Oviposition substrate location by the invasive woodwasp Sirex noctilio: the combined effect of chemical cues emitted by its obligate symbiont Amylostereum areolatum and different host-tree species.

BACKGROUND: Sirex noctilio is an invasive forest wasp that affects pines and can result in severe economic losses. The use of semiochemicals offers an opportunity to develop sensitive and specific capturing systems to mitigatenegative impacts. Previous research showed that female S. noctilio would use volatiles emitted by its fungal symbiont, Amylostereum areolatum, but little is known about how these modulate behaviour when combined with pine-wood emissions. Our aim was to understand the relevance of fungal volatiles grown on artificial media and wood from two hosts trees, Pinus contorta and Pinus ponderosa, on behavioural and electroantennographic responses of wasp females. Because background odours can modify an insect's response towards resource-indicating semiochemicals, we propose that the behaviour towards the symbiont (resource) will be modulated by host pine emissions (background odours). RESULTS: Olfactometric assays showed that both host species with fungus were attractive when contrasted against air (P. contorta versus Air, χ2 = 12.19, P < 0.001; P. ponderosa versus Air, χ2 = 20.60, P < 0.001) and suggest a clear hierarchy in terms of female preferences towards the tested stimuli, with response highest towards the fungus grown on P. contorta (olfactory preference index: 5.5). Electrophysiological analyses indicate that females detect 62 volatile compounds from the tested sources. CONCLUSION: Results indicate a strong synergy between symbiont and host semiochemicals, suggesting that the pine species could play a fundamental role in the interaction. Further understanding of the chemical basis of this, could guide the development of specific and attractive lures, in order to maximize attraction of wasps in surveillance programmes. © 2023 Society of Chemical Industry.

Effect of Fruit Volatiles from Native Host Plants on the Sexual Performance of Anastrepha fraterculus sp. 1 Males.

Anastrepha fraterculus sp.1 males are sexually stimulated by the aroma of fruit of its native host Psidium guajava (guava). Other hosts, which are exotic to A. fraterculus, do not enhance male sexual behavior. Here we evaluate the effects of fruit volatile exposure on male A. fraterculus sp. 1 sexual performance using other native hosts, under the hypothesis that male improvement derives from a common evolutionary history between A. fraterculus sp. 1 and its native hosts. Four species were evaluated: Eugenia myrcianthes, Juglans australis, Psidium cattleianum, and Acca sellowiana. Guava was used as a positive control. Males were exposed to fruit from 12:00 pm to 4:00 pm, from day 8 to day 11 post-emergence. On day 12, we evaluated their calling behavior and mating success. Both guava and P. cattleianum enhanced calling behavior. Mating success was enhanced only by guava and a trend was found for P. cattleianum. Interestingly, the two hosts belong to the Psidium genus. A volatile analysis is planned to identify the compounds responsible for this phenomenon. The other native fruits did not improve the sexual behavior of males. Implications of our findings in the management of A. fraterculus sp. 1 are discussed.

Identification and characterization of soluble binding proteins associated with host foraging in the parasitoid wasp Diachasmimorpha longicaudata.

The communication and reproduction of insects are driven by chemical sensing. During this process, chemical compounds are transported across the sensillum lymph to the sensory neurons assisted by different types of soluble binding proteins: odorant-binding proteins (OBPs); chemosensory proteins (CSPs); some members of ML-family proteins (MD-2 (myeloid differentiation factor-2)-related Lipid-recognition), also known as NPC2-like proteins. Potential transcripts involved in chemosensing were identified by an in silico analysis of whole-body female and male transcriptomes of the parasitic wasp Diachasmimorpha longicaudata. This analysis facilitated the characterization of fourteen OBPs (all belonging to the Classic type), seven CSPs (and two possible isoforms), and four NPC2-like proteins. A differential expression analysis by qPCR showed that eleven of these proteins (CSPs 2 and 8, OBPs 2, 3, 4, 5, 6, 9, 10, and 11, and NPC2b) were over-expressed in female antenna and two (CSP 1 and OBP 12) in the body without antennae. Foraging behavior trials (linked to RNA interference) suggest that OBPs 9, 10, and 11 are potentially involved in the female orientation to chemical cues associated with the host. OBP 12 seems to be related to physiological processes of female longevity regulation. In addition, transcriptional silencing of CSP 3 showed that this protein is potentially associated with the regulation of foraging behavior. This study supports the hypothesis that soluble binding proteins are potentially linked to fundamental physiological processes and behaviors in D. longicaudata. The results obtained here contribute useful information to increase the parasitoid performance as a biological control agent of fruit fly pest species.

Mating-induced changes in responses of female Queensland fruit fly to male pheromones and fruit: A mechanism for mating-induced sexual inhibition.

