In this study, we gave a comprehensive description of the mating behavior of the greenhouse whitefly, T. vaporariorum. In particular, we defined the strict association between vibrational signals and behavioral steps of the pair formation process, from the male call to the final mating. We also described some social interactions between two or more individuals of both sexes, confined to a small portion of leaf, thus simulating a natural occurring aggregation. In this regard, we found that males tend to modify the quality of their vibrational signals, by changing some spectral features, according to either the social context or the behavioral step. For example, they tend to increase the fundamental frequency of their signals (i.e., chirps and PT) when in the presence of potential rivals. A possible explanation of this behavior could be associated with the male competition for food and/or mating. In fact, species that live in high population densities are subjected to strong male-male competitions and a male needs to show his quality to females but also to be clearly recognizable from the others24. The higher quality can be witnessed by the emission of specific aggressive calls which are characterized by lower frequencies, like in some anurans25 or in Chiropteran where the relative frequency of the social calls increases when more individuals compete for a food source. An example of individual recognition behavior is the change of frequency of the calling song to avoid signal overlapping thus allowing an individual to perceive the presence of more potential partners. Frequency overlapping, in general, can be noxious to animal communication, and male responsiveness can be reduced when background noise from conspecific signals obscure the species-specific temporal pattern of a female song26. In the southern green stink bug, Nezara viridula (Heteroptera, Pentatomidae), females were found to change their calling song frequency to let the males recognize them when exposed to a disturbance stimulus27. Even if small variations of the frequency pattern may potentially affect the partner responsiveness to a call28, overlapping frequencies can seriously compromise the signal reception29. In this way, the change to a different value of frequency in presence of other calling males seems to be a more desirable solution.
Another signal variation that we observed in GW males, in the presence of another male (i.e., male duos trials), regarded the chirp duration. By increasing the duration of a mating signal, some species also increase the chances to elicit the female response at the earliest stage of the mating behavior30. In various acoustic insects, females prefer longer calls and males can vary their length by adding or subtracting call elements31. However, a limit of our study was that we could not associate the signal emissions to specific individuals, therefore we did not determine not only if one or both the males were actually singing but also whether this change of chirp duration involved one or both the individuals. A definitive explanation about male-male calling interactions and how males regulate their calling activities should be provided with additional experiments with the use of playbacks to stimulate single specimens.
In general, we need to consider that the alteration of the signal features is a common strategy in animals with a complex mating behavior in which different stages can alternate in a non-linear sequence29,32,33. Such an intricate behavior is on the one hand, at the basis of a species-specific mate recognition system, on the other hand, is a result of the sexual selection that worked to shape signals with certain characteristics that are able to elicit the female acceptance to mate34. Despite the considerable knowledge about vibrational signal production in the family Aleyrodidae19, we still have little information about the importance of the courtship and of the female choice in driving the reproductive isolation and speciation in this family. Aleyrodid species are known to be morphologically similar and to form a species complex (i.e. Bemisia tabaci) with several biotypes35, where the characterization of the mating behavior can be an important tool to discriminate among them. For instance, variations in the courtship behavior between different B. tabaci biotypes demonstrated the presence of pre-copulation barriers36,37. Moreover, the analysis of male vibrational signals during the courtship, combined with genetic and morphological analysis, allowed to discriminate between the camellia spiny whitefly Aleurocanthus camelliae and the citrus spiny whitefly Aleurocanthus spiniferus18. In such a context, knowing the characteristics of the mating ritual may lead to distinguish, not only among different species, but also among different populations. For example, before this study, the GW mating behavior was described only from Japanese populations where the pair formation process started with the male approaching a female before emitting any vibrational signal (i.e. courtship stage)17. Instead, in our study with European populations of GW, we observed that the male, before starting the approach, emits calling signals which can elicit the female response from a certain distance. Such a difference between geographically distant GW populations seems to suggest a different strategies of mating behavior, likely associated to distinct populations or biotypes. On this regard, it would be interesting to test them with crossed mating trials (Japanese vs Europeans) to assess the effects of the observed differences on the mating success rate.
In our study, we also measured a difference of male signal parameters between different behavioral phases of the pair formation process and in particular between the courtship stage and the call and alternated duet stages. We found a significant increase of signal duration, fundamental frequency and pulse repetition rate. The duration of the courtship stage was very variable in our trials, from zero (it was skipped when females replied immediately to the male signals) up to 78 min. This means, in first instance, that the role of the courtship is to elicit the female response and thus promoting her acceptance to mate. Indeed any single behavioral step is functional to elicit the female’s acceptance and in fact, whenever females showed high responsiveness since the early stage of the mating process, males could skip whole stages and even go directly from the call to the final precopula stage, the alternated duet. It also indicates that males are available to spend a remarkable amount of energy to perform the courtship38. The use of elaborated and energetic signals during the courtship is rather common in animals34. For example, the leafhopper S. titanus and the glassy-winged sharpshooter Homalodisca vitripennis have a mating strategy that reminds the GW’s, starting with a call which is followed by the location of the partner and by the courtship. While during call and location males make use of extremely simplified signals, during the courtship they emit the most elaborated (and energetically demanding) signals, through which they try to convince the female to accept the mating21,39. A study of Las (1980) demonstrated that the GW courtship persistence (i.e., duration) is an important trigger to address the female choice. A fast and prolonged male “cycling rate” (alternation of wing flicking and antennation) during the courtship is preferred by females who become even more selective after the first mating. On the other hand, in our tests, males showed a remarkable perseverance in courting the females. The ethogram showed that after a failed mating attempt, a male always restarted from the courtship. This means that the courtship phase is the key part of the mating process but also that the female choice drives the selection in favor of “stubborn” males that persist in courting the potential partner, performing a prolonged courtship, even if the first mating attempt fails. Stubbornness affects male’s survival for its energetic cost and risk of eavesdropping. Such character fits the handicap theory model, in which condition dependent and costly traits are honest indicators of male quality40,41. On the other hand, the option of an easy surrender, and the search for another available female, after investing so many energies in courting the first one, seems to be not convenient for the male in that it would mean to spend more energy in searching for/courting a new partner also risking the possibility of dealing with competitors42.
