Chaos of Wolbachia Sequences Inside the Compact Fig Syconia of Ficus benjamina (Ficus: Moraceae)

Figs and fig wasps form a peculiar closed community in which the Ficus tree provides a compact syconium (inflorescence) habitat for the lives of a complex assemblage of Chalcidoid insects. These diverse fig wasp species have intimate ecological relationships within the closed world of the fig syconia. Previous surveys of Wolbachia, maternally inherited endosymbiotic bacteria that infect vast numbers of arthropod hosts, showed that fig wasps have some of the highest known incidences of Wolbachia amongst all insects. We ask whether the evolutionary patterns of Wolbachia sequences in this closed syconium community are different from those in the outside world. In the present study, we sampled all 17 fig wasp species living on Ficus benjamina, covering 4 families, 6 subfamilies, and 8 genera of wasps. We made a thorough survey of Wolbachia infection patterns and studied evolutionary patterns in wsp (Wolbachia Surface Protein) sequences. We find evidence for high infection incidences, frequent recombination between Wolbachia strains, and considerable horizontal transfer, suggesting rapid evolution of Wolbachia sequences within the syconium community. Though the fig wasps have relatively limited contact with outside world, Wolbachia may be introduced to the syconium community via horizontal transmission by fig wasps species that have winged males and visit the syconia earlier.     

Consistently high incidence of Wolbachia in global fig wasp communities

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Comparisons of host mitochondrial, nuclear and endosymbiont bacterial genes reveal cryptic fig wasp species and the effects of Wolbachia on host mtDNA evolution and diversity

Background  

Figs and fig-pollinating wasp species usually display a highly specific one-to-one association. However, more and more studies have revealed that the "one-to-one" rule has been broken. Co-pollinators have been reported, but we do not yet know how they evolve. They may evolve from insect speciation induced or facilitated by Wolbachia which can manipulate host reproduction and induce reproductive isolation. In addition, Wolbachia can affect host mitochondrial DNA evolution, because of the linkage between Wolbachia and associated mitochondrial haplotypes, and thus confound host phylogeny based on mtDNA. Previous research has shown that fig wasps have the highest incidence of Wolbachia infection in all insect taxa, and Wolbachia may have great influence on fig wasp biology. Therefore, we look forward to understanding the influence of Wolbachia on mitochondrial DNA evolution and speciation in fig wasps.     

Wolbachia prevalence patterns: horizontal transmission,recombination, and multiple infections in chestnut gall wasp-parasitoid communities

The Oriental chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae), is a global invasive pest that causes serious damage to almost all chestnut species belonging to the Castanea genus (Fagaceae). Dryocosmus zhuili Liu et Zhu is a recently described sibling species of D. kuriphilus, which induces galls on Castanea henryi (Skan) Rehd. et Wils. There are many indigenous parasitoid species in China which play an important role in the natural regulation of their population dynamics. Wolbachia is a maternally inherited α-proteobacterium widely found in arthropods. This study screened for the presence of Wolbachia in the two chestnut gall wasps and in six parasitoid species from 12 populations, to investigate the prevalence patterns of Wolbachia in the chestnut gall wasp-parasitoid communities. We found that D. zhuili and four parasitoid species were infected with Wolbachia; among them, all individuals of the two populations of Megastigmus sp. had multiple Wolbachia infections. By using multilocus sequence types to characterize bacterial strains, three new sequence types were identified. The Wolbachia strains infecting D. zhuili (ST-507), Torymus sinensis Kamijo (ST-508), and Sycophila variegata (Curtis) (ST-508) belonged to supergroup A, whereas the Wolbachia strain infecting Megastigmus nipponicus Kamijo (ST-503) belonged to supergroup B. Our results also suggested that horizontal transmission of Wolbachia occurs between chestnut gall wasps and their parasitoids. Moreover, multiple Wolbachia infections of Megastigmus sp. may be due to gene recombination and horizontal transmission.     

Natural occurrence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal

Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.     

Pollinators entering female dioecious figs: why commit suicide?

