New phase proposed in the relationship between figs and wasps

The study was published in a special issue of Acta Oecologica with four articles by scientists from Brazil. Another paper analyzes morphological differences in the ovipositors of several parasitoid wasp species.

By Peter Moon  |  Agência FAPESP – The mutualistic relationship between figs and the fig wasps that pollinate them is one of the most fascinating in nature. Their existence is so intertwined that one cannot survive without the other. Fig trees cannot reproduce if there are no fig wasps (Agaonidae) and vice versa.

The fig wasp’s reproductive cycle takes place only inside figs, which have changed so much due to their interaction with fig wasps over the course of tens of millions of years of evolution that today, they are thought to be fruit. However, a fig is not actually a fruit; it is an inverted inflorescence, a cluster of hundreds of tiny flowers contained inside a bulbous stem. The flowers produce seeds internally after being pollinated by fig wasps.

The lifecycles of figs and fig wasps are studied as a way of understanding the evolution of mutualism. Fifty years ago, in the late 1960s, when the fig-wasp mutualism began to be elucidated, it was divided into five biologically based developmental phases (A, B, C, D and E) describing how the ripening fig becomes attractive to female wasps, which enter the inflorescence to lay their eggs, and how a new generation of fertilized female wasps eventually emerges from the fig to renew the cycle.

Half a century after the initial description of this development cycle, Brazilian biologist Luciano Palmieri Rocha has proposed a new phase, which he calls the F phase; this phase encompasses the ecological interactions that occur after the wasps leave, involving the ripe figs that fall and rot on the ground.

The study was published in the journal Acta Oecologica as part of a special volume compiled to commemorate the 50th anniversary of the original discovery of the fig-wasp mutualism. The research was supported by FAPESP.

The special issue of the journal contains 20 papers, four of them by researchers at the Insect-Plant Interaction Laboratory belonging to the Biology Department of the University of São Paulo’s Ribeirão Preto School of Philosophy, Science and Letters (FFCLRP-USP). The laboratory is headed by Professor Rodrigo Augusto Santinelo Pereira.

“There’s a lot of competition in nature. This is why fig-wasp mutualism is so interesting. The two species coexist and mutually adapt to survive. If one dies, the other will also disappear,” Palmieri told Agência FAPESP. 

This mutualism is not confined to the interaction between the species that produces edible figs (Ficus carica, the common fig) and its specific pollinators, fig wasps of the species Blastophaga psenes. The genus Ficus comprises more than 750 species, and for each, there is a species of pollinating agaonid wasp.

The mutualism is ancient, Palmieri explained. The oldest fossils of fig wasps date from 34 million years ago. They closely resembled the species alive today, indicating that the symbiotic relationship evolved early and has not changed fundamentally since then. Molecular evidence shows that the relationship existed 65 million years ago, suggesting that it might be even older, perhaps going back to the age of dinosaurs. 

“At some point, the ancestors of fig wasps began laying eggs in the flowers of ancestral fig trees. We believe these inflorescences were still open, so they could be pollinated by various insects,” Palmieri said. 

Over the course of at least 65 million years of evolution, the fig’s inflorescence became an enclosure sealed off from the outside world, and only the fig wasp was able to penetrate it.

“Initially, the wasp parasitized the fig, but then through some unknown evolutionary mechanism, the plant ended up co-opting the wasp’s parasitism and making it part of its own reproductive cycle,” Palmieri said. 

The fig-wasp lifecycle begins when the female wasp enters the fig. “The fig is an urn that preserves and protects hundreds of tiny flowers. The flowers open inside it, so they need a special pollination process. They cannot rely on wind or bees to carry their pollen. That’s where the fig wasp comes in,” he explained. 

Inside the fig, there are female and male flowers that develop at different times. The A phase occurs when the female flowers are not yet mature. They soon mature and are ready to be fertilized. They become receptive to the wasps and release a scent made up of a huge amount of volatile compounds, triggering the B phase. 

“The scents are chemical signals serving to attract only the specific wasps that pollinate the flowers of that fig species. It’s all synchronized,” Palmieri said.

Each fig receptacle is not entirely closed but has a small hole called an ostiole, through which the female wasp penetrates its interior. As it does so, it loses its wings and its antennae are broken, so that it cannot get out again. It lays its eggs and dies. “The female must force its way inside through the ostiole. After that it’s harder for others to get in, but not unusual,” he noted.

Synchronized actions

Once inside the fig, the female wasp lays eggs in many of the flowers but not all. At the same time, it fertilizes the flowers with pollen stored in a pouch on the underside of its thorax. The flowers on which the eggs are laid now undergo a transformation to become hardened structures call galls. 

Now begins the C phase, which lasts two to three months. The flowers that receive pollen but no eggs develop into seeds. Flowers that receive eggs and harden into galls become nurseries with food and shelter for wasp larvae. 

The D phase occurs at the end of larval incubation. This is also when the male flowers start to mature, opening up to expose pollen containers known as anthers. 

“The opening of the male flowers is synchronized with the end of larval development. The first wasps to emerge from the galls are wingless males with reduced eyes but large strong mandibles,” Palmieri said. “They crawl over the female flowers until they find the galls containing the females, their sisters, now ready to emerge. The male penetrates the female with a telescopic penis and fertilizes the female inside the gall. Once they have mated in this way, the males use their mandibles to bite through the fig wall. They then go out through the hole, fall to the ground and die.”

The D phase ends when the female wasps emerge from their galls. “As they crawl toward the hole made by the male wasps, they pass over the male flowers and fill their pouches with pollen, with which they will pollinate other fig trees,” Palmieri said. 

Leaving the receptacle through the hole made by their brothers, the fertilized females fly away in search of other fig trees, and the cycle begins again. The E phase consists of seed dispersal. 

“A large fig tree is capable of producing more than 1 million figs in a single flowering. The figs are eaten by monkeys, rodents, bats, peccaries and many other animals. Almost all forest-dwelling vertebrates feed on figs as part of their diet. By eating ripe figs that are still attached to branches or have fallen to the ground, the animals scatter the seeds through the environment in their droppings,” Palmieri said. 

F phase

Palmieri has now proposed a new phase in addition to the five phases of the classic fig-wasp lifecycle, which has been studied for 50 years. 

“The F phase is an ecological phase, which doesn’t relate directly to the development of the fig but rather to its role in the development of dozens of other insect species aside from fig wasps,” he said.

“Many organisms including acarid mites and nematodes as well as insects are able to parasitize figs, but most are wasps belonging to groups that are closely related to fig wasps. They manage to insert their eggs into figs without performing the biological role of pollination.”

Evidence of the new F phase began to appear over the course of years of observation. “While studying the interaction between figs and fig wasps, we often found larvae of other creatures that played no role in the development cycle. These figs were discarded and left out of the research. In some cases, larvae that were almost the same size as the fig had eaten almost its entire contents. That’s when we decided to investigate what was going on,” Palmieri said. 

“As some of these larvae reached adulthood, creatures no one recognized would start coming out of rotten figs on the ground. In the article just published, I describe 129 insects belonging to five orders and 24 different families that are not fig wasps but that also interact with figs, performing different functions.” 

Palmieri identified ten species of wasps (Hymenoptera), 39 species of flies (Diptera), 46 species of beetles (Coleoptera), 17 species of cicadas and other bugs (Hemiptera), and 18 species of butterflies or moths (Lepidoptera). 

These insects may colonize figs during different phases of the tree’s lifecycle. Some rely on fallen figs to complete their development. Palmieri divided the insects into