site (17% of

sample) but for most of the study period, S. variegatumwas dominant. In May, S. aureum appeared in the

upstream sample (6%) and in June S. aureum, S. intermedi-

um and S. agyreatum together comprised 34%, 8% and 4%

of fue bioassayed samples for fue upstream, central and

downstream sites, respectively. For fue samples of July,

August and September, S. variegatum was highly domi-

nant and S. intermedium appeared at levels of 4-10% in

September. S. variegatum larvae were distinctive in having

a paJe head capsule with virtually no markings, in contrast

to fue other species for which fue head capsule markings

were well developed, even in fue middle instars. It is pos-

sible, however, that newly moulted larvae mar have been

mistaken for S. variegatum and so fue proportion of other

species in fue samples mar have been slightly underesti-

mated.Larvae taken from the Ystwyth in May, June and

September showed good survival during laboratory rear-

Table 2 Summary of sampling data and bioassay results showing incidence of patent and covert infection during the sampling period inrelation to published findings for the same period

Patent infectedlarvae foundduringsampling

Mean (:1: SE)density oflarvae (m-2)(x 103)

No. of larvaeassayed forcovertinfection

No. of larvaepositive forcovertinfection

Water temp.at time oísample (OC)

Mean (:tSE)length of larvae

bioassayed (mm)Month oi sample

5.OOOOOO

6.5

11

18

18

15

16

10

0.21.34.4

30.814.83.94.3

ND6.7:1: 0.164.6 :1:0.165.5 :1: 0.205.9 :1: 0.166.5:1: 0.105.9 :1: 0.20

30505050505050

oo

oo5+n

71118201812.510.5

1.1 % 0.0922.0 % 3.367.4 % 6.61

131.2 % 12.9124.0 % 12.126.5 %4.125.6 % 2.0

ND6.8 :t 0.145.6 :t 0.094.4 :t 0.204.7 :t 0.175.4 :t 0.196.8 :t0.15

30505050505050

11*2 (+ 8)*OOOO

10 o

Upstream (Blaenycwm)March

AprilMayJuneJulyAugustSeptember

Central (Abermagwr)March


Downstream (Pentre-llyn)March


oo2+OOO1+

0.3 :t18.2:t22.7:t

191.6 :t74.9:t11.2 :t10.4:t

ND5.5 :t 0.165.4:t 0.144.7 :t 0.194.7 :t 0.166.7 :t 0.126.0 :t 0.26

30505050505050

7"ooo1O4

7

11.5

19

21

19.5

1711

ND: No Data. -Data from PCR ~alysis of covert infection with sample size of 30 larvae (Williams 1993); +Patent infections described byWilliams & Cory (1993).

:0.04:0.2:0.7:3.3:1.2:0.8:0.6

:0.05:2.6:3.4: 18.1:6.2:1.7:1.7

452 T. WILLIAMS

1 234 56 7 8 91°11121314151617181 2 3 4 5 6 7 8 9 1011 12131415 161718

(b)(a)

1 2 3 4 5 6 1 891°1112131415161118 Fig. 2 (a) EcoRI restriction endonuclease pro-files for IV isolates from covertly infectedblackfly larvae during fue study compared toAberystwyth IV (Batson et al. 1976). Lanes ofgel as folIows, 1 = Aberystwyth IV, 2-3 =Abermagwr April sample, 4 = Pentre-lIyn Julysample, 5-14 - Abermagwr September sam-pIe, 15-18 = Pentre-lIyn September sample. (b)High stringency Southem blot of EcoRI gel(above) probed with major capsid proteingene fragment of Aberystwyth IV showingstrong hybridization of probe only to variantsof Aberystwyth IV. (c) Low stringencySouthem blot of EcoRI gel (above) reprobedwith Aberystwyth gene probe showinghybridization to variants of September(Pentre-lIyn lile variants) and to a novel virusin lane 3 (Abermagwr, April). Arrowheadindicates fue position of weak band for clarity.

(c)

infections were detected in all stages of fue larvae tested(3rd to final-instar larvae). Control larvae from Oxfordproduced no viral infections in G. mellonella larvae indicat-ing that G. mellonella cultures did not harbour inapparentviral pathogens and that bioassay procedures were suffi-ciently rigorous to prevent cross-contamination amongsamples. To assist in understanding fue incidence of covertinfection early in fue season, Table 2 includes fue data oncovert IV infections detected by PCR in samples of 30 lar-vae from March 1992 (Williams 1993). The incidence of

6.8 mm for fue sample as a whole. During fue high popu-lation density periods of the summer (May, June, July), asingle covertly infected larva was detected. This larvafrom fue downstream site (Pentre-Ilyn) in July was 3 mmcompared to fue sample mean of 4.7 mmo Covertly infect-ed larvae from fue September sample were sized between6 and 7.6 mm (n = 10) compared to fue sample mean of6.8 mm at fue central site (Abermagwr) and 3.5-8 mm long(n = 4) at fue downstream site compared to 6.0 mm for fuesample mean. This range of sizes indicates that covert


