Entomologia Experimentalis et Applicata 101: 123–129, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Oviposition in Culicoides impunctatus under laboratory conditions

S. Carpenter, A. J. Mordue (Luntz) & W. MordueDepartment of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen, AB24 2TZ, UK

Accepted: September 13, 2001

Key words: Culicoides impunctatus, oviposition, colonisation, site selection

Abstract

We present a laboratory-based examination of oviposition preference in the Scottish biting midge, Culicoidesimpunctatus (Diptera: Ceratopogonidae). A variety of oviposition substrates were screened in no-choice andchoice bioassays for efficacy in eliciting an egg-laying response. Both upper-layer photosynthetic Sphagnumspp. moss and Juncus articulatus infusions were identified as producing increased oviposition under no-choiceconditions. During choice trials against a control of damp cottonwool, upper-layer Sphagnum spp. moss produceda significantly greater egg-laying response. These conclusions are interpreted in terms of possible cues involved inoviposition site selection and assessed for future use in colonisation of this troublesome species.

Introduction

Culicoides impunctatus causes economically signifi-cant disruption to both forestry and tourist industriesthroughout the Scottish Highlands (Hendry & God-win, 1988). Despite recent progress in the study ofC. impunctatus in the field (see Blackwell, 1997 forreview), laboratory study and eventual colonisationof C. impunctatus is necessary to allow study of alllifecycle stages without the restrictions imposed bya relatively short adult season. In addition colonisa-tion would allow further exploration of host locationand aggregation cues in adults of defined age andphysiological state, a field that has already yieldedcompounds that increase trapping efficiency of parousfemales (Bhasin et al., 2000a, b).

Oviposition by C. impunctatus has never been ob-served under natural conditions due both to smalladult size and the vast breeding areas available tothis species. This combination has made Culicoidesoviposition investigations notoriously difficult to con-duct in the field (Kettle, 1977). Significantly, the onlystudies on oviposition in Culicoides have concernedspecies that lay eggs in localised conditions such as an-imal dung (e.g., Culicoides brevitarsis Kieffer; Bishopet al., 1996). Work examining the larval distribution ofC. impunctatus, however, suggests associations with

low soil pH, high organic and water content, and thepresence of mosses (Sphagnum spp.), rushes (Juncusspp.) and bog myrtle (Myrica gale) (Kettle, 1961;Blackwell et al., 1994, 1999). Oviposition in C. im-punctatus under laboratory conditions had not beeninvestigated since M.A. Hill’s study (1947), whichmaintained that eggs could be obtained from gravid fe-males using damp filter paper as a substrate, a methodlater used in the culture of many other Culicoidesspecies (e.g., Boorman, 1974).

A wide range of exogenous cues are utilised byfemale Diptera in the location of suitable oviposi-tion sites and, indirectly, larval habitats. Mosquitoes,for example, have been found to utilise visual cuessuch as the colour and optical density of the substrate,tactile factors such as substrate texture and chemi-cal stimuli such as those utilised by adult flies layingpreferentially in the presence of conspecific larvae(Bentley & Day, 1989) or habitat infusions (Milleret al., 1992; Mordue (Luntz) et al., 1992; Blackwellet al., 1993). These factors tend to constitute a highlycomplex system of synergistic relationships within theoverall process of oviposition. Further complexity hasalso been observed in the behaviour of phytophagousflies such as Cecidomyid midges which often requirea series of long range and contact cues in specific

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sequences to effect oviposition (Galanihe & Harris,1997; Crook & Mordue (Luntz), 1999).

The aim of the study was to investigate ovipositionpreferences of C. impunctatus with a view to futurecolonisation. The experiments performed utilised awide range of substrates of increasing complexity inorder that deductions could be made concerning thecues required for oviposition.

Materials and methods

The study site was located on the Ormsary Estate, nearLochgilphead, Argyllshire, NR 743 724 (56◦ N 5◦ W).A room measuring 2.2 m width × 2.8 m length witha sloping roof giving an effective height of 2.2 m to3 m, was utilised as an isolated constant temperature,humidity and photoperiod area for the project (23 ±4 ◦C/60–80% r.h./L18:D6 photoperiod).

