Exploration and assessment of the oviposition substrate by the cabbage root fly , Delia radicum ( Dip ter a : Anthomyiidae )

Oviposition behaviour of Delia radicum is not only influenced by host plant quality but also by the quality of the substrate in which the plant grows. Direct behavioural observations showed that the females partition their visits to a host plant (cauliflower) into ovipositional bouts separated by exploration of the host plant surface. Ovipositional bouts were further partitioned into acts of egg deposition separated by exploration of the substrate. While the mean number of ovipositional bouts per visit (2.6), and eggs laid per egg deposition event (1.4) were stable, the mean number of egg deposition events per ovipositional bout significantly varied (from 2.1 to 7.3) with the quality of the substrate and the physiological state of the female (egg load). Ovipositing females adjusted the final number of eggs laid around the plant during the behavioural stage of substrate exploration. Additional experiments using plant surrogates treated with methanolic extract of Brassica leaves mounted in different substrates showed that: (a) the presence of living Brassica, Hordeum or Allium roots in a substrate enhances the number of eggs laid into this substrate, but females do not dis­ criminate between the different plants; (b) females avoid both wet and dry substrates and prefer the substrates with a dry surface and moist particles directly accessible at a depth of about 5 mm; (c) substrates rich in organic matter are preferred to sand; (d) olfactory perception of volatile chemicals from the substrate must at least partially be responsible for the differences in oviposition in various substrates.


INTRODUCTION
The chain o f activities o f a herbivorous insect foraging for a suitable host plant can be viewed as a sequence of behaviours including finding, examining and consuming (sensu Miller & Strickler, 1984).All o f these behaviours have been studied in some detail in the cabbage root fly, Della radicum (L.) (Diptera: Anthomyiidae) (reviewed by Nottingham, 1988;Roessingh et al., 1992).The finding behaviour is dominated by the optomotor-guided anemotaxis elicited by olfactory signals which emanate from host plants o f the Cruciferae family (Finch & Skinner, 1982;Nottingham, 1988) and, closer to the plant, also by its visual characteristics (Prokopy et al., 1983).N on volatile, host plant-specific chemicals play a major role during examination o f the plant surface (Traynicr, 1967;Roessingh et al., 1992;1997;Baur et al., 1996aBaur et al., , 1998;;Hurter et al., 1999).However, acceptance is also signifi cantly influenced by other plant characteristics such as plant height, stem presence, leaf form, size, colour, vena tion and surface structure (Zohren, 1968;Roessingh & Stadler, 1990;Košťál, 1993a;Degen & Stadler, 1997) and also by the plant's surroundings (Košťál & Finch, 1994).The behavioural sequence performed by females during plant examination (landing on leaf, walking on leaf, descending the stem, circling the stem base) has been described in detail by Zohren (1968) and further quantified by Hopkins (1994).Stadler & Schoni (1990) found that the same behavioural sequence is performed by females on a plastic model plant coated with paraffin wax and sprayed with ethanolic extract o f cabbage leaves.
The female cabbage root fly normally oviposits into the soil-substrate adjacent to the host plant.It has been shown that the decision to accept a plant as host is reached mainly during examination o f the plant's leaves and stem (Zohren, 1968;Städler & Schöni, 1990;Hopkins, 1994).Egg deposition behaviour, though receiving much less at tention than plant examination, has been previously de scribed and it was observed that the females explore the soil surface with their ovipositor extended (Zohren, 1968).It was found that females do evaluate some traits o f the substrate, prefering to lay eggs into sand with a structure loose enough to allow for easy penetration with their ovipositors.They also prefer specific moisture con ditions and shaded areas (Traynier, 1967;Zohren, 1968;Havukkala, 1982).Furthermore, Baur et al. (1996b, c) found that sensory cues originating front the substrate, in cluding the plant roots, allow the ovipositing cabbage root Hies to discriminate plants with undamaged roots from those with roots damaged by conspecific larvae.Thus, oviposition substrate and sensory cues not originat ing from the aerial plant parts might considerably influ ence egg deposition behaviour and modify the final output o f the host-plant foraging process, the number o f eggs laid by an individual cabbage root fly female around the stem-base o f the plant.Nevertheless, the behaviour o f cabbage root fly females when on the substrate surround ing the plant stem was, according to Zohren (1968), diffi cult to quantify exactly and for this reason studies have remained incomplete.
In the present study, we assessed the substrate-related egg deposition behaviour o f cabbage root fly females un der laboratory conditions.The behavioural mechanism by which females adjust the num ber o f deposited eggs to the quality o f the substrate is described.Plant surrogates treated with methanolic surface extracts o f host-plant leaves and mounted on various modified substrates were used in dual-choice oviposition assays to characterize the effect o f different features o f the substrate such as the presence o f host or non-host roots, organic matter, sub strate moisture and to distinguish between olfactory and contact cues.