In the GW, the male courtship can be considered successful when the mating moves to the overlapped duet stage in which the female emits the Female Responding Signal (FRS). The FRS is produced in synchronous with the courtship chirp and PT and, for this reason, it requires high degree of coordination between male and female. The presence of female acceptance signals synchronized with the male’s is known for the whitefly species Aleurothrixus floccosus (Maskell)43, in which the female signal can partially overlap the male’s one, but it was unknown in the GW, until now.
Another signal that we found for the first time in the GW is the male rivalry signal (MRS). Males exhibit aggressiveness towards other males. A random encounter on the leaf is enough to trigger the expression of rivalry behavior in presence of a female. Such interaction has never been observed in duos, but only in groups with responsive females, thus suggesting that the presence of receptive/active females is required to trigger the MRS production and thus provoking a context of aggressiveness and competition between males. Another male rivalry behavior that we observed in the presence of a receptive female is the silent approach (satellite behavior) to intercept a female while duetting with another male44. This behavior is known in other aleyrodids like in B. tabaci. In this species, rival males interrupt the ongoing courtship of the duetting male by approaching the female from the opposite side. In response to the competitor, the first male spreads the wings and beats the rival on the head45. In GW, the rivalry behavior is associated with the continuous production of the MRS, which is the male signal at highest frequency. Such finding strengthens the hypothesis that the frequency shift has a role in competitor’s deterrence. The rivalry behavior of GW seems to be extraordinarily strong, as much to push females to abandon the interaction with both males. In our experiments, none of the females, even those that had already established a duet with a male, eventually mated. On the contrary, they left the arena before the end of the trial. Our findings are consistent with previous observations of GW behavior, in which the contended female always walked away when two males were competing15. Therefore, we can speculate that the adaptive advantage of the male rivalry behavior in GW is not immediate and the disruption of another male’s attempt could provide more chances in the future to the intruder, by leaving a receptive female unmated. Beside the effects of the male’s rivalry, we also observed females that refused to mate and rejected approaching males with the emission of specific vibrational signals. There are several reasons to refuse mating: immature females are not yet available to mate, and recently mated females must undergo to a refractory period before they accomplish other copulations15. On the other hand, a mature female can choose whether to accept or not a courting male depending on the level of his fitness which is, very likely, testified by the courtship performance. Females can evaluate the male’s quality based on the courtship persistence, so that they need to let males perform the whole ritual before choosing whether to mate or not46. In fact, we observed both females that rejected approaching males and females that rejected them at the end of the courtship performance. The latter, in particular, was associated to wing flicking and/or male’s aedeagus parrying with the legs. Similar behaviors were also observed in B. tabaci, in which the female can either walk or fly away from approaching males, flap the wings or push the male’s abdomen away with the middle pair of legs45. What seems to be a peculiar treat of GW is the use of a specific rejective signal (FRjS). The emission of FRjS seems to reinforce the motivation of the female to reject the male. However, it is not clear to us why the FRjS signal has been observed only in the group (males and females together) trials and never in pairs (one male and one female). Our hypothesis is that in case of groups, males can approach the “wrong” female, who was close the receptive one. This implies that males are not capable of precisely locating the responding female and that the emission of FRjS by an unreceptive female would help the males to not waste too much time (and energy) with them.
To conclude, this study unveiled many aspects of the mating behavior of the GW that were previously overlooked and thus it contributes to fill several gaps of knowledge that will be important to start a program in the field of applied biotremology10. The question, from which originally arose this research study, was whether the use of vibrational signals could be suitable to manipulate the mating behavior of the GW. We can say that the vibrational communication is fundamental to accomplish mating and, in our trials, with pairs and groups, we never observed mating without the exchange of vibrational signals between male and female. This means that the interruption or the disruption of this communication could be potentially useful to reduce the rate of mating success. Manipulation of intraspecific communication by means of vibrational signals has been already developed for other insect species both in the lab and in the field10. For example, the male rivalry signal has been exploited for the development of a vibrational mating disruption strategy against the grapevine leafhopper Scaphoideus titanus29, while the female playback has been used to attract and trap males in the brown marmorated stink bug Halyomorpha halys47. The use of playbacks that cover the fundamental frequencies of the male and female signals could be used to mask their communication2. Another possible approach could be to use signals that mimic the natural signals of the species48. In the case of the GW, the FRS could be employed to disrupt males and induce them in courting unreceptive females. This would lead to a substantial reduction of the mating success rate but also to a considerable increase of wasted energy caused by the male persistence in courting unreceptive females. Another possible outcome could be a change of the gender balance in the population. GW females reproduce by arrhenotokous parthenogenesis in which unfertilized eggs develop into males49. Delays in mating could lead to a sex bias that could eventually mine the population structure. Another option is the use of the MRS to generate an aggressive and stressful environment. The transmission of MRS into the plant tissues in loop could eventually negatively affect the development of GW populations. All these approaches are potentially effective and could be in the future considered as tools for IPM and/or organic protection programs. Further applied research will provide a final answer to our question and will test the effectiveness of behavioural manipulation strategies for the control of the GW. Finally, considering that the GW uses a short range sexual pheromone emitted by females50 olfactory and vibratory cues could be potentially integrated to develop new pest control technologies10.
Source: Ecology - nature.com