Parous female midges were caught from the sur-rounding area and fed on the collector at ambienttemperature in darkness. Midges were then chilledand fully replete individuals selected and stored foruse in oviposition and survival trials. Cuboid cages(30 cm3 volume) with top, left and right faces net-ted, base and back faces plywood and an entrance facemade of perspex, were used to house cylindrical pill-boxes (diameter 6 cm: height 5.5 cm) containing fiftymidges each. Humidity was maintained at 95–100%by placing two layers of cotton sheeting measuring120 cm by 60 cm wet with 100 ml boiled, warm tapwater on all sides except the base and entrance faces.Evaporation from the cotton was reduced by the useof a plastic sheet as the outer layer on all sides ofthe cages. Midges were supplied with 20% sucrosesolution (w/v) during the post-blood meal period viaa cotton wool pad measuring 2 cm length by 1.5 cmwidth.

No-choice bioassays. In no-choice bioassays gravidfemale midges were introduced into a pillbox that hada 2.5 cm diameter cylindrical plastic vial attached tothe base (see Figure 1). Cotton wool was packed intothe vial to just below the base of the pillbox and wetwith 6.5 ml of distilled water, forming a standarddesign to which alterations could be made to test avariety of oviposition cues.

In the first trial experiments were designed usingthe damp cotton wool and filter paper approach pre-viously utilised in Culicoides colonisation (Boorman,1974) but with variations in design to investigate the

possibility that reduced egg laying on standard filterpaper was due to increased adult mortality throughdrowning. To this end vials containing the standardbase described above, or with a circular covering offilter paper on top of the wet cottonwool, or withan additional arch of filter paper over the filter pa-per disc, were tested as suitable oviposition sites. Itwas thought that the latter arrangement would allowmidges to approach the oviposition substrate moreeasily without becoming trapped by surface tension.Ten midges were introduced into each pillbox at fivedays post-blood meal (PBM) and the total eggs laid ineach treatment recorded after seven days PBM. Eightreplicates of each treatment were completed in total.Differences between treatments in the mean numberof eggs laid were then examined using a one wayANOVA.

In addition to this experiment a second trial ex-amined eight separate substrates which were screenedin no-choice bioassays for oviposition acceptability.Control substrates in this trial consisted of (1) the stan-dard cottonwool base described above and (2) the stan-dard base plus circular filter paper treatment. Six otheradditions or alterations to the standard base were thenmade to allow investigation of various cues. Theseconsisted of preparing standard bases with (3) drops ofa 0.5% solution of India ink to darken the cottonwoolsubstrate, or (4) breeding site water, or (5) Juncus ar-ticulatus, or (6) Sphagnum spp. infusions instead ofdistilled water. Additionally, substrate (7) had a layerof green, photosynthetic, upper layer Sphagnum spp.moss placed on top of the standard damp cottonwoolbase and (8) a layer of lower layer, dead Sphagnumspp. moss similarly added. Sphagnum spp. moss andbreeding site water used during the trials were takenfrom C. impunctatus breeding areas as previously de-fined (Blackwell et al., 1994, 1999). Infusions ofJ. articulatus were prepared by first washing 2 g of theplant with distilled water and then cutting spears into1cm lengths. These were placed in a bottle with 200 mldistilled water, shaken and left for four days to fermentin order to mimic the natural decay process as exam-ined in previous studies using mosquitoes (e.g., Milleret al., 1992; Mordue (Luntz) et al., 1992; Blackwellet al., 1993). Before use, the resulting mixture wasfiltered through a sieve and solid material discarded. Asecond infusion was prepared in an identical fashionusing an equal weight of Sphagnum spp. (layers notdistinguished).

Eight replicates of each substrate were performedduring each trial using a single midge in each replicate

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Figure 1. Bioassay system used to investigate oviposition by C. impunctatus under (a) no-choice and (b) choice conditions.

Table 1. Variation in the percentage of female C. impunctatus laying eggs in no-choicebioassays according to acceptability of oviposition substrate. Where oviposition oc-curred, the percentage of laid eggs in relation to the total egg-batch was calculated(see Figure 3 for further details)

Treatment n % (n) midges Mean % egg batch

laying eggs laid by females (±SE)

Standard damp cottonwool base 24 29.2 (7) 49.3 ± 15.9

Filter paper disc 24 16.6 (4) 61.1 ± 13.2

Dyed standard base 24 25.0 (6) 61.3 ± 17.6

Breeding site water 24 12.5 (3) 41.6 ± 18.6

Juncus articulatus infusion 24 54.2 (13) 78.7 ± 12.6

Sphagnum spp. infusion 24 25.0 (6) 58.5 ± 22.9

Upper layer Sphagnum spp. 24 87.5 (21) 97.9 ± 1.7

Lower layer Sphagnum spp. 24 20.8 (5) 83.6 ± 17.0

with three complete trials being carried out. Midgeswere introduced at five days PBM with numbers ofeggs laid being recorded each day post-introductionuntil the seventh day PBM. At this point all midgeswere killed, dissected and the retained eggs counted.The proportion of total eggs laid was calculated andanalysed using a Kruskal–Wallis ANOVA due to anarcsine transformation failing to normalise the dataset. Conover’s (1999), post-hoc test was then em-ployed to separate out differences between substrates.In addition, following dissection, the proportion ofeggs laid of the total batch developed was also cal-culated for those midges ovipositing in each trial.Mean values were calculated for each substrate. Thesedata were then examined using G-statistic contingencytable analysis.