In order to reproduce, female tephritid fruit flies need both mates for fertilization and fruit for oviposition. Virgin females are prone to mating and approach males, attracted by their pheromones. Mated females, however, may experience an abrupt reduction of mating propensity and prioritise the search for suitable fruit rather than additional mates. Accordingly, mating in fruit flies may induce a switch in olfactory preferences of females from pheromones to fruit stimuli, and this switch may also be an important mediator of mating-induced sexual inhibition. To test for mating-induced switches in olfactory preference of female Queensland fruit fly, Bactrocera tryoni, we used wind tunnel assays to assess attraction of mated and virgin females to (1) male sex pheromone delivered through a perforated glass sphere or (2) an entire fruit. Electroantennogram (EAG) responses were also used to test for mating-induced changes in olfactory sensitivity to pheromones and fruit odours. Pheromones elicited quicker upwind responses in virgin females than in mated females; during the first six minutes of trials more virgin females than mated females were observed in the upwind end of the wind tunnel where pheromone odours were released. Fruit cues, in contrast, elicited stronger association with the upwind end of the wind tunnel in mated females than in virgin females from the fifth minute onwards. Also, mated females were observed on the fruit for longer periods than virgin females. EAG responses to pheromones and fruit odours were similar in virgin and mated females, indicating that changes in preferences are not a consequence of changes in peripheral sensitivity of antennae to odours but instead appear to be mediated by post-receptor processing. Our results show that mating reduces attraction to male-produced pheromones and increases attraction to fruit stimuli in B. tryoni females. We propose that this behavioural switch from mating stimuli to oviposition stimuli is an important mediator of mating-induced sexual inhibition in this species.

Host discrimination in the fruit fly parasitoid Diachasmimorpha longicaudata: evidence from virgin female behaviour and egg distribution patterns.

Many parasitoid species discriminate already parasitized hosts, thus avoiding larval competition. However, females incur in superparasitism under certain circumstances. Superparasitism is commonly observed in the artificial rearing of the parasitoid Diachasmimorpha longicaudata, yet host discrimination has been previously suggested in this species. Here, we addressed host discrimination in virgin D. longicaudata females in a comprehensive way by means of direct and indirect methods, using Ceratitis capitata and Anastrepha fraterculus which are major fruit fly pests in South America. Direct methods relied on the description of the foraging behaviour of females in arenas with parasitized and non-parasitized host larvae. In the indirect methods, healthy larvae were offered to single females and the egg distributions were compared to a random distribution. We found that D. longicaudata was able to recognize parasitized host from both host species, taking 24 h since a first parasitization for A. fraterculus and 48 h for C. capitata. Indirect methods showed females with different behaviours for both host species: complete discrimination, non-random (with superparasitism), and random distributions. A larger percentage of females reared and tested on A. fraterculus incurred in superparasitism, probably associated with higher fecundity. In sum, we found strong evidence of host discrimination in D. longicaudata, detecting behavioural variability associated with the host species, the time since the first parasitization and the fecundity of the females.

Symbionts do not affect the mating incompatibility between the Brazilian-1 and Peruvian morphotypes of the Anastrepha fraterculus cryptic species complex.

The South American fruit fly, Anastrepha fraterculus, is clearly undergoing a speciation process. Among others, two of their morphotypes, the Brazilian-1 and Peruvian, have accumulated differences in pre- and post-zygotic mechanisms resulting in a degree of reproductive isolation. Both harbor a different strain of Wolbachia, which is a widespread endosymbiotic bacterium among many invertebrates producing a range of reproductive effects. In this paper, we studied the role of this bacterium as one of the factors involved in such isolation process. Infected and cured laboratory colonies were used to test pre- and post-zygotic effects, with special emphasis in uni- and bi-directional cytoplasmic incompatibility (CI). We showed that Wolbachia is the only known reproductive symbiont present in these morphotypes. Wolbachia reduced the ability for embryonic development in crosses involving cured females and infected males within each morphotype (uni-directional CI). This inhibition showed to be more effective in the Peruvian morphotype. Bi-directional CI was not evidenced, suggesting the presence of compatible Wolbachia strains. We conclude that Wolbachia is not directly involved in the speciation process of these morphotypes. Other mechanisms rather than CI should be explored in order to explain the reduced mating compatibility between the Brazilian-1 and Peruvian morphotypes.

Mate choice confers direct benefits to females of Anastrepha fraterculus (Diptera: Tephritidae).