Composition of substrates
The substrate in the pot was divided into two layers: (1) a top layer into which the flies oviposited and, (2) a bottom layer which filled most of the pot volume (Fig. 1).Both layers were variously modified.Fig. 1.The oviposition top layer (dotted area) in each pot (containing either the plant or plant surrogate) was arranged in three different set-ups: (A) "paper" -moist filter paper sheet covered with a layer (< 0.5 cm deep) of dry, sterilized sand; (B) "plastic" -two plastic foils covered with a layer (2 cm deep) of dry, sterilized sand; (C) "olfactory" -plastic wire mesh (0.5 mm) situated 2 cm above the bottom layer and cov ered with a layer (< 0.5 cm deep) of dry, sterilized sand.The bottom layer (black area) was formed (depending on experi ment) by either watered growth substrate with or without roots of three different plants, or sterilized sand which was either wa tered or not.Further details arc in the text.
Three different set-ups of the top layer were used for experi ments (Fig. 1); these will be referred to as "paper" (A), "plastic" (B) and "olfactory" (C): (A) "paper" -the bottom and top layers were separated by one sheet of moist filter paper.A thin (<0.5 cm) layer of a dry, sterilized sand (limestone, particles 1-5 mm) was used as the top layer.In this set-up the filter paper was continuously sup plied with moisture from the bottom layer and thus stayed moist throughout the assay while the sand particles forming the top layer remained dry.The females could directly touch (by tarsi, proboscis or ovipositor) the moist filter paper between the sand particles.
(B) "plastic" -the bottom and top layers were separated by two layers of thick polyethylene plastic foil tightly surrounding the stem.A thick (2 cm) layer of dry, sterilized sand was used as the top layer.In this set-up, the females could directly touch only dry sand while the penetration of volatile cues from the substrate bottom layer was prevented/minimized by the plastic foil.
(C) "olfactory" -the bottom and top layers were separated by a 2 cm-wide empty space.A plastic wire mesh (hole diam.0.5 mm) was positioned (supported by a plastic ring) 2 cm above the bottom layer.A thin layer of dry, sterilized sand was used as the top layer.In this set-up the females were allowed to touch directly only dry sand, but perception of any volatiles released from the bottom layer of the substrate was possible.
Ten different modifications of the substrate (combinations of various top and bottom layers) were used in dual-choice ovipo sition assays.These are briefly summarized in Table 1 but some more details follow below: (I), ( 2), (3) The plants [(1), one cauliflower; (2), densely grown barley; or (3), two leeks, respectively] were cut and the above-ground plant parts were removed.The remaining rootsubstrate complexes formed the bottom layer in the pot.The top layer was arranged as for "paper".
(4) The same substrate as that used for growing plants.
(5) , ( 6), (7) The bottom layer was formed by sterilized sand, either watered (5), (6) or not (7).The sand used as top layer in (6) was dipped into water before it was filled into a pot.Such a set-up assured that not only the filter paper, but also the top layer of sand stayed wet throughout the assay [(contrast with (5)].
Bottom layers of all the substrates except (7) were watered close to the maximum sorption capacity of the substrate prior to the experiments.Watering, together with high relative humidity of the air in the climate controlled chamber and standardized top layer set-up, minimized potential differences in water evapora tion rates from the different substrates.

Plant surrogates
The design by Roessingh & Stadlcr (1990) was used.The sur rogates were made of green paper with vertical folds (projection area 45 cm2) coated with a thin layer of paraffin wax and sprayed with 2 glc (gram leaf equivalents) of the methanolic surface extract of kale leaves prepared according to the method of Stiidler & Roessingh (1991).