In the dioecious fig/pollinator mutualism, the female wasps that pollinate figs on female trees die without reproducing, whereas wasps that pollinate figs on male trees produce offspring. Selection should strongly favour wasps that avoid female figs and enter only male figs. Consequently, fig trees would not be pollinated and fig seed production would ultimately cease, leading to extinction of both wasp and fig. We experimentally presented pollinators in the wild (southern India) with a choice between male and female figs of a dioecious fig species, Ficus hispida L. Our results show that wasps do not systematically discriminate between sexes of F. hispida. We propose four hypotheses to explain why wasp choice has not evolved, and how a mutualism is thus maintained in which all wasps that pollinate female figs have zero fitness.     

高榕雌花期传粉榕小蜂和欺骗性小蜂的繁殖特点

榕树及其专一性传粉榕小蜂组成了动植物界最为经典的协同进化关系,传粉榕小蜂演化出欺骗性是非常罕见的。在雌雄同株的高榕隐头果内,共存着一种传粉榕小蜂Eupristina altissima和一种欺骗性的小蜂Eupristina sp.,两种小蜂在雌花期进入隐头果内繁殖,但有不同的繁殖特点。对比研究了两种小蜂从成虫羽化到产卵和传粉这个阶段的雌蜂个体大小、孕卵量及繁殖差异,结果表明:羽化期两种雌蜂的平均个体小,经飞行小个体的雌蜂易死亡,大个体雌蜂到达接受树,但通过苞片通道,一些个体较大的传粉榕小蜂被夹死导致进入果腔的雌蜂相对小,而欺骗性小蜂易通过苞片以至进入果腔的雌蜂个体较大。两种未产卵雌蜂均表现为个体大者孕卵量较多,但两种雌蜂的平均孕卵量没有差异。即使有充足雌花资源产卵,两种雌蜂均未产完所有卵,产卵后两种雌蜂卵巢中的卵量均显著减少,遗留下的卵量两种小蜂间没有差异。传粉榕小蜂只有部分个体传完所携带花粉,并表现为传粉越成功的雌蜂,产卵越多。存在种内竞争时,两种小蜂的产卵量均减少,传粉榕小蜂的传粉效率也降低。在种间竞争背景下,欺骗性小蜂产卵更成功,传粉榕小蜂的产卵和传粉量均受到极大抑制。研究结果说明雌花期隐头果内传粉榕小蜂只适量利用雌花资源产卵繁殖后代,更有效地传粉繁殖榕树种子,这可能是维持榕-蜂互惠系统稳定共存的重要机制之一;欺骗者稳定存在需降低与传粉者的直接竞争,而欺骗者和传粉者分散在不同果内,甚至是不同的树上繁殖是理想的繁殖策略。     

Deep mtDNA divergences indicate cryptic species in a fig-pollinating wasp

Background  

Figs and fig-pollinating wasps are obligate mutualists that have coevolved for ca 90 million years. They have radiated together, but do not show strict cospeciation. In particular, it is now clear that many fig species host two wasp species, so there is more wasp speciation than fig speciation. However, little is known about how fig wasps speciate.     

Dispersal of adult female fig wasps: 2. Movements between trees

Fig wasps (Chalcidoidea, Agaonidae, Agaoninae) are the exclusive pollinators of fig trees (Ficus spp., Moraceae). Fig development on the African fig tree, F. burtt-davyi, is normally synchronised on individual trees, but not between trees. Consequently the females of each generation of the pollinating species (Elisabethiella baijnathi) have to disperse to other trees to find ‘receptive’ figs which are suitable for oviposition. This paper examines this aspect of fig - fig wasp biology. The flight speed of insects is closely linked to their size, and directional flight is difficult for small insects, such as fig wasps, in all but the lightest of winds. We investigated the movements of fig wasps between trees using sticky traps placed around fig trees or near cotton bags containing figs. Away from the trees, the densities of flying wasps at different heights was also determined. When the wasps disperse from their natal figs they take off near-vertically. They are unable to exert directional control once they enter the air column and are subsequently blown downwind. Near receptive host trees the wasps appear to lose height and then fly upwind at speeds of around 25 cm/sec.     

