Many aphid species exhibit a life cycle in which parthenogenetic viviparous generations are followed by a single sexual generation and such species are called holocyclic. However, many aphid species have lost the sexual phase of their life cycle and are termed as anholocyclic. These tiny insect pests multiply rapidly through a series of parthenogenetic generations and in the sexual phase of their life cycle, genetic recombinations occur.

 Mating behaviour in aphids has been studied only in a few species. In aphids, it has been reported that oviparous females attract winged males through releasing pheromones produced by their hind tibiae (1). (2) reported that sexual reproduction has advantages under circumstances like intense sibling competition, vulnerability to parasites and other enemies, accumulation of detrimental mutations or a saturated and heterogeneous environment. The oviparae of most species bear circular plaques or pseudosensoria on their swollen hind tibiae. Various functions have been attributed to these organs (3). Several workers have suggested that they produced sex pheromone which attracts the males (4-8).

 In aphids in general, the occurrence of scent plaques on the hind tibiae of oviparae seems to be correlated with the flight and/or migration in the males, for they are most numerous in species having winged males (5) and in those showing host alteration (24). Several workers suggested that a female scent is involved in long-range attraction of migrating male aphids (5,6). The males of many aphid species may react to oviparae from a relatively close range, perhaps having located the host plant in much the same way as do gynoparae (11). Other factors such as host plant chemicals and non–chemical stimuli from other aphids may be important in effecting the aggregation of males in situations where they are likely to meet oviparae (20) reported that males of Megoura viciae are greatly out numbered by their sexual partners.  Under ideal conditions in the laboratory, an average of 10 oviparae are produced for every one male. (3) stated that females of all ages begin daily bouts of pheromone release about two hours before males become spontaneously active. In other insects, maximal responsiveness to the sex pheromone is attained when the gonads are mature and the insects are willing to copulate (13, 19 & 22).       

(20) stated that the presence of the female pheromone was essential if mating attempts were to occur, for males deprived of their antennae refused to mate. (20) reported that for successful development, mating females should be mobile and have the capacity to judge leaf senescence. Mating females were found in very low numbers on leaves that were about to be shed. (20) further reported that mating females abandoned abscising leaves in favour of leaves at an earlier stage of senescence, increasing the time available for mating. It was concluded that mating females are mobile searchers for optimum feeding and mating sites.

(15) studied copula duration and sperm economy in the large thistle aphid, Uroleucon cirsii. According to him, the male aphids have a limited amount of sperm at their disposal and that the female’s capacity to store sperm is limited as could be expected when males were not able to displace sperm from former copulas. (15) found that in Uroleucon cirsii, the pattern of sex–ratio adjustment indicated local mate competition among males, while the pattern in Rhoplaosiphum padi suggested local resource competition among sexual females. Local mate competition among males meant that fitness returns were limited by the availability of females as mates with local breeding groups, whereas local resource competition meant that fitness returns are limited by the availability of resources for females competing within local groups. These workers discussed how the life cycles of both species fitted to these hypotheses. (21) performed series of experiments on Myzus persicae (Sulzer) to investigate aspects of male maturation and semen transfer that should be considered in planning or interpreting experiments on aphid sexual strategies. (21) further reported that it was necessary to set up multiple replicates to compensate for the small number of eggs laid per female when investigating sexual behaviour of aphids.

In India, Cavariella aegopodii has been reported from Northwestern and Northeastern Himalayas (39). However, its detailed mating behaviour have not been reported so far from India. Since the behavior and biology of this insect pest differ considerably in different regions due to different environmental conditions, it is desirable to study its mating behaviour in Himachal Pradesh. Studies on the mating behaviour of Cavariella aegopodii aphids would be helpful in understanding complex life cycles of these aphids in this region as far mating behaviour is concerned. Such studies may prove useful in developing the effective strategies for the management of willow aphids. 