Behavioural observations
Females of two physiological categories were used: 1) naive flics, 5 to 6 days old, without previous contact with a host plant, 2) experienced flies 7 to 14 days old, continuously exposed to a cauliflower plant before the trials.Females in the former cate gory most likely carried a first complete load of mature eggs in All substrates except (7) were watered close to the maximal sorption capacity of the substrate prior to the experiment.
Those in the latter category had probably already laid some of their eggs on the cauliflower plant (Košťál, 1993b; see also Fig. 1B for developmental profile of oviposition rate on cauliflower).Twenty-five females, marked on five thorax positions using five different colours ("Bee marks"), were introduced into an empty acrylic plastic cage (50 x 50 x 50 cm) 30 min before the start of observation.The observational session always started at 07:30 h, when a plant (cauliflower) was introduced into the cage, and ended at 09:30 h.To minimize any plant effects, only three cauliflower plants of similar age, size and architecture were subsequently used to collect all behavioural data.The be haviour of individual females was recorded without disturbance from the moment they landed on the plant until they spontane ously flew away from the plant and landed on the cage wall.Particular attention was paid to the female's behaviour close to or directly on the substrate surface where four typical behav ioural stages, first described by Zohren (1968), were distin guished: (1) stem base circling -female, heading down, makes more or less complete circles around the stem base; (2) substrate run -simple, relatively straight and rapid move ment across the substrate; (3) substrate exploration -slow and curved movement fre quently interrupted by a short stop lasting a few seconds when ovipositor is inserted into a crevice; (4) egg deposition -motionless for a few minutes with ovi positor inserted into a crevice.
The duration of each act of egg deposition was recorded using stop-watches.When the female left the plant after having fin ished ovipositing, the sand from around the plant stem was transferred to a plastic beaker, the eggs were separated by flota tion and counted.The data come from different individuals, since no female was used more then once.During a single 211long observational session, only a few (3-6) complete behav ioural sequences could be collected because (a) only a low proportion of females landed on the plant, (b) the duration of single complete sequence was too long (10-40 min approxi mately) and (c) only one single female was observed at a time; the other females which landed on the plant and were about to start their preoviposition behaviour were removed out from the cage and excluded from further experiments in order to avoid interactions between flies.The data collected during 24 observa tional sessions were pooled to yield 81 complete behavioural se quences including egg deposition.

Oviposition assays
A no-choice assay was performed in two cages (50 x 50 x 50 cm) made of acrylic plastic.Twenty five pairs of one-day old flies were placed into each cage.On the third day after emer gence, at 16:00 h, one plant with either the "paper" or "plastic" substrate was introduced into each cage and females were al lowed to oviposit for the next 24 h.Every day, at 16:00, the eggs were collected, counted after which the substrate set-up was renewed.The assays were terminated after day 11, before females began to die.The experiment was repeated with new flies and the same plants but with a switch in the substrate set ups (i.c. the plant that had a "plastic" in the first run, received a "paper" in the second run and vice versa).
Dual-choice assays were performed in large (70 x 70 x 70 cm) metal wire cages (Stadler & Schoni, 1990) that contained ap proximately 300 of 7-14 day-old (emerged during one week) cabbage root flies of both sexes.Prior to the assay, the flies were continuously exposed to a cauliflower plant.One dual choice assay was performed with real plants.Six cauliflower plants were selected to obtain three pairs of plants similar in size and form.First plant of each pair was assigned to the "paper" and second plant to the "plastic" substrate.All six plants were arranged in hexagon on the bottom of the cage and females of D. radicum were allowed to oviposit for 22-23 h, after which the plants were removed, the eggs counted and the plants re turned to the cage with a renewed substrate set-ups.The com plete experiment lasted for four consecutive days (eggs counted daily) and the set-ups of the substrates within each pair were al ternated after two days ("paper" changed to "plastic" and vice versa) to minimize any influence exerted by the plant.
In addition, oviposition rates on plant surrogates attached to various modified substrates (listed in Table 1) were compared in the dual-choice assays.Two or three replicates (depending on the assay) of the treatments to be tested were placed in a square or hexagon on the bottom of the cage.Females were allowed to oviposit for 22-23 h and then the treatments were removed and the eggs counted.A cauliflower plant was introduced into the cage for 1-2 h between the end of an assay and the installation of a new set of treatments (positions were alternated in a sys tematic manner).Depending on the assay, the experiments took three or four consecutive days and data for a total of 8 to 12 re plicates were thus collected.

