Netherlands Journal of Zoolo 30(4) : 611-621 (1980) COURTSHIP BEHAVIOUR, SIZE DIFFERENCES BETWEEN THE SEXES AND OVIPOSITION IN SOME ACHRYSOCHAROIDES SPECIES (HYM., EULOPHIDAE) by G. BRYAN (Department of Zoology, University of Manchester, England) SUMMARY Courtship behaviour of three Achrysocharoides species was observed. No visible differences in the courtship displays of males of these species were apparent. The display consists of a side-to-side rocking movement performed by the male whilst he stands to one side of the female and not as in most chalcids in a mounted position. Mating is also accomplished while the male is alongside the female, but during copulation one set of the male’s legs rest on the female’s dorsum. A few pairs of A. atys and A. niveipes mated under the conditions provided, but 4. cilla did not and A. latreillii males showed no interest in conspecific females. In A. afys the male always moved away from the female immediately after copulation, but A. niveipes males reorientated and performed a short post-copulatory rocking display. Various aspects of the courtship behaviour are discussed, including the possible reason for a post-copulatory display in A. niveipes. Suggestions are made as to how the courtship display of Ackrysocharoides males could be species-characteristic and as to why males of some species are larger than conspecific females. It is postulated that the unusual locomotory behaviour of Achrysocharoides may increase the efficiency of host-searching behaviour by females. Females of A. cilla and A. niveipes were observed searching for and ovipositing in hosts. They were found to temporarily paralysc the host larva. INTRODUCTION The world-wide genus Achrysocharoides Girault (= Enaysma Delucchi) is part of the Chrysocharis complex in the chalcidoid family Eulophidae, subfamily Entedontinae. There are eleven species found in Britain, all known to be partially gregarious endoparasistes of leaf-mining larvae, almost exclusively larvae of Phyllonorycter Hiibner (Lepidoptera, Gracil- lariidae) (Askew & Ruse, 1974; Bryan, 1980). In contrast with other leaf-miner parasite genera, Achrysocharoides species have a relatively high degree of host-tree specificity and most of the British species are each confined to attacking hosts on a single tree species or genus (Askew & Suaw, 1974). Typically leaf-miner parasite larvae are solitary, but in Achrysocha- rides more than one adult commonly emerges from a single mine. BA612 G. BRYAN Askew & Rusz (1974) found that species could be grouped into three categories on the basis of brood composition: (i) males and females in separate broods; both sexes sometimes gregarious—A. cilla (Walker) (ii) males and females in separate broods; females usually gregari- ous; males solitary—including A. latreillii (Curtis) and A. niveipes (Thomson) (iii) males and females sometimes in the same brood; both sexes sometimes gregarious—including A. alys (Walker). For a more detailed analysis of brood composition in the above- mentioned species see Fig. 1. A,cilla { Fagus) Sex ratio Nz403 12439 (9:07 2 3 1.80 x o- A.lotreitlii (Quercus) nst2 eNO ; 243 x A piveipes| (Betula) nz 432 ns344 i 182 x o- A.atys (Rosaceae) ‘Ns 58 ns20t 2288 2 Be ar & x 123 122450 GheY4%% No.5 No. 99 No. 3/9 Fig. 1. Percentages of complete broods of different size and composition in four Achrysocharvides species (host trees in parentheses; percentages <1% excluded) and sex ratios, calculated from all recorded emergences. (Data from Bryan, 1978).COURTSHIP BEHAVIOUR IN ACHRYSOCHAROIDES 613 The behaviour of Achrysocharoides adults is practically unstudied. ‘They are generally very active and rather than walking normally as close relatives do, they move about in a waltzing manner, reminiscent of the locomotory behaviour of the red spider mite, Tetranychus urticae Koch. Some species of Pediobius Walker (Eulophidae, Entedontinae) perform a similar curious “dance”, hence the synonym Pleurotropis Forster, a name referring to the sideways movement. The “danza strana ed elegante” of Achrysocharoides is very effectively described by Arrv (1957), with reference to A. parva (Delucchi), but he was unable to ascertain whether the behaviour was important in the commence- ment of courtship. MATERIAL AND METHODS Observations were made of males and females of four Achrysocharoides species: A. alys, A. niveipes, A. cilla and A. latreillii. All specimens were reared from Phyllonorycter mines, stored individually in corked glass specimen tubes. Initially pairs were kept in small glass tubes without success. The adults were, therefore, introduced into small plastic Petri dishes or boxes (13 x 7 x 5.5 cm) with at least one of the sides replaced by fine nylon netting. Small pieces of host plant were sometimes intro- duced into the mating chambers. The age of the females used was varied, as was the number of males and females introduced into a container at any one time, also the time of day. Observations were made using a binocular head-set with low magnification lenses. Similar methods were employed in attempts to observe oviposition in A. cilla and A. niveipes. Leaves with Phyllonorycter mines were placed or suspended in an appropriate container