Mating behaviour of Cavariella aegopodii was studied under laboratory as well as field conditions. To observe pre-copulation, copulation and postcopulation behaviour of males and oviparae, continous observations were made.  Four different sets of experiment were conducted to study the mating behaviour of Cavariella aegopodii. These sets were, one male and one ovipara sexual, one male and three oviparae sexuals, three males and one ovipara sexual and one male, one virgin ovipara sexual and other ovipara sexual from set 3. The photographs of different morphs and their stages were taken with the help of digital still camera SONY cyber-shot DSC-W35 7.2 mega pixels.

Mating behaviour was studied in Cavariella aegopodii under laboratory conditions as well as in nature. To observe pre-copulation, copulation and post-copulation behaviour of males and oviparae, continous observation were made with the help of magnifying lens both in the field and under laboratory conditions.

Ovipara sexuals and male sexuals occur in autumn season (September-October). The adult ovipara sexual (Fig. 1) is dark orange brown in colour and has elongated but swollen body. Males are winged and black in colour (Fig. 2). The external male genitalia are heavily pigmented. Males are smaller in size in comparison to winged parthenogenetic female and winged female sexuparae. Males generally have a pattern of body markings similar to that of parthenogenetic winged females and have many more secondary sensoria on the antennae. Males have well developed wings and are usually black coloured. Their body size is short in comparison to ovipara. Adult ovipara sexual after mating usually searches for a suitable place to lay eggs in stem crevices, nodes and leaves (Fig. 3). While laying eggs, it raises posterior part of abdomen. Adult ovipara stop feeding after laying the eggs. Freshly laid eggs are creamish green in colour. The colour changes to light brown, and then to dark brown and finally the eggs turn black (Fig. 4). Eggs are laid in clusters and hatch in next spring.Observations on the mating behaviour were made under different sets of experiments under laboratory conditions.

Fig. 1-4:    Adult of  ovipara sexual of Cavariella aegopodii (1), Winged male sexual of Cavariella aegopodii (2),  Eggs of ovipara sexual of  Cavariella aegopodii on crevices and  nodes of stem (3)  Black coloured eggs of ovipara   sexual of Cavariella aegopodii (4)

One male and one ovipara sexual

In this set of experiments, one male and one ovipara sexual were placed (Fig. 5). Ovipara sexual did not show much movement, but male was active enough and immediately searched the whole area (Fig. 6 a, b). Male approached the ovipara sexual many times, but initially did not mate (Fig. 6 d, e). At times, it climbed over the ovipara sexual from posterior end of abdomen (Fig. 6 h, i), moved over the thoracic and head region.  The male also touched the abdomen of female with its rostrum and antennae. It appeared from the behaviour of male, as if it was inspecting the female. When ovipara sexual showed no resistance, the male moved towards the posterior end of ovipara sexual and raised the posterior end of female slightly with its right foreleg. Thereafter, male mounted over ovipara sexual from posterior end (Fig. 6 j). It grasped the ovipara sexual by placing its fore legs on middle part of abdomen, middle legs on lateral sides and hind legs on the ventral side of abdomen (Fig. 6 k). Ovipara sexual also showed slight movement at this time raising its abdomen slightly. Male supports its body by keeping itself on the body of ovipara sexual. For copulation, male bends its posterior part of abdomen (Fig. 6 l, m). After finding the vulva of ovipara sexual with its aedeagus, it copulates for 5 to 8 minutes. After copulation when male has to separate, it moves both antennae and wings and female again raises its abdomen slightly. Male remains active after mating but ovipara sexual remains motionless (Fig. 6 o, p, q).

Fig. 5   Mating behavior (One male sexual and one female sexual) of Cavariella aegopodii

Fig. 6   Mating behavior (pre-copulation, copulation and post copulation phases) of Cavariella aegopodii

One male and three ovipara sexuals

One male and three oviparae sexual of different sizes were placed together in another set of experiments (Fig. 7). The idea was to see the behavior of males and ovipara sexual and to find out which ovipara sexual and male is selected for mating.