RESULTS
Fig. 2 illustrates the effect o f two different substrates on oviposition by the cabbage root fly.In the choice assay (Fig. 2A), the females laid significantly greater numbers o f eggs on the "paper" than on the "plastic" substrates.This preference was less pronounced but was still signifi cant in the no-choice assay (Fig. 2B) where the daily in creases in the number o f eggs laid by 25 females to two different treatments were compared.Since the same cauli flower plants were alternately used with the "plastic" and "paper" substrates, the resulting differences in oviposition must be entirely due to features o f the substrates, namely the differences in moisture and/or the accessibility o f ol factory and contact chemical cues.

Behavioural observations
Observations o f ovipositing individual cabbage root fly females yielded data that give an indication o f the behav ioural mechanisms that underlie the differences in egg numbers oviposited in the two different substrates, "paper" and "plastic" .N ot all females that touched the substrate were directly involved in a search for a suitable oviposition site.Some females landed on the substrate and, without touching the plant, rested or moved around (substrate run) for some time and then flew away.Other females landed on the plant surface, remained motionless for some time and then jum ped on the substrate, per formed a substrate run and left it again.These females, showing no apparent pre-ovipositional activity, were re garded as not performing the host plant selection and con sequently were not included in the statistical analysis (Table 2).
Stem base circling and substrate exploration behaviours were taken as clear indications that the female was searching for a suitable oviposition site.All females that finally oviposited first performed substrate exploration and stem base circling.Not all behavioural sequences containing the stages o f substrate exploration and stem base circling ended with oviposition, however.In some cases, the stage o f stem base circling was expressed as a mere stop at the base o f a stem heading down, but usually the females made a more or less complete circle(s) around the stem.During circling, females would stop and almost push their heads into the sand, remaining in that position for a few seconds.Similarly, during substrate exploration, females would stop and push their heads into the pits or crevices.As we observed no proboscis probing during these stops, we suspected that this behaviour might be re lated with olfactory perception o f cues emanating from the root-substrate complex.
The behavioural sequences in 400 females were re corded in total.Only 129 o f the 400 cases included con tact with the substrate ("substrate run"), only 110 cases included the "stem base circling" (and those were used for statistical analysis in Table 2) and only 81 cases in cluded oviposition.
More than 90% o f experienced females that reached the stem base circling stage continued to explore the substrate and finally laid eggs on the "paper" substrate (Table 2).In contrast, significantly fewer experienced females (only 50% o f those performing stem base circling), continued to oviposit on the "plastic" substrate (Table 2).Most impor tantly, 10 o f the 28 experienced females that began ex ploring the "plastic" substrate finally left the otherwise Tabu; 2. Behaviour of Delia radicum females in contact with the substrate in which the cauliflower plant grows.Substrate top layer was arranged as either "plastic" or "paper".Females were either naive (no previous oviposition on or contact with plant) or experienced (previous oviposition on plant).