and adult females intro- duced. OBSERVATIONS Courtship and mating in A. atys The following is a general account of the normal courtship sequence, which is depicted in Fig. 2. Males only started to approach or chase a female when she was within close range and reacted to moving females more readily than they did to stationary ones. Often the female would be chased for some distance before she stopped, usually to groom. The male would then orient to the female, generally in the position indi- cated by the male in Fig. 3. There was no apparent preference for courting from the left or the right side. Once in this position, the male immediately begins to rock laterally.614 G. BRYAN MALE FEMALE Ignores female <-~—~=-————- Walks within 2em. of male including all latreillij observed) — Notices and Unreceptive, tlies or ‘approaches female —— ~~ oF walks away ‘Stops walking and _— ‘often grooms. Orientates to temate and £~ > Unreceptive, flies or ocks from side toside ——_ ‘walks away _——Z Remains quiescent, usually ‘ceases grooming Unreceptive,tlies oF ‘walks away. Continues rocking and gradually moves alongside ~ female: rocking increases. in amplitude iene Unreceptive, but Attar some time, stops, p77” Sage" Famalnsquiescont courting ndrmcves way K2—~ Crctuaing at eta observed) One set of legs onthe = <7 substrate and other legs fon fernale, curls gaster underneath female’ | COPULATION Moves away from female, 7 ‘and grooms (all atys onetved) Becomes receptive: raises abdomen to ‘expose genital aperture Reorientates to female ‘and rocks several times before moving away and ‘grooming (all niveipes ‘observed ) Fig. 2. Courtship sequence of Achrysocharoides species, depicted as a reaction chain. Solid lines depict the normal or typical sequence; alternative directions are indicated by broken lines. If the female remains quiescent, the male gradually moves closer, rocking after every few steps, until their heads are almost touching and the male is in the position indicated by the solid arrow in Fig. 3. A receptive female remains quiescent and the rocking movements by the male become more rapid, increase in amplitude slightly and a back- ward-forward component appears to be introduced. After a variable amount of time (a few seconds to about 20 or 30COURTSHIP BEHAVIOUR IN ACHRYSOCHAROIDES 615 seconds), the male springs into a position astride the female whereby their heads are almost touching, the male has one ipsolateral set of legs on the female’s dorsum and the other set on the substrate; the gaster is curled round and underneath the female’s in order to establish <€------~ AN A 1 i 1 Fig. 3. Orientation of Achrysocharoides male during courtship. The male is figured in the most commonly observed position for initial courtship display; the short arrows indicate the direction of rocking movements. Broken arrows indicate other ap- proximate observed orientations of the courting male. The solid arrow shows the position of the male just prior to copulation. (The left-hand side of the diagram would be identical, but is omitted for convenience). genital contact. Copulation lasts for between 15 and 25 seconds and is accompanied by a pumping action of the male’s body with the wings usually extended backwards. After copulation the male walks some distance away and grooms; the female generally remains quiescent for a short time, sometimes grooming also. Males did perform courtship behaviour in small specimen tubes, but all the females observed were unreceptive and usually ran or flew away when a male approached, or if grooming, continued to do so. Many courtships were observed, but only three matings: two immediately after one another between newly emerged insects; the other pair was nine days old. All matings took place in plastic Petri dishes without host plant foliage.616 G. BRYAN Courtship and mating in A. niveipes The basic pattern of courtship was exactly as described for A. atys above and indeed there were no visibly discernable differences between the courtship displays of A. niveipes and A. atys males. Male A. niveipes, however, showed no interest in females when confined with them in small tubes or even Petri dishes with host tree leaves (Betula). The three matings observed took place in small plastic boxes. All individu- als were several days old at the time of mating. Copulation duration was from approximately 40 to 80 seconds. Invariably following copu- lation the male immediately returned to the courting position along- side the female and performed a short rocking display for several seconds before walking away. Courtship in A. cilla This species did not mate under the condition provided, but in small plastic boxes with host tree leaves (Fagus), males courted females. The courtship behaviour of A. cilla males appeared identical to that of A. atys and A. niveipes. Behaviour in A. latreillit A. latreillii males and females were repeatedly introduced into various containers, but on no occasion did the males show any interest in females. Neither were they observed to interact with conspecific males in any way. DISCUSSION In the Achrysocharoides species observed, males appeared to react to visual stimuli from females at close range and often reacted