Parasites and mutualism function: measuring enemy‐free space in a fig–pollinator symbiosis

Mutualisms involve cooperation between species and underpin several ecosystem functions. However, there is also conflict between mutualists, because their interests are not perfectly aligned. In addition, most mutualisms are exploited by parasites. Here, we study the interplay between cooperation, conflict and parasitism in the mutualism between fig trees and their pollinator wasps. Conflict occurs because each fig ovary can nurture either one seed or one pollinator offspring and, while fig trees benefit directly from seeds and pollinator offspring (pollen vectors), pollinators only benefit directly from pollinator offspring. The mechanism(s) of conflict resolution is debated, but must explain the widespread observation that pollinators develop in inner, and seeds in outer, layers of fig flowers. We recently suggested a role for non‐pollinating figs wasps (NPFWs) that are natural enemies or competitors of the pollinators and lay their eggs through the fig wall. Most NPFW offspring develop in outer and middle layer flowers, suggesting that inner flowers provide enemy‐free space for pollinator offspring. Here, we test the hypothesis that NPFWs cannot reach inner flowers, by measuring wasp and fig morphology at the species‐specific times of NPFW attack in the field. We found that three species of Sycoscapter and Philotrypesis wasps that parasitise pollinators could reach 34–73%, 75–92% and 82–97% of fig ovaries, respectively. Meanwhile, Eukobelea and Pseudidarnes gall‐formers, despite having shorter ovipositors, can access almost all fig flowers (93–99% and 100%), because they attack smaller (younger) fig fruits. Our mechanistic results from ovipositing wasps support spatial patterns of wasp offspring segregation within figs to suggest that inner ovules provide enemy‐free‐space for pollinators. This may contribute to mutualism stability by helping select for pollinators to avoid laying eggs where they are likely to be parasitised. These outer flowers then remain free to develop as seeds, promoting mutualism persistence.     

Measuring the discrepancy between fecundity and lifetime reproductive success in a pollinating fig wasp

Lifetime reproductive success in female insects is often egg‐ or time‐limited. For instance in pro‐ovigenic species, when oviposition sites are abundant, females may quickly become devoid of eggs. Conversely, in the absence of suitable oviposition sites, females may die before laying all of their eggs. In pollinating fig wasps (Hymenoptera: Agaonidae), each species has an obligate mutualism with its host fig tree species [Ficus spp. (Moraceae)]. These pro‐ovigenic wasps oviposit in individual ovaries within the inflorescences of monoecious Ficus (syconia, or ‘figs’), which contain many flowers. Each female flower can thus become a seed or be converted into a wasp gall. The mystery is that the wasps never oviposit in all fig ovaries, even when a fig contains enough wasp females with enough eggs to do so. The failure of all wasps to translate all of their eggs into offspring clearly contributes to mutualism persistence, but the underlying causal mechanisms are unclear. We found in an undescribed Brazilian Pegoscapus wasp population that the lifetime reproductive success of lone foundresses was relatively unaffected by constraints on oviposition. The number of offspring produced by lone foundresses experimentally introduced into receptive figs was generally lower than the numbers of eggs carried, despite the fact that the wasps were able to lay all or most of their eggs. Because we excluded any effects of intraspecific competitors and parasitic non‐pollinating wasps, our data suggest that some pollinators produce few offspring because some of their eggs or larvae are unviable or are victims of plant defences.     

Convergent incidences of Wolbachia infection in fig wasp communities from two continents

Wide surveys suggest that Wolbachia bacteria infect ca. 20% of all insect species, but particular taxonomic or ecological groups may display significantly higher or lower incidences. We studied 61 fig wasp species in Australia and found the highest known incidence (67%) of infection in a targeted study of this nature. A comparable study in Panama reported a similar figure (59%), confirming the exceptionally high incidence of Wolbachia in fig wasps. Importantly, these are two independent estimates of Wolbachia incidence in fig wasp communities, because no host species, or even genera, are shared between localities. The high level of infection may reflect enhanced opportunities for horizontal transmission inside fig fruits. Although incidence was similar in Panama and Australia, the actual strains involved were different and region-specific. Local strains were shared by several host species, although there was often no obvious (direct) ecological link between two hosts with the same infection.     