Category of females
Step in behavioural sequence Numbers of females observed performing the different behaviours on the two substrates Physiological category of the fly Fig. 3. Oviposition of naive (no previous oviposition on or contact with plant) and experienced (previous oviposition on plant) Delia radicum females in two substrate set-ups: "plastic" (black columns) or "paper" (white columns).See Material and methods for explanation of the terms used.Each column repre sents a mean ± SEM response obtained from data analysis of 15-33 individual ovipositing females whose preoviposition be haviour is summarized in Table 2. Different letters above the columns indicate that the means are significantly different (P < 0.05, Kruskal-Wallis followed by Mann-Whitney U-test).
suitable host plant without ovipositing.These females performed long and intensive substrate exploration, in cluding numerous subsurface ovipositor probings but, ap parently, the stimuli present in the "plastic" substrate were not sufficient to elicit egg deposition behaviour.A similar tendency was only slightly suggested for naive fe males (88.2% vs. 80.5%) but this was not statistically sig nificant (Table 2).
By measuring the time spent by individual females at the egg deposition stage and relating this to the number of eggs counted after departure from the plant, we found that the time needed for deposition o f one egg is constant.This time was very close to 1 min (1 min 3 sec ± 8 sec, mean ± SD, N = 81).We could use this result to estimate the numbers o f eggs laid during individual acts o f egg deposition and ovipositional bouts (description follows), assuming that each minute spent by a female in the egg deposition posture resulted in one egg being laid.During egg deposition, the female remained sitting still with the ovipositor inserted into the substrate crevice and was not sensitive to disturbance, even by direct touch.Individual acts o f egg deposition were always preceded and sepa rated by substrate exploration.Each ovipositional bout consisted o f one or more acts o f egg deposition.Individ ual ovipositional bouts were separated by return to the plant surface.On return, females would re-explore the plant's surface qualities by more or less intensive probos cis probing, running and spiral flights.
The analysis o f ovipositing female behaviour showed that the number o f ovipositional bouts performed by one female during each visit to a plant (2.6 in average) de pended neither on the physiological state o f the fly nor on the substrate (Fig. 3A).The number o f eggs laid during one act o f egg deposition was also relatively constant (1.4 in average); females always deposited one or two eggs ir respective o f their physiological state and the substrate (Fig 3B).On the other hand, the numbers o f eggs laid during one ovipositional bout significantly differed in re lation to substrate qualities (Fig. 3C).Thus, the differ ences in the numbers o f egg depositions within one bout were almost exclusively responsible for the differences in the numbers o f eggs laid (Fig. 3D).Naive females laid twice as many eggs on the "paper" as on the "plastic" substrate and the same trend, though not statistically sig nificant (P = 0.054, Mann Whitney U test), was seen in the experienced females.Naive females laid more eggs than experienced females both per one ovipositional bout (Fig. 3C) and per one visit to plant (though this difference was not significant on "plastic" substrate; Fig. 3D).
The moisture content o f the substrate had a clear effect on the number o f eggs laid.Both wet and dry substrates were avoided [(5) vs. ( 6)], [(5) vs. ( 7)] (Table 3c).Fe males preferred to lay eggs into substrate that had a dry surface but contained at the same time wet particles not deeper than 0.5 cm (i.e.directly accessible) (called moist sand in this study).
Volatile chemical stimuli originating from the substrate were at least partly responsible for the observed differ ences in oviposition.This was clear from the assays [(8) vs. ( 9)] and [(9) vs. ( 10)] (Table 3d) in which the females were allowed to directly touch only dry sand and the treatments differed in the volatiles released from the vari ous bottom layers spatially separated from the top sand layer (Fig. 1C).The treatment using Brassica roots (8) was preferred to the organic substrate (9) and the latter was preferred to the sand treatment (10) (Table 3d).