Choice bioassays. Four experiments were performedto assess levels of choice in C. impunctatus when dif-ferent substrates were available. In these experiments,ten female midges were introduced into pillboxes thathad two vials attached to the base to provide a choiceof oviposition substrate (Figure 1). Comparisons weremade against the standard damp cottonwool base(Substrate 1 in no-choice bioassays), using India inkdarkened cottonwool (Substrate 3), J. articulatus infu-sion (Substrate 5) or upper layer Sphagnum spp. moss(Substrate 7) treatments. These were prepared as forthe no choice bioassays. A double blank standard cot-tonwool control was also run to test for intrinsic biasin the trials. Dissections of midges were in this casenot made and comparisons were based simply on thenumber of eggs laid.

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Figure 2 Variation in the number of eggs laid by ten gravid females of C. impunctatus according to filter paper presence and arrangement on astandard base of cottonwool wet with 6.5 ml distilled water (n = 8 in all cases).

In these experiments the number of eggs laid ontreatment and control substrates were converted toproportions of total eggs laid. These proportions werethen arcsine transformed, if required, and analysedusing paired t-tests.

Results

No-choice bioassays. Dissection of all females (n =192) in the second no-choice trial revealed the av-erage number of eggs developed by parous femaleC. impunctatus to be 20.1 (range 8–39; SE ± 0.5). Dif-ferences were observed in both the number of midgeswhich laid eggs and the proportion of the egg batchlaid in those that did, according to substrate (Table 1).No significant differences were found between the cot-tonwool and filter paper arrangements used (F2,24 =0.140; P = 0.870) (Figure 2). No significant differencewas found between the proportion of available eggslaid in the three sets of replicates completed (t = 2.67;P = 0.263), so results were pooled for analysis. A

significant difference was found in the proportions ofeggs laid on the substrates utilised in the second trial(t = 57.65; P =≤ 0.0001) (Figure 3). Post-hoc test-ing identified upper layer Sphagnum spp. as having asignificantly higher proportion of eggs laid than anyother substrate (P =≤ 0.001 in all cases). J. articulatuswas identified as having a significantly higher propor-tion of eggs laid than all other substrates (P =≤ 0.05),with the exception of upper layer Sphagnum spp. moss(Figure 3). G-statistic analysis comparing the propor-tions of eggs laid on each substrate by only thosemidges laying eggs showed also that, on inclusionof all substrate data there was a highly significantdifference between substrates (G = 47.36; P < 0.001).When the data from upper layer Sphagnum was re-moved P remained significant (G = 12.881; P < 0.05)but marginally so. Further removal of J. articula-tus infusion data led to a non-significant result beingobtained (G = 2.78; P > 0.05).

Choice bioassays. Significant differences were foundbetween the number of eggs laid on upper layer Sphag-

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Figure 3 Variation in the proportion of eggs laid by single, gravid female C. impunctatus in relation to the oviposition substrate used, inno-choice bioassays (a vs b: P≤ 0.05; b vs c: P≤ 0.001; a vs c: P≤ 0.001) (n = 24 in all cases).

num spp. moss in comparison to cottonwool in thepill box choice tests (t = 20.37; P =≤ 0.001). Nosignificant difference was found between the doubleblank control (t = 0.32; P = 0.758), dyed and controlcottonwool (t = 0.478; P = 0.640) or J. articulatusinfusion treated and control cottonwool (t = −0.79;P = 0.441) (Table 2).