Male approached each female, vibrating its antennae and at times it climbed over sexual females and inspected them. Male did not copulate with small sized and large sized ovipara sexual and selected middle-sized ovipara sexual for first mating. Copulation lasted for about 10 minutes. It was found that the male rejected the large sized ovipara sexual because it showed resistance and did not allow male to mount. Males at times rejected large sized ovipara sexual because it is difficult to take over them.

It was observed that for a male, middle-sized ovipara sexual was the ideal mate. It was easy for the male to copulate with her and she also showed no resistance. After 3 hours, male mated with the same ovipara sexual but this time copulation lasted for 5 minutes only. The duration of second copula is always shorter than the first copulation. 

Fig. 7-8:   Mating behaviour (One male sexual and three females ovipara sexuals) of        Cavariella aegopodii(7)  Mating behaviour (Three  male sexuals and one females ovipara sexual) of Cavariella aegopodii.(8)

Three males and one ovipara sexual

In this experiment, 3 males and 1 ovipara sexual were placed together in a Petri plate (Fig. 8). The main purpose was to see whether the males fight for ovipara sexual and also to see any kind of guarding behavior in males.

One male did not show much interest in the ovipara sexual. The behaviour of other two males was very interesting. Both males approached and moved around the ovipara sexual, at times touching the ovipara sexuals with antennae and legs. As one male climbed over the sexual female from posterior end, the other male tried to kick it with its fore legs. The first male tried to avoid, the other male and started climbing on the female. The first male kicked the second male with its hind leg and also vibrated its antennae and wings. The second male soon gave up and the first male succeeded in mating with the oviparous female

One male, one virgin ovipara sexual and other ovipara sexual from previous experiment 

Male first preferred virgin ovipara sexual for copulation. Male mated with her for about 11 minutes. When the male mated with other ovipara sexual, the copulation lasted for 4 minutes only. This shows that males prefer virgin female and copulate with a virgin female for a longer time in comparison to the one that has just been mated.

In the present study, maximum number of oviparous females and males was observed during autumn season. It is interesting to note that sexuales were collected from September to October when the day length is relatively longer and temperature is higher than during winter. The occurrence of sexuales during colder part of the year (winter), when the day length is rather short and temperature low, is in conformity with the observations of earlier workers on other aphid species (26,27, 28, 31, 32 &40), although these have not been considered as the only determining factor for the production of sexuales by other workers (7). The mechanism of photoperiodic response has not been studied in Cavariella aegopodii. In Cavariella aegopodii, sexual morphs occurred in autumn season confirming the early reports by (10), that the short day length of autumn season is conducive for sexual morph production.

There is no clear evidence that any environmental factors except photoperiod and temperature have a direct influence on this aphid life cycle. Oviparous females are apterous and orange brown in colour while males are winged and black in colour. Production of sexuals is attributed to some intrinsic timing mechanism (10) or interval timer as reported by Lees (28).

So far the mating behaviour in Cavariella aegopodii is concerned, middle-sized females were found to be more preferred. These observations are in agreement with the earlier reports in other aphid species where the size of female is reported to be important (14). As reported in Myzus persicae (Sulzer) (18) and in other aphid species, Cavariella aegopodii shows no striking courtship behaviour. The males of Cavariella aegopodii inspect the dorsal side of the female with its antennae and rostrum. The courtship lasts for 2 to 5 minutes and after this, normal copulation occurred. (20) observed similar courtship behaviour in aphids of Schizaphis dubia and S. borealis.

Position of eggs in the reproductive tract of females is believed to play some role in the mating behaviour of males. Females that are about to lay eggs are never preffered by male aphids. Oviparous females with blocked genital opening and sticky honeydew were also rejected by male aphids.

Fighting behaviour among males for a single female has been observed in Cavariella aegopodii. The male aphids show no guarding behaviour after copulation. Similar behaviour was reported by (15) in males of Uroleucon cichorii (Koch), which leaves the female after copulation. However, male Euceraphis betulae Koch is reported to guard their females after copulation (15).