DISCUSSION
Earlier studies o f host plant acceptance by the cabbage root fly concentrated mainly on the chemical (Traynier, 1967;Städler & Schöni, 1990;Baur et ah, 1996aBaur et ah, , 1998;;Roessingh et ah, 1997) and physical (Prokopy et ah, 1983;Roessingh & Städler, 1990;Košťál, 1993a;Degen & Städler, 1997) properties o f the aerial plant parts.It was concluded that the decision about whether to start with oviposition or not is mainly taken by females during their exploration o f the plant surface (Zohren, 1968;Städler & Schöni, 1990).Flopkins (1994) showed that the likelihood that a female, after having reached the stage o f 3 Mann-Whitney U test was used to test for significant differences between the compared treatments (ns, P > 0.05; ***, P < 0.001).
stem base circling or substrate exploration, will go on to lay eggs is 0.92 or 0.94, respectively.In fact, our values for the "paper" substrate confirms this.But when we of fered the females two substrates differing in quality, clear differences appeared in ovipositional rates on these sub strates.The "plastic" substrate allowed the females to touch only dry sand, while the penetration o f contact or volatile signals from the root substrate was prevented or minimized by the plastic cover-foils.The "paper" sub strate was designed to allow direct perception o f moisture and chemical signals from the substrate.In a choice situa tion, females clearly discriminated between these two substrates and preferred the "paper" substrate for oviposi tion.More importantly, the difference in ovipositional rates was apparent also in a no-choice situation and was further documented by the observation o f the oviposi tional behaviour o f individual females.
The lower numbers o f eggs laid on the "plastic" sub strate could be explained by considering two potential mechanisms: (1) females interrupted the behavioural se quence leading to oviposition after intensive substrate ex ploration before any egg was laid; (2) females started oviposition but laid fewer eggs.Although the two mecha nisms may represent ju st two extreme ends o f a contin uum we will discuss them separately for the sake of clarity.(1) The first mechanism occurred only with ovipositionally experienced females.Experienced females had laid a certain proportion o f their eggs on cauliflower plants prior to the trial.Thus the greater selectiveness (with respect to substrate quality) o f the experienced fe males might be due to the presence o f an incomplete egg load in their ovaries which might increase their selective ness as has been shown for Delia radicum (Zohren, 1968, Košťál, 1993b) and other insects (Fitt, 1986;Minkenberg et al., 1992).(2) The second mechanism was clearly noted in naive females but was less pronounced in experi enced females.W hile similar proportions o f females started laying eggs on both "plastic" and "paper" sub strates, the females stopped oviposition earlier on the "plastic" substrate, perhaps owing to an enhanced selec tiveness gained gradually during oviposition and a de crease o f their actual egg load.Detailed analysis o f the behavioural sequence performed on the substrate during oviposition revealed that the females repeatedly re explore the substrate between individual egg deposition events (each yielding invariably 1-2 eggs) and either con tinue to the next deposition (continue the ovipositional bout) or return to the plant to re-explore its surface (end the ovipositional bout) or leave the substrate and plant (ending the visit to the plant).While the numbers o f ovi positional bouts remained stable throughout the different substrates and physiological states, the number o f eggs laid during one bout varied significantly and, as a result, so did the total number o f eggs laid around the plant stem.Thus, during the substrate exploration stage which sepa rates individual acts o f egg deposition, the females assess the substrate qualities and adjust the number o f eggs laid accordingly, and also with respect to their current physio logical state (egg load).
We attempted to characterize the factors which influ ence substrate quality and, subsequently, oviposition by the cabbage root fly.Physical structure o f the substrate has been earlier shown to play a clear role in oviposition.Females tended to lay more eggs into substrates which al lowed penetration by their ovipositor (Traynier, 1967;Zohren, 1968;Havukkala, 1982).Similar results have been reported for the related seedcorn fly, D. platura (Barlow, 1965), and the onion fly, D. antigua (Mowry et al., 1989).
Females strongly preferred moist sandy substrates with a dry surface and wet particles at not deeper than 0.5 cm (called moist sand in our study) to the wet (also wet on the surface) and completely dry sands.Zohren (1968) found a weak preference for dry rather than moist sub strate in the ovipositing cabbage root fly females.His ar rangement o f the "moist" treatment was, however, different from the one presented here, having moist sand also in the surface layer (thus resembling the "wet sand" treatment here), which most likely explains the differ ences between his and our results.
Delia radicum females laid almost three times as many eggs in response to the growth substrate containing Bras sica roots than to the growth substrate without roots and seem thus to be able to perceive the presence o f plant roots in a substrate and respond positively to it.However, the females did not prefer Brassica roots to two non-host root-substrate complexes, barley and leek.It remains to be clarified whether non-specific stimuli affect the cab bage root fly's response to the root-substrate complex.
Cabbage root fly females laid almost eight times as many eggs in organic growth substrate than in moist sand.The organic substrate harboured abundant populations of organisms (Colembolla, mites, dipteran larvae, earth worms and microorganisms).Microorganisms associated with host plant decomposition are known to considerably influence both host plant acceptance and larval survival in related species, D. platura (Eckenrode et al., 1975) and D. antiqua (Ellis et ah, 1979;Hausmann & Miller, 1989).Doane & Chapman (1964a, b) have shown that D. radi cum also transmits various bacteria associated with the decay o f healthy tissues o f Brassica roots.The authors failed, however, to find any positive effect o f these bacte ria on larval survival or development.On the other hand, Ellis et al. (1982) have shown that D. radicum females laid three or four times more eggs around radish seedlings grown from untreated seeds than from seeds treated with various sterilizing agents to eliminate or reduce microor ganisms.
We attempted to exclude the possibility that the results o f the above mentioned experiments with roots from dif ferent plants and with different substrates (organic sub strate, sand) might have been influenced by different moisture contents in the different treatments.Prior to the experiment, all substrates were watered close to their maximum sorption capacity.Relative humidity was kept high (> 80%) which would minimize potential differences in evaporation rates from the different substrates.The ar rangement o f the oviposition top layers were similar in all the treatments ("paper"), which excluded any effect o f different physical structure and further minimized poten tial differences in moisture contents and evaporation rates.
Volatile chemical signals originating from the substrate were found to considerably influence the number o f eggs laid by the cabbage root flies.In fact, when the perception o f contact chemicals was eliminated and only the volatiles released by different substrates could have been perceived by ovipositing females, the differences in oviposition rates to volatiles o f Brassica roots vs. organic substrate or to organic substrate vs. sand were clear cut and similar to when the treatments included the contact chemicals (com pare results in Table 3b and 3d).Although these results prove that the volatile chemicals originating from the sub strate influence oviposition, we cannot estimate the rela tive influence o f volatile and contact chemicals.This would require the identification o f the chemical nature of the compounds responsible for the behavioural changes.
Volatile chemicals can be perceived by the antennae or the labial palpi when females stop for short intervals (up to a few seconds) with their head close to or directly in serted into the substrate, a behavioural pattern identified in this study as typical for either the stem base circling or the substrate exploration stages.
In a parallel study, Baur et al. (1996b, c) found that plants damaged by cabbage root fly larval feeding are preferred to undamaged plants and that the major stimuli responsible for that preference apparently originate from the root-substrate complex.Thus, the root-substrate com plex (including microorganisms) seems to have a strong influence on the final output o f the host-plant foraging process in the cabbage root fly.This should not be sur prising in an insect that deposits its eggs into the substrate and whose ancestors probably used decaying plant mate rial as the main medium for larval development (Griffiths, 1991).
In conclusion, the results confirm that the number o f eggs laid around host plants by the cabbage root fly is considerably influenced by the physical and chemical na ture o f the substrate in which the plant grows.The whole behavioural sequence perfonned by the female foraging for an oviposition site, first on the plant and then on the substrate around its stem, may be regarded as consisting o f a series o f three behavioural sub-sequences differing in their hierarchical level (ordered from the highest to the lowest): (1) plant exploration -assessment o f the suitabil ity o f the plant as detailed in earlier studies (Zohren, 1968;Städler & Schöni, 1990;Hopkins, 1994); (2) sub strate exploration -assessment o f the suitability o f the substrate for egg laying as described in this paper; (3) egg deposition -deterministic behaviour producing invariably 1 or 2 eggs per deposition.The completion/disruption o f one sub-sequence leads either to the start o f a following sub-sequence or to the return to a previous one or to the departure from the plant, and thus produces variation in the number o f eggs laid by individual females, depending on their physiological state (egg load) and the "quality" o f both the plant and the substrate surrounding its stem.