1 2 3 4 5 6 1 8 9 10 11 12 1314 15 1611 18

1 2 3 456 7 891°1112131415161118

(c)

Fig. 3 (a) HindIlI restriction endonuclease profilesfor IV isolates from covertly infected blackf1y lar-vae as given in Fig. 2: 1 = Aberystwyth Iv; 2-3 =Abermagwr April sample, 4 = Pentre-l1yn Julysample, 5-14 = Abermagwr September sample,15-18 = Pentre-l1yn September sample. (b) High

stringency Southem blot of HindIlI gel (above)probed with major capsid protein gene fragmentof Aberystwyth IV showing strong hybridizationof probe only to variants of Aberystwyth IV. (c)Low stringency Southem blot of HindIlI gel ,,/reprobed with Aberystwyth IV gene probe show-ing hybridization to variants of September(Pentre-l1yn like variants) and very weakhybridization to apparently novel virus in lane 3(Abermagwr, April) indicated by arrowhead.

patently infected blackfly larvae found during fue sam-pling period and reported by Williams & Cory (1993) isalso noted in Table 2.

variation among isolates (Figs 2a and 3a). There were threeisolates which appeared to be variants of Aberystwyth IV(Kelly et al. 1976): from Abermagwr April, Pentre-llyn Julyand Abermagwr September (lanes 2/ 4/ and 12/ respective-Ir; on fue gels/blots). These isolates had clear restrictionprofile similarities to Aberystwyth IV (coefficients of simi-larity 70-82%) and to one another (coefficients of similari-ty 70-75%) but not to other isolates obtained in the study(Table 3). These Aberystwyth IV variants showed a highaffinity for fue Aberystwyth IV MCP gene probe, but all

Characterization and comparison 01 covert IV isolates

Restriction endonuclease analysis and Southern blotanalysis of fue various covert isolates detected in thebioassays gave very similar results for both HindIlI andEcoRI enzymes. There was 'a marked degree of genetic

454 T

. W

ILLIAM

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differed from Aberystwyth IV in fue size of the restrictionfragment to which the probe hybridized. At high strin-gency, the probe hybridized to a 7-kb Hindlll triplet frag-ment and a 15-kb EcoRI fragment of Aberystwyth Iv; con-sistent with a previous study (Williams 1994) whereas forfue variants, hybridization occurred to 2.4-kb Hindlll and15-kb or 19-kb EcoRI fragments depending on fue variant(Figs 2b and 3b).\

With one exception (Pentre-llyn, April, lane three ofgels) the remaining 13 isolates were all variants of a second(tentative) virus species (coefficients of similarity 76-98%for Hindlll and 72-1000;0 for EcoRI, Table 3) and all dis-played a common low affinity for fue Aberystwyth IVMCP gene probe. Clear hybridization to fue probe wasonly seen at low stringency in fuese variants (Figs 2c and3c). The hybridizing fragments were slightly variable insize but were '" 2.8 kb for fue Hindlll and usually spanned

two fragments of c. 24 kb and c. 21 kb of the EcoRI blots. Bycomparison of fue Hindlll profile of fuese variants withother profiles published previously it is evident that theseare all variants of an isolate from a patently infected black-fly larva found at Pentre-llyn (downstream site) inSeptember 1991 (Williams & Cory 1993): coefficients ofsimilarity of 81-950;0 (Table 2).

The single remaining isolate (Pentre-Ilyn Aprillane 3)showed no restriction profile similarities with any otherisolates in this study, with other isolates from blackflies, orwith any of fue viruses which have been characterized infue same laboratory previously. Therefore, it cannot be alaboratory contaminant. This isolate also showed lowaffinity to fue MCP gene probe which hybridized to a 10.6-kb EcoRI fragment and very weakly to a 5.4-kb Hindlllfragment on the low stringency blots (Figs 2c, 3c).Consequently it appears that this is a novel virus fromblackflies in fue River Ystwyth.

Discussion

Monthly samples of blackfly larvae taken from fue RiverYstwyth, Wales, showed marked changes in fue incidenceof covert IV infection. The incidence of covert infectionwas initially high in March (previous data) but fell in Apriland remained extremely low « 0.5%) during the surnmermonths (May-August) when population densities were attheir highest. Covert infections reappeared at appreciablelevels (5-300;0) in low density over-wintering blackfly pop-ulations in the autumn. Because of fue lack of winter sam-pling, fue relationship between fue generation sampled inSeptember and fue generation sampled fue followingspring is not known, although it is likely that larvae sam-pled in September would over-winter and emerge fue fol-lowing March, given fue lack of blackfly reproduction infue winter months. Another intriguing aspect of fue studywas fue observation that three distinct groups of virus iso-

lates were present in covertly infected blackfly larvae dur-ing fue sampling periodo For two of fuese viruses, high lev-els of genetic variability were evident among variants.Both of fuese viruses have previously been isolated caus-ing overt (lethal) disease in blackfly larvae in fue Ystwyth.Nothing is known about fue ability of fue novel (third)virus to cause patent infections in blackflies. The role ofspatial heterogeneity in fue distribution of larvae was notaddressed here but mar be an additional factor of impor-lance in fue dynamics of fue host-pathogen interaction asreported in other insect virus systems (Dwyer 1991). Thebioassay procedures used were highIy sensitive. Possiblyless than 10 particles are required to initiate a patent infec-tion in this host (Day & Gilbert 1967), a level of detectioncomparable with PCR (Williams 1993). Moreover, bioassayhas an advantage in that large quantities of fue virus resultwhen covert infections are detected (i.e. when patent infec-tions develop in G. mellonella larvae).