Discussion

In both choice and no-choice trials upper layer Sphag-num spp. moss was found to stimulate the greatestproportion of females to lay eggs. Additionally, inthose females that did oviposit, few eggs were re-tained. The two types of Sphagnum spp. substrateoffered in trials to C. impunctatus represent a highlycomplex combination of possible cues in comparisonto the other, relatively simple substrates used. Whileit is not possible to make deductions regarding the

large variety of cues probably being used in selectionof upper layers of Sphagnum spp. it is interesting tonote that midges did not consistently oviposit uponthe lower layers of Sphagnum spp. that are normallynot exposed to C. impunctatus adults. It is likely that,in the laboratory at least, visual stimuli such as colouror substrate architecture, perhaps in combination withan olfactory component of a stimulatory or inhibitorynature, combine to make one substrate acceptable andthe other not. Damp filter paper substrates yielded feweggs in relation to the number of insects introducedand would not provide a viable means of initiatinglaboratory colonies. Arranging filter paper to allowmidges to orientate themselves for oviposition did notsignificantly increase the number of eggs laid on thesubstrate by gravid females.

Breeding site water did not stimulate ovipositionwhen used in place of distilled water to wet cotton-wool in no-choice trials. Similarly, Sphagnum spp.

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Table 2. Variation in the number of eggs laid by ten gravid females per replicate in choicebioassays between treatment and control vials

Treatment (a) vs Control (b) Mean number of Treatment % n P

Oviposition Substrates eggs laid/substrate Eggs Laid (a) vs (b)

(Mean±SE) (Mean±SE)

Damp cottonwool control (a) 6.9 ± 1.8 46.6 ± 14.3 8 0.758

Damp cottonwool control (b) 7.9 ± 2.6

Dyed cottonwool (a) 8.0 ± 2.1 53.1 ± 8.0 16 0.653

Damp cottonwool control (b) 7.1 ± 1.6

Juncus articulatus infusion (a) 23.1 ± 5.2 42.9 ± 8.6 16 0.441

Damp cottonwool control (b) 27.2 ± 5.3

Upper layer Sphagnum spp. (a) 118.1 ± 10.8 90.0 ± 7.9 16 ≤0.001

Damp cottonwool control (b) 13.6 ± 2.9

infusions made up with a combination of both up-per and lower layers of Sphagnum spp. failed toproduce an increased oviposition response. Juncus ar-ticulatus infusions, however, significantly increasedthe proportion of eggs laid in no-choice trials whencompared to distilled water/cottonwool controls andin addition caused an increase in the proportion of fe-males ovipositing. This could be due to the influenceof olfactory rather than contact components used foroviposition site location which, in the small arenasused in the trials, could explain why no significant dif-ference was found during choice bioassays. The originof these cues may be found in the process of bacterialgrowth and subsequent production of metabolites intreatment vials containing the infusion. This has beenfound to occur in mosquito oviposition sites in both thelaboratory (Miller et al., 1992; Mordue (Luntz) et al.,1992; Blackwell et al., 1993) and the field (Mboeraet al., 1999). The large variation found within the J. ar-ticulatus infusion data set and the lack of responserecorded for breeding site water and Sphagnum spp.infusions may be due at least in part to variation in theconcentration of specific active components. Isolationand characterisation of these components would allowquestions of both concentration and plant specificity tobe addressed.

Darkening substrate colour was not found to affectoviposition in comparison to untreated cottonwool.Dyed substrates have previously been shown to stim-ulate oviposition in many species of biting flies thatcommonly lay their eggs on open water (e.g., Culexquinquefasciatus; Beehler et al., 1993). Culicoides im-punctatus showed no such preference, however, in

either choice or no-choice bioassays. This togetherwith its small size, vulnerability to drowning on sur-face films under laboratory conditions and the lackof correlation between larval density and open watermakes it unlikely that C. impunctatus utilises openwater for oviposition.

Extrapolations to the oviposition behaviour ofC. impunctatus in its natural environment from lab-oratory evidence must be made tentatively. The factthat C. impunctatus larval distribution has been foundto follow that of Sphagnum spp. moss and J. ar-ticulatus both of which have been shown to induceincreased oviposition in different formulations doesindicate, however, that these substrates may play a rolein natural oviposition. To establish a definite causallink in the degradation process of J. articulatus andpresence of upper layer Sphagnum spp. moss withC. impunctatus oviposition will require further study.

Ultimately the identification of upper layer Sphag-num spp. moss and additionally J. articulatus infusionas highly successful oviposition substrates will allowas yet unexplored areas of the biology of C. impunc-tatus to be examined and represents a step forwardstowards eventual colonisation of this species.

Acknowledgements

SC was supported by the Ormsary estate, Argyllshire& University of Aberdeen.

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References

Beehler, J. W., J. G. Millar & M. S. Mulla, 1993. Synergism betweenchemical attractants and visual cues influencing ovipositionof the mosquito, Culex quinquefasciatus (Diptera: Culicidae).Journal of Chemical Ecology 19: 635–644.