only to moving females. BARRASS (1960) made a similar observation for Nasonia vitripennis (Walker) (Pteromalidae). Also, male A. alys and A. niveipes approached and courted conspecific males and the former even courted other congeneric females and a female Chrysocharis phryne (Walker). In the Achrysocharoides species studied courtship is very simple, with the rocking body movement the only obvious element. Performance of such a rocking movement is common in a great number of chalcids and in pteromalids is mostly performed before mounting (VAN DEN ASSEM, 1974); it is thought to provide visual stimulation. The position of the courting Achrysocharoides male is somewhat unusual since in most chalcids the male approaches the female, mounts and continues to court from a position on top of her. However, in some Encyrtidae theCOURTSHIP BEHAVIOUR IN ACHRYSOCHAROIDES 617 male courts from a position alongside the female or vis-a-vis (VAN DEN Asse, 1976) and males of Chrysocharis laricinellae (Ratzeburg) perform a dance round the female and then stand alongside with one front leg on her prothorax (Quepnav, 1967). Some female eulophids have been observed to signal receptivity by stretching the head forwards and raising the antennae vertically (vAN DEN AsseM, 1974). Unfortunately such a signal was not detected in the above observations. Achrysocharoides males copulate from a position astride the female. A lateral position of the male during copulation seems to be rare among parasitic Hymenoptera. In Chrysocharis larici- nellae the male stands to one side of the female, but grips her prothorax with one or both of his forelegs (Quepnau, 1967). There is considerable evidence that sperm competition is a real threat to many male insects; sperm of the last male to mate with a female may predominate in fertilising the offspring (PARKER, 1970). VAN DEN Assem & Visser (1976) concluded from experiments with Nasonia vitripennis that the performance of a post-copulatory display by the male renders a second mating by the female more unlikely. Gre- garious mixed sex broods of chalcids commonly mate almost immedi- ately after emergence, many within their pupation chamber (AsKEw, 1968). Such sib-mating is probably of common occurrence in A. atys (see Fig. 1). In A. niveipes, however, males are solitary and being much larger than A. atys males (see Table I) are likely to live much longer. ‘These two factors must increase the likelihood of sperm competition if A. niveipes females are receptive to more than one male. This may account for the fact that a post-copulatory display by the male was observed in A. niveipes, but not in A. atys. VAN DEN Assem (1974) states that only by the performance of a species-characteristic courtship repertoire is a male able to induce a female to mate and since males often do not restrict their attention to conspecific females, the females have to distinguish between these patterns of courtship. However, the courtship displays of the three Achrysocharoides species observed appeared indistinguishable. The possibility of the females recognising conspecific males by means of species-specific male sex pheromones must be considered. Such a pheromone was first discovered in parasitic Hymenoptera by Kuasi- muppin & De Bacu (1975) in the aphelinid genus Aphytis Howard. The courtship behaviour in Aphytis species follows a more or less set pattern, but the male sex pheromone can only be perceived by con- specific females. The production by the male of species-characteristic noises can also not be dismissed and these may play a more important role in courtship than previously thought (cf. VAN DEN ASsEM, in press). Achrysocharoides species are unusual amongst the guild of leaf-miner618 G. BRYAN parasites in that they are remarkably host-tree specific and it is probable that in those species with separate sex broods adults immedi- ately upon emergence seek out host-trees where mating takes place. ‘Adult field-capture data supports this idea: males and females of those species are almost invariably captured on their host-trees (Askew & Ruse, 1974). The apparent unimportance of host-tree foliage in mating experiments would, however, seem to argue against it, but adult females may need to actively search for their host tree before they are willing to mate. (This would account for the difficulties in getting pairs to mate in laboratory conditions). It is interesting to note that in the three european species associated with Quercus the males are very distinctive: A. latreillii with a very transverse head and truncate forewings and A. bulus (Walker) and A. albiscapus (Delucchi) with characteristic antennae (Askew & Ruse, 1974). VAN DEN AssEM (1976) discusses the evolution of a frontal position of courtship by the male and an antennal receptivity signal by the female. He argues that once this is achieved, a reduction in the size of the male is advantageous: in gregarious parasites with mixed sex broods, small males can be tolerated since they are not at such a disadvantage and the resultant increase in female size means increased fitness. A similar situation is also found in solitary parasites in which the female can discriminate and lay unfertilised