Stronger males and the ones with larger size than the average copulate more frequently because they can gain more females by take overs. Similar observations were reported by Parker and other workers (14) in other aphid species.  The reduction of the duration of the copula of individual males suggests a limitation in the amount of sperm a male has at its disposal (43).

In the present study, the duration of second copulation was shorter than the first one. Similar findings were reported in the large thistle aphid, Uroleucon cirsii by Dagg (2003). A male gives preference to a virgin female and the time of copulation with a virgin female is always longer in comparison to a female that has just been mated. Possibly the fertilization success decreased with the age of the female as reported by Doherty and Hales (2002). 

Acknowledgement

One of us (ND) is thankful to CSIR, New Delhi for granting research fellowship.

1. Dawson, G. W., et al. "Aphid Semiochemicals: A Review and Recent Advances on the Sex Pheromones." Journal of Chemical Ecology, vol. 16, 1990, pp. 3019-3030. DOI: 10.1007/BF00982102.

2. Stearns, S. C. "Why Sex Evolved and the Difference It Makes." The Evolution of Sex and Its Consequences, edited by S. C. Stearns, Birkhäuser, Basel, 1987, pp. 15-31. DOI: not available.

3. Marsh, D. "Sex Pheromone in the Aphid Megoura viciae." Nature New Biology, vol. 238, 1972, pp. 31-32. DOI: 10.1038/newbio238031a0.

4. Flogel, J. H. L. "Monographie der Johannisbeeren-Blattlaus, Aphis ribis L." Insektenbiologie, vol. 1, 1905, pp. 145-55. DOI: not available.

5. Weber, H. "In Hemiptera. III. Biologie der Tiere Deutschlands." Biologie der Tiere Deutschlands, vol. 38, 1935, pp. 210-286. DOI: not available.

6.  Smith, L. M. "Biology of the Mealy Plum Aphid, Hyalopterus pruni (Geoffroy)." Hilgardia, vol. 10, 1936, pp. 167-211. DOI: 10.3733/hilg.v10n07p167.

7. Bodenheimer, F. S., and E. Swirski. The Aphidoidea of the Middle East. The Weizmann Science Press, Jerusalem, 1957. DOI: not available.

8. Stroyan, H. L. G. "Contribution to the Taxonomy of Some British Species of Sappaphis matsumura (Homoptera: Aphididae)." Journal of the Linnean Society of London, Zoology, vol. 43, 1958, pp. 644-713. DOI: 10.1111/j.1096-3642.1958.tb01470.x.

9. Birkhead, T. R., and G. A. Parker. "Sperm Competition and Mating Systems." Behavioural Ecology: An Evolutionary Approach, edited by J. R. Krebs and N. B. Davis, Blackwell Science, Oxford, 1997, pp. 121-145. DOI: not available.

10. Bonnemaison, L. "Contribution à l'étude des Facteurs Provoquant l'Apparition des Formes Ailées et Sexuées Chez les Aphidinae." Annales des Épiphyties (Paris), vol. 2, 1951, pp. 1-380. DOI: not available.

11. Broadbent, L. "The Grouping and Overwintering of Myzus persicae Sulz. on Prunus Species." Annals of Applied Biology, vol. 36, 1949, pp. 334-40. DOI: 10.1111/j.1744-7348.1949.tb06440.x.

12. Brodel, C. F., and G. A. Schaefers. "The Influence of Temperature on the Production of Sexuals by Aphis rubicola under Short-Day Conditions." Entomologia Experimentalis et Applicata, vol. 27, 1980, pp. 27-132. DOI: 10.1111/j.1570-7458.1980.tb02900.x.

13. Calvert, I., and S. A. Corbett. "Reproductive Maturation and Pheromone Release in the Flour Aphid Toxoptera aurantii (Boy)." Bulletin of Entomological Research, vol. 60, 1973, pp. 559-63. DOI: 10.1017/S0007485300034553.

14. Charnov, E. L., and G. A. Parker. "Dimensionless Invariants from Foraging Theory’s Marginal Value Theorem." Proceedings of the National Academy of Sciences of the United States of America, vol. 92, 1995, pp. 1446-1450. DOI: 10.1073/pnas.92.5.1446.