Fig. 2 .
Fig. 2. Oviposition of Delia radicum females on the two sub strates with different top layer set-ups, "plastic" and "paper" (see Material and methods for explanation) either in a dual choice (A) or no-choice (B) assay.Each pot with particular sub strate set-up (treatment) contained one cauliflower plant.Either three pairs of plants with two different substrates (A) or single plants with "plastic" or "paper" substrate (B) were exposed to groups of ovipositing females: ca 150 females per cage for 4 subsequent days in (A), and 25 females per cage for 11 con secutive days (repeated twice with different females).Vertical bars show variation of the mean: SEM in (A) and SD in (B) Differences between the numbers of eggs laid in the two sub strates were significant in both choice (P <0.01) and no-choice (P < 0.05) assays [Wilcoxon signed rank tests for: (A) mean numbers of eggs laid per replication per treatment and, (B) mean daily oviposition rates per treatment, grouped by days af ter eclosion].

Table 1 .
List and brief description of the substrates used for the oviposition dual-choice assays with Delia radícími.Surrogate plants treated with methanolic surface extract of the Brassica leaves were mounted on top of each substrate.

Table 3 .
Oviposition of Delia radicum females in dual-choice assays; comparison between different substrates supplied in pots with a plant surrogate sprayed with methanolic host-plant extract.Letters preceding treatment names refer to detailed descriptions in Material and methods. 2 Numbers of eggs were recalculated to percentages to allow easier comparisons between various experiments.