The dominant exploiters of the fig/pollinator mutualism vary across continents,but their costs fall consistently on the male reproductive function of figs

1. Fig trees (Moraceae: Ficus) are keystone species, whose ecosystem function relies on an obligate mutualism with wasps (Chalcidoidea: Agaonidae) that enter fig syconia to pollinate. Each female flower produces one seed (fig female reproductive function), unless it also receives a wasp egg, in which case it supports a wasp. Fig male reproductive function requires both male flowers and pollinator offspring, which are the only vectors of fig pollen. 2. The mutualism is exploited by other wasps that lay eggs but provide no pollination service. Most of these non‐pollinating fig wasps (NPFWs) do not enter syconia, but lay eggs through the wall with long ovipositors. Some are gall‐makers, while others are parasitoids or lethal inquilines of other wasps. 3. Ficus is pan‐tropical and contains >750 fig species. However, NPFW communities vary across fig lineages and continents and their effects on the mutualism may also vary. This provides a series of natural experiments to investigate how the costs to a keystone mutualism vary geographically. 4. We made the first detailed study of the costs of NPFWs in a fig (Ficus obliqua G. Forst) from the endemic Australasian section Malvanthera. In contrast to the communities associated with section Americana in the New World, wasps from the subfamily Sycoryctinae (Chalcidoidea: Pteromalidae) dominated this community. 5. These sycoryctine wasps have a negative impact on pollinator offspring numbers, but not on seed production. Consequently, while the NPFW fauna varies greatly at high taxonomic levels across continents, we show that the consistent main effect of locally dominant exploiters of the mutualism is to reduce fig male reproductive function.     

Egg deposition patterns of fig pollinating wasps: implications for studies on the stability of the mutualism

1. Fig pollinating wasps (Agaonidae) enter Ficus inflorescences (figs), oviposit in some of the flowers, and pollinate in the process. Each larva completes its development within a single flower. In most cases, an inflorescence entered by a wasp will represent its only egg‐laying site. The mechanisms that prevent pollinating wasps from exploiting all the flowers inside a fig are not understood. In this study, hypotheses about flower use by pollinating fig wasps were tested by investigating egg deposition patterns in three species. 2. Either one or three wasps were introduced into figs. The figs were then harvested. Serial sections allowed assessment of the presence or absence of a wasp egg in a sample of flowers in each fig. The overall proportion of flowers with eggs and the spatial distribution of eggs were then compared in single wasp figs and three foundress figs. 3. In all species, the proportion of flowers with a wasp egg increased with foundress number but less than three‐fold. 4. In all species, at least in single foundress figs, flowers near the fig cavity were more likely to receive a wasp egg than were flowers near the fig wall. 5. In two species, when the number of foundresses was multiplied by three, there was an increase in the use of flowers near the fig wall, while in the third species, the increase was spread evenly among flowers. 6. Factors affecting wasp egg deposition patterns and the potential of investigating such patterns for studying the stability of the mutualism are discussed.     

Bacteria Endosymbiont,Wolbachia, Promotes Parasitism of Parasitoid Wasp Asobara japonica

Wolbachia is the most widespread endosymbiotic bacterium that manipulates reproduction of its arthropod hosts to enhance its own spread throughout host populations. Infection with Wolbachia causes complete parthenogenetic reproduction in many Hymenoptera, producing only female offspring. The mechanism of such reproductive manipulation by Wolbachia has been extensively studied. However, the effects of Wolbachia symbiosis on behavioral traits of the hosts are scarcely investigated. The parasitoid wasp Asobara japonica is an ideal insect to investigate this because symbiotic and aposymbiotic strains are available: Wolbachia-infected Tokyo (TK) and noninfected Iriomote (IR) strains originally collected on the main island and southwest islands of Japan, respectively. We compared the oviposition behaviors of the two strains and found that TK strain females parasitized Drosophila melanogaster larvae more actively than the IR strain, especially during the first two days after eclosion. Removing Wolbachia from the TK strain wasps by treatment with tetracycline or rifampicin decreased their parasitism activity to the level of the IR strain. Morphological and behavioral analyses of both strain wasps showed that Wolbachia endosymbionts do not affect development of the host female reproductive tract and eggs, but do enhance host-searching ability of female wasps. These results suggest the possibility that Wolbachia endosymbionts may promote their diffusion and persistence in the host A. japonica population not only at least partly by parthenogenesis but also by enhancement of oviposition frequency of the host females.     