15. Dagg, J. L. "Copula Duration and Sperm Economy in the Large Thistle Aphid, Uroleucon cirsii (Hemiptera: Aphididae)." European Journal of Entomology, vol. 100, 2003, pp. 201-203. DOI: 10.14411/eje.2003.031.

16. Dagg, J. L., and S. Scheurer. "Observations on Some Patterns of the Males Sexual Behaviour of Certain Aphid Species." Aphids in Natural and Managed Ecosystems, Univ. Leon Publication, 1998, pp. 167-171. DOI: not available.

17. Dagg, J. L., and S. Vidal. "Sex Ratio Adjustment and Maternal Condition in Two Aphid Species." Behavioral Ecology and Sociobiology, vol. 55, 2004, pp. 231-235. DOI: 10.1007/s00265-003-0710-9.

18. Doherty, H. M. R., and D. Hales. "Mating Success and Mating Behaviour of Aphid, Myzus persicae (Hemiptera: Aphididae)." European Journal of Entomology, vol. 99, 2002, pp. 23-27. DOI: 10.14411/eje.2002.004.

19. Fletcher, B. S., and A. Giannakakis. "Factors Limiting the Response of Females of the Queensland Fruit Fly, Dacus tryoni, to the Sex Pheromone of the Male." Journal of Insect Physiology, vol. 19, 1973, pp. 1147-55. DOI: 10.1016/0022-1910(73)90235-8.

20. Glinwood, R. T., and J. Pettersson. "Movement by Mating Females of a Host Alternating Aphid: A Response to Leaf Falls." Oikos, vol. 90, 2000, pp. 43-49. DOI: 10.1034/j.1600-0706.2000.900106.x.

21. Hales, D. F. "Physiological Determinants of Male Mating Performance in Aphids (Hemiptera: Aphididae)." European Journal of Entomology, vol. 102, no. 4, 2005, pp. 641-646. DOI: 10.14411/eje.2005.090.

22. Happ, G. M. "Maturation of the Response of Male Tenebrio molitor to the Female Sex Pheromone." Annals of the Entomological Society of America, vol. 63, 1970, pp. 1782. DOI: 10.1093/aesa/63.6.1782.

23. Hardie, J. J., et al. "Sex Pheromones and Visual Trap Location Strategies and Aggregation by Host Alternating Aphids in the Field." Physiological Entomology, vol. 21, 1996, pp. 93-96. DOI: 10.1111/j.1365-3032.1996.tb00840.x.

24. Hille Ris Lambers, D. "Polymorphism in Aphididae." Annual Review of Entomology, vol. 11, 1966, pp. 47-78. DOI: 10.1146/annurev.en.11.010166.000403.

25. Kennedy, J. S., et al. "Host Finding by Aphids in the Field. Gynoparae of Myzus persicae (Sulzer)." Annals of Applied Biology, vol. 47, 1959, pp. 410-33. DOI: 10.1111/j.1744-7348.1959.tb02534.x.

26. Kenten, J. "The Effect of the Photoperiod and Temperature on Reproduction in Acyrthosiphon pisum Harris and on the Forms Produced." Bulletin of Entomological Research, vol. 46, 1955, pp. 599-624. DOI: 10.1017/S0007485300040790.

27. Lamb, R. J., and P. J. Pointing. "Sexual Morph Determination in the Aphid, Acyrthosiphon pisum." Journal of Insect Physiology, vol. 18, 1972, pp. 2029-2042. DOI: 10.1016/0022-1910(72)90214-1.

28. Lees, A. D. "The Role of Photoperiod and Temperature in the Determination of Parthenogenetic and Sexual Forms in the Aphid, Megoura viciae Buckton. I. The Influence of These Factors on Apterous Virginoparae and Their Progeny." Journal of Insect Physiology, vol. 3, 1959, pp. 92-177. DOI: 10.1016/0022-1910(59)90002-5.