eggs on small hosts and fertilised eggs on large hosts. CHARNov (1979) has built a mathematical model and laid out rules to which an ovipositing female must adhere in order to gain the maximum fitness possible under specified circumstances (for example: what proportion of male eggs should be laid on small hosts). There may have been a similar trend in the Eulophidae in move- ment of the courting position of the male from the rear to the front and evolution of an antennal receptivity signal by the female. In A. atys, with large numbers of mixed sex broods, as in most parasitic Hymen- optera, the female is the larger sex. However, in several Achrysocha- roides species with separate sex broods, the males are generally larger (Askew & Ruse, 1974; see Table I). I have evidence (BRYAN, 1978), TABLET " ‘No.in ‘Mean length of thorax ++ propodean (mm). : — ‘Males Females cilla 7 053 £0.07(n—= 30) 0.46 + 0.06 (n = 10) leareilis 1 0.63 £0.09 (n= 10) (0.52 + 0.06 (n = 10) niveipes 1 0.66 + 0.05 (n= 20) 0.65 4: 0.10 (n = 13) ays I 0.50 + 0.10 *n = 9) 0.57 + 0.08 (n = 26) 4 Cah OUT 054 £008 mat)COURTSHIP BEHAVIOURIN ACHRYSOCHAROIDES 619 that the males of these species emerge earlier than the females, killing on average earlier host instars, but since they develop solitarily or less gregariously than the females (see Fig. 1), they remain generally larger. In fact the differences given in Table I will obviously be greater when mean male and female sizes are considered. In these species some of the disadvantages of a reduction in male body size listed by VAN DEN Assem (1976) may be important: for example, a reduction in longevity and number of possible inseminations. It is, however, likely that the size differences of the sexes must be considered in the light of the wider ecological strategies of these species before a full understanding can be gained. OVIPOSITION BEHAVIOUR Observations On the leaf females move about as described in the introduction. Females exploring the lower leaf surface with their antennae immedi- ately show increased activity when they come into contact with a mine, moving quickly from side to side and appearing to use the antennae to determine the extent of the mined area. Soon after the commencement of examination (about 10 seconds), she begins to twist the gaster, making rapid horizontally directed thrusts into the mine with the ovipositor. Once the larva has been located with the antennae and ovipositor, the female stands over the larva with the antennae touching the mine surface and then moves forwards a few steps and inserts the ovipositor more or less vertically through the part of the mine the antennae had examined. If the ovipositor does not contact the larva, the female twists the gaster and thrusts horizontally as described above. When the ovipositor penetrates the Phyllonorycter larva, it makes convulsive move- ments and moves away. The parasite then relocates the larva and the procedure is repeated. Most of these ovipositor thrusts are very rapid (less than a second to several seconds). For about three to five minutes the ovipositor insertions by the parasite and escape reaction by the host larva continue, but gradually the larva’s movements slow and eventually it ceases activity. Some of the ovipositor thrusts are then longer: 60 to 90 seconds. Females spent from about five to over 30 minutes on a single mine. When mines were removed from containers in which the above oviposition behaviour was observed, the Phyllonorcyter larva was in- variably found to be motionless and flaccid. However, after about 20 minutes the larva began to move and eventually resumed “normal activity”.620 G. BRYAN Discussion ‘Twisting and turning movements are commonly used during searching for hosts by parasitic Hymenoptera and it is possible that the curious gait of Achrysocharoides species may increase the efficiency of searching behaviour by females for host mines on the surface of a leaf. The females appear to be stimulated only by direct contact with the mined surface of the leaf as in other leaf-miner parasites (QuEDNAU, 1967; Quepnau & GuevreMont, 1975). Asxew (1971) records that chalcids that attack larvae frequently paralyse their victim prior to oviposition, achieved by injection of venom through the ovipositor. He also states that paralysis is the general rule for ectophagous chalcids (its principal purpose apparently being to eliminate host movements which might crush or dislodge the parasite eggs or larvac), but that endophagous chalcids do not usually paralyse the host. However, it may be that any paralysis of the host by endoparasites is more difficult to detect since it is merely temporary, especially in those parasites such as Achryosocharoides that exploit very young growing larval hosts. Quepwav (1967) records that female Chrysocharis laricinellae temporarily paralyse the host coleophorid and oviposit in the motionless larva before it recovers. In such cases it would appear that the purpose of paralysis is to enable the female paraiste to successfully lay one or more eggs in the larva with less difficulty (RoTHERAY, 1979). It also possibly allows host feeding to take place. 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