Exposure to plant compounds and analogues of juvenile hormone (JH) increase male mating success in several species of tephritid fruit flies. Most of these species exhibit a lek mating system, characterized by active female choice. Although the pattern of enhanced male mating success is evident, few studies have investigated what benefits, if any, females gain via choice of exposed males in the lek mating system. In the South American fruit fly, Anastrepha fraterculus, females mate preferentially with males that were exposed to volatiles released by guava fruit or treated with methoprene (a JH analogue). Here, we tested the hypothesis that female choice confers direct fitness benefits in terms of fecundity and fertility. We first carried out mate choice experiments presenting females with males treated and non-treated with guava volatiles or, alternatively, treated and non-treated with methoprene. After we confirmed female preference for treated males, we compared the fecundity and fertility between females mated with treated males and non-treated ones. We found that A. fraterculus females that mated with males exposed to guava volatiles showed higher fecundity than females mated to non-exposed males. On the other hand, females that mated methoprene-treated males showed no evidence of direct benefits. Our findings represent the first evidence of a direct benefit associated to female preference for males that were exposed to host fruit odors in tephritid fruit flies. Differences between the two treatments are discussed in evolutionary and pest management terms.

Innate Host Habitat Preference in the Parasitoid Diachasmimorpha longicaudata: Functional Significance and Modifications through Learning.

Parasitoids searching for polyphagous herbivores can find their hosts in a variety of habitats. Under this scenario, chemical cues from the host habitat (not related to the host) represent poor indicators of host location. Hence, it is unlikely that naïve females show a strong response to host habitat cues, which would become important only if the parasitoids learn to associate such cues to the host presence. This concept does not consider that habitats can vary in profitability or host nutritional quality, which according to the optimal foraging theory and the preference-performance hypothesis (respectively) could shape the way in which parasitoids make use of chemical cues from the host habitat. We assessed innate preference in the fruit fly parasitoid Diachasmimorpha longicaudata among chemical cues from four host habitats (apple, fig, orange and peach) using a Y-tube olfactometer. Contrary to what was predicted, we found a hierarchic pattern of preference. The parasitism rate realized on these fruit species and the weight of the host correlates positively, to some extent, with the preference pattern, whereas preference did not correlate with survival and fecundity of the progeny. As expected for a parasitoid foraging for generalist hosts, habitat preference changed markedly depending on their previous experience and the abundance of hosts. These findings suggest that the pattern of preference for host habitats is attributable to differences in encounter rate and host quality. Host habitat preference seems to be, however, quite plastic and easily modified according to the information obtained during foraging.

Male Sexual Behavior and Pheromone Emission Is Enhanced by Exposure to Guava Fruit Volatiles in Anastrepha fraterculus.

BACKGROUND: Plant chemicals can affect reproductive strategies of tephritid fruit flies by influencing sex pheromone communication and increasing male mating competitiveness. OBJECTIVE AND METHODOLOGY: We explored whether exposure of Anastrepha fraterculus males to guava fruit volatiles and to a synthetic blend of volatile compounds released by this fruit affects the sexual performance of wild and laboratory flies. By means of bioassays and pheromone collection we investigated the mechanism underlying this phenomenon. RESULTS: Guava volatile exposure enhanced male mating success and positively affected male calling behavior and pheromone release in laboratory and wild males. Changes in male behavior appear to be particularly important during the initial phase of the sexual activity period, when most of the mating pairs are formed. Exposure of laboratory males to a subset of guava fruit volatiles enhanced mating success, showing that the response to the fruit might be mimicked artificially. CONCLUSIONS: Volatiles of guava seem to influence male mating success through an enhancement of chemical and physical signals related to the communication between sexes. This finding has important implications for the management of this pest species through the Sterile Insect Technique. We discuss the possibility of using artificial blends to improve the sexual competitiveness of sterile males.

Evaluating mating compatibility within fruit fly cryptic species complexes and the potential role of sex pheromones in pre-mating isolation.

The study of sexual behavior and the identification of the signals involved in mate recognition between con-specifics are key components that can shed some light, as part of an integrative taxonomic approach, in delimitating species within species complexes. In the Tephritidae family several species complexes have received particular attention as they include important agricultural pests such as the Ceratitis fasciventris (Bezzi), Ceratitis anonae (Graham) and Ceratitis rosa Karsch (FAR) complex, the Bactrocera dorsalis (Hendel) complex and the Anastrepha fraterculus (Wiedemann) complex. Here the value and usefulness of a methodology that uses walk-in field cages with host trees to assess, under semi-natural conditions, mating compatibility within these complexes is reviewed, and the same methodology to study the role of chemical communication in pre-mating isolation among Anastrepha fraterculus populations is used. Results showed that under the same experimental conditions it was possible to distinguish an entire range of different outcomes: from full mating compatibility among some populations to complete assortative mating among others. The effectiveness of the methodology in contributing to defining species limits was shown in two species complexes: Anastrepha fraterculus and Bactrocera dorsalis, and in the case of the latter the synonymization of several established species was published. We conclude that walk-in field cages constitute a powerful tool to measure mating compatibility, which is also useful to determine the role of chemical signals in species recognition. Overall, this experimental approach provides a good source of information about reproductive boundaries to delimit species. However, it needs to be applied as part of an integrative taxonomic approach that simultaneously assesses cytogenetic, molecular, physiological and morphological traits in order to reach more robust species delimitations.