Oogenesis and ovarian morphology in pollinating and non-pollinating fig wasps: evidence from adult and immature stages

Hymenopteran insects have meroistic polytrophic ovaries characterised by trophocytes associated with oocytes inside the follicles. In pro-ovigenic species, all oocytes mature before emergence and no trace of oogenesis is visible in adult females. Pro-ovigeny is a rare condition among Hymenoptera, but common in pollinating fig wasps. In the present investigation, we studied adult and pupa females of three fig wasp species with different trophic strategies. We demonstrated that females of Pegoscapus aerumnosus and Idarnes spp. have an unusual ovarian organisation (i.e. each ovariole has only one mature egg and no oocyte) that has led to misleading interpretation of fig wasp reproductive anatomy. The ovaries of these studied species have several ovarioles, recognisable by the presence of nuclei of tunica propria cells surrounding them. Each adult wasp ovariole had one mature egg. None of the pupae had mature eggs, but all of them had follicles with oocytes in different developmental stages. The studied fig wasps are pro-ovigenic, irrespective of their trophic strategy, since there were no signs of ovigeny in adult females. We discuss ecological and phylogenetic factors that could play a role in fig wasps reproductive strategies.     

Virginity in haplodiploid populations: a study on fig wasps

ABSTRACT.

• 1 The fig wasps inhabiting the figs of Ficus hispidioides S. Moore in New Guinea were investigated. The galls formed by the pollinating agaonid were also inhabited by two other non-pollinating species of wasp; a third non-pollinating wasp caused the production of a different type of gall, and was itself parasitized by a fourth species. In all species, males were wingless and all matings occurred within the fig.
• 2 It was estimated that 2% of the pollinating fig wasps left the fig unmated. The equivalent figures for three of the non-pollinating wasps were 2%, 4% and 23%. The significance of oviposition by virgin females to the sex allocation strategy of mated females is discussed.
• 3 The absence of fighting and male wing dimorphism were studied in the context of the predictions of their occurrence by Hamilton (1979).

     

Fighting in fig wasps: do males avoid killing brothers or do they never meet them?

1. In many fig wasp species, armoured wingless males regularly engage in lethal fights for access to females inside figs, which act as discrete mating patches. 2. Kin selection generally opposes killing brothers, because their reproductive success provides indirect genetic benefits (inclusive fitness). However, siblicide may be avoided if (i) brothers do not occur in the same figs, or (ii) males avoid fighting brothers in the same fig. Alternatively, (iii) siblicide may occur because intense mate competition between brothers at the local scale overcomes kin selection effects, or (iv) males do not recognise kin. 3. A fig may also contain wasps from other closely related species and it is not known if males also fight with these individuals. 4. Nine microsatellite loci were used in the first genetic analysis of fighting in fig wasps. We assigned species and sibling identities to males and tested alternative fighting scenarios for three Sycoscapter wasp species in figs of Ficus rubiginosa. 5. Approximately 60% of figs contained males from more than one Sycoscapter species and approximately 80% of fights were between conspecifics, but a surprising 20% were between heterospecific males. 6. Within species, few figs contained brothers, suggesting that females typically lay one son per fig. Overall, most males do not compete with brothers and all fights observed were between unrelated males.     

Do Fig Wasps Produce Mixed Paternity Clutches?

Pollinating fig wasps (Hymenoptera, Agaonidae) have been the focus of numerous studies examining sex ratio evolution. Recently, molecular genetic techniques have been introduced that assume single matings in fig wasps, yet their mating biology has not been investigated genetically. We used recently developed microsatellite markers to investigate whether a pollinating fig wasp (Liporrhopalum tentacularis Grandi) produces single or mixed paternity clutches. The clutches of 12 females which had had the opportunity to mate with males of different genotypes were investigated. The results suggest that, at least in this species of fig wasp, single paternity clutches are the norm. Based on our behavioural observations, this appears to be due to mating with a single male rather than sperm competition.     

Diversification in the use of resources by Idarnes species: bypassing functional constraints in the fig–fig wasp interaction

Mutualisms such as the fig–fig wasp mutualism are generally exploited by parasites. We demonstrate that amongst nonpollinating fig wasps (NPFWs) parasitic on Ficus citrifolia, a species of Idarnes galls flowers and another species feeds on galls induced by other wasps killing their larvae. The galling wasp inserts its ovipositor through the fig wall into the fig cavity. The ovipositor then follows a sinuous path and is introduced through the stigma and style of the flower. The egg is deposited between the integument and nucellus, in the exact location where the pollinating mutualistic wasp would have laid its egg. Gall induction is a complex process. In contrast, the path followed by the ovipositor of the other species is straightforward: attacking a larva within a developed gall poses different constraints. Shifts in feeding regime have occurred repeatedly in NPFWs. Monitoring traits associated with such repeated evolutionary shifts may help understand underlying functional constraints. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 114–122.