This study aimed to answer three questions concerningfluctuations in fue incidence of covertinfections ayer time,fue ability to correlate fuese fluctuations with biotic andabiotic factors and concerning fue number of speciesinvolved in the host-virus interaction. Changes in fue inci-dence of IV infection in blackflies were concurrent withchanges in biotic and abiotic factors: elevated levels ofcovert infection occurred at low population densities, highwater flow rates, low temperatures and presumably slow-er growth rates, as reflected in fue larger larvae of fuespring and autumn samples, although there are insuffi-cient data for correlation. ActualIy, fue study has raisedmore questions than it has answered. Whereas changes infue incidence of covert infection are we11 d~ed, fue rea-"sons behind such changes remain far from clear. High lev-els of covert infection in March and April preceded theobservation of patently infected larvae in May (a11 of theseisolates, covert and patent, were variants of AberystwythIv; bar one). Similarly, covert infections in September werea11 variants of an isolate causing patent disease found atPentre-11yn in September of fue previous year. There is asubstantial part of the story sti11 missing however; whathappens during fue winter months, wherein fue Pentre-11yn like variants of September disappeared and theAberystwyth IV variants re-appeared the fo11owingspring? During fue winter, fue river becomes greatlyswo11en and sampling would be both difficult and haz-ardous. At fue outset of fue study, fue winter was not con-sidered to be particularly interesting to sample, a dormantperiod for the over-wintering stages, therefore no suchsampling was performed. This was unfortunate in retro-

spect.Whereas previous work based on a springtime sample

from fue Ystwyth (Wi11iams 1993) had suggested that fuesystem was a simple S. variegatum - Aberystwyth IV inter-

action, further sampling showed this to be at least a one

456 T. WILLIAMS

Geraldine Gillespie for their diverse help, and Sonya Watts andChris Rowlands of fue National Rivers Authoriiy, Wales for fuewater data.

host - and possibly a three virus system. The specific sta-

tus of each type of virus detected here cannot be deter-mined until proper characterization and comparativestudies are performed. One possible explanation for fueobserved patterns of infection in this study is that otheraquatic species, possibly of completely different inverte-brate taxa such as Crustacea, are involved in the persis-tence of iridescent viruses in the river and in fue transmis-sion of fuese infections to susceptible blackfIy populationsat different times of fue year. Various sympatric aquaticinsect species (e.g. caddis fijes as Simulium predators) werebioassayed for covert IV infections during fue course ofthe study, but not in large numbers and not in a systemat-ic way; rather out of curiosity to find out if any alternativelines of study were worth developing. None proved posi-tive for IV infection. However, the results of this studysuggest that systematic sampling and appraisal of possiblealternative host species in fue river could be worthwhile.Such studies mar reveal additionallayers of complexity inthe blackfIy IV interaction, or mar confirm that fue virus-es in this system persist and are transmitted within black-fIy populations alone.

It appears that in this blackfly system patent and covertinfections are not linked with distinct classes of iridescentviruses. Rather it seems probable that there exists sometriggering mechanism, be it' environmental or innate,which switches fue cornmon covert form of infection intothe patent (lethal) form (Williams 1993). In baculoviruses,stress factors such as food quality, crowding, coinfectionby heterologous viruses are cornmonly cited as supposedswitches for fue expression of latent infections (Entwistle& Evans 1985; Hughes et al. 1993). It is not known if covertinfection has a detrimental effect on the viability or repro-duction ofblackflies. In laboratory experiments with a lep-idopteran-baculovirus system, the presence of virus hasbeen shown to alter both fue magnitude and periodicity ofhost population cycles (Sait et al. 1994a). It is assumed thatthis was due to sublethal effects of virus infection on mothdevelopment rate and reproductive capacity which weremeasured separately (Sait et al. 1994b).

Because of fue great interest in pathogens for insect bio-control, wherein high virulence is fue most desired feature,very few studies address dynamics of host-parasite sys-tems wherein the infection is mostly innocuous.Understanding fue ecology of invertebrate pathogensmeans recognizing that duality mar exist in the strategy ofhost exploitation by fue virus. Such duality is particularlyevident in iridescent virus infections of blackflies.

Acknowledgements1 am very grateful to fue following people who have assisted meduring fue course of this study, namely: Tlffi Carty for rus worksupplying Galleria larval)!, Chris Hatton for rus quick photo-graphic service, Simao VaSconcelos, Rosie Hails, Pablo Liedo, and

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