29. Lees, A. D. "The Role of Photoperiod and Temperature in the Determination of Parthenogenetic and Sexual Forms in the Aphid, Megoura viciae Buckton. II. The Operation of the “Interval Timer” in Young Clones." Journal of Insect Physiology, vol. 4, 1960, pp. 154-175. DOI: 10.1016/0022-1910(60)90033-0.

30. Lees, A. D. "The Role of Photoperiod and Temperature in the Determination of Parthenogenetic and Sexual Forms in the Aphid, Megoura viciae Buckton. III. Further Properties of the Maternal Switching Mechanism in Apterous Aphids." Journal of Insect Physiology, vol. 9, 1963, pp. 153-154. DOI: 10.1016/0022-1910(63)90017-4.

31. Macrovitch, S. "The Migration of the Aphididae and the Appearance of Sexual Morphs as Affected by the Relative Length of Daily Light Exposure." Journal of Agricultural Research, vol. 27, 1924, pp. 513-522. DOI: not available.

32. Matsuka, M., and T. E. Mittler. "Production of Males and Gynoparae by Apterous Viviparae of Myzus persicae Continuously Exposed to Different Scotoperiods." Journal of Insect Physiology, vol. 25, 1979, pp. 587-593. DOI: 10.1016/0022-1910(79)90122-3.

33. Parker, G. A., et al. "Optimal Copulation Duration in Yellow Dung Flies: Effects of Female Size and Egg Content." Animal Behaviour, vol. 57, 1999, pp. 795-805. DOI: 10.1006/anbe.1998.1035.

34. Pettersson, J. "Preliminary Report on Ethological Observations of the Copulatory Act Among Schizaphis dubia Huc. and Schizaphis borealis Tambs-Lyche (Homoptera: Aphididae)." Opuscula Entomologica, vol. 33, 1968, pp. 359-361. DOI: not available.

35. Pettersson, J. "Studies on Rhopalosiphum padi (L.). I. Laboratory Studies on Olfactometric Responses to the Winter Host Prunus padus L." Lantbruks högskolans Annaler, vol. 36, 1970, pp. 381-99. DOI: not available.

36. Pettersson, J. "An Aphid Sex Attractant. II. Histological, Ethological and Comparative Studies." Entomologica Scandinavica, vol. 2, 1971, pp. 81-93. DOI: 10.1163/187631271X00288.

37. Pickett, J. A., et al. "The Chemical Ecology of Aphids." Annual Review of Entomology, vol. 37, 1992, pp. 67-90. DOI: 10.1146/annurev.en.37.010192.000435.

38. Raychaudhuri, D. N. Aphids of North-East India and Bhutan. The Zoological Society, Calcutta, 1980. DOI: not available.

39. Sharma, M. L., et al. "Séquences de Descendence des Formes Sexuées Chez le Puceron du Pois Acyrthosiphon pisum (Homoptera: Aphididae) en Relation avec la Durée des Photopériodes." Canadian Entomologist, vol. 107, 1975, pp. 1063-1067. DOI: 10.4039/Ent1071063-10.

40. Stevens, N. M. "A Study of the Germ Cells of Aphis rosae." Proceedings of the Academy of Natural Sciences of Philadelphia, vol. 57, 1905, pp. 167-176. DOI: not available.

41. Tamaki, G., et al. "Arrest and Aggregation of Male Myzus persicae (Hemiptera: Aphididae)." Annals of the Entomological Society of America, vol. 63, 1970, pp. 955–960. DOI: 10.1093/aesa/63.3.955.

42. Van Embden, H. F., et al. "The Ecology of Myzus persicae." Annual Review of Entomology, vol. 14, 1969, pp. 197-270. DOI: 10.1146/annurev.en.14.010169.001213.

43. Wedell, N., et al. "Sperm Competition, Male Prudence and Sperm Limited Females." Trends in Ecology & Evolution, vol. 17, 2002, pp. 313-320. DOI: 10.1016/S0169-5347(02)02533-8.