Bentley, M. D. & J. F. Day, 1989. Chemical ecology and behaviouralaspects of mosquito oviposition. Annual Review of Entomology34: 401–421.

Bhasin, A., A. J. Mordue (Luntz) & W. Mordue, 2000a. Electro-physiological and behavioural identification of host kairomonesas olfactory cues for Culicoides impunctatus and C. nubeculosus.Physiological Entomology 25: 6–16.

Bhasin, A., A. J. Mordue (Luntz) & W. Mordue, 2000b. Re-sponses of the biting midge Culicoides impunctatus to acetone,CO2 and 1-octen-3-ol in a wind tunnel. Medical and VeterinaryEntomology 14: 300–307.

Bishop, A. L., H. J. McKenzie, I. M. Barchia & A. M. Harris, 1996.Effect of temperature regimes on the development, survival andemergence of Culicoides brevitarsis Kieffer (Diptera: Cerato-pogonidae) in bovine dung. Australian Journal of Entomology35: 361–368.

Blackwell, A., 1997. Recent advances on the Scottish bitingmidge, Culicoides impunctatus Goetghebuer (Diptera: Cerato-pogonidae). Memoirs of the Entomological Society of Washing-ton 18: 78–83.

Blackwell, A., A. J. Mordue (Luntz), B. S. Hansson, L. J. Wad-hams & J. A. Pickett, 1993. A behavioural and electrophysi-ological study of oviposition cues for Culex quinquefasciatus.Physiological Entomology 18:343–348.

Blackwell, A., M. R. Young & W. Mordue, 1994. The microhabitatof Culicoides impunctatus (Diptera: Ceratopogonidae) larvae inScotland. Bulletin of Entomological Research 84: 295–301.

Blackwell, A., K. A. Lock, B. Marshall, B. Boag & S. C. Gordon,1999. The spatial distribution of larvae of Culicoides impunc-tatus biting midges. Medical and Veterinary Entomology 13:362–371.

Boorman, J., 1974. The maintenance of laboratory colonies of Culi-coides variipennis (Coq.), C. nubeculosus (Mg.) and C. riethi

Kieff. (Diptera: Ceratopogonidae). Bulletin of EntomologicalResearch 64: 371–377.

Conover, W. J., 1999. Practical Non-parametric Statistics, 3rdedition. Wiley, New York.

Crook, D. J. & A. J. Mordue (Luntz), 1999. Olfactory responses andsensilla morphology of the blackcurrent leaf midge Dasineuratetensi. Entomologia Experimentalis et Applicata 91: 37–50.

Galanihe, L. D. & M. O. Harris, 1997. Plant volatiles mediatehost-finding behaviour of the apple leafcurling midge. Journalof Chemical Ecology 23: 1639–1655.

Hendry, G. & G. Godwin, 1988. Biting midges in Scottish forestry:a costly irritant or trivial nuisance? Scottish Forestry 42: 113–119.

Hill, M. A., 1947. The life-cycle and habits of Culicoides impunc-tatus Goetghebuer and Culicoides obsoletus Meigen, togetherwith some observations on the life-cycle of Culicoides odibilisAusten, Culicoides pallidicornis Kieffer, Culicoides cubitalisEdwards and Culicoides chiopterus Meigen. Annals of TropicalMedical Parasitology 41: 55–115.

Kettle, D. S., 1961. A study of the association between moorlandvegetation and breeding sites of Culicoides (Diptera, Cerato-pogonidae). Bulletin of Entomological Research 52: 381–411.

Kettle, D. S., 1977. Biology and bionomics of bloodsucking Cerato-pogonids. Annual Review of Entomology 22: 33–51.

Mboera, L. E. G., K. Y. Mdira, F. M. Salum, W. Takken & J. A.Pickett, 1999. Influence of synthetic oviposition pheromone andvolatiles from soakage pits and grass infusions upon oviposi-tion site-selection of Culex mosquitoes in Tanzania. Journal ofChemical Ecology 25: 1855–1867.

Miller, J. G., J. D. Chaney & M. S. Mulla, 1992. Identifica-tion of oviposition attractants for Culex quinquefasciatus fromfermented Bermuda grass infusions. Journal of the AmericanControl Association 8: 11–17.

Mordue (Luntz), A. J., A. Blackwell, B. S. Hansson, L. J. Wadhams& J. A. Pickett, 1992. Behavioural and electrophysiological eval-uation of oviposition attractants for Culex quinquefasciatus Say(Diptera: Culicidae). Experientia 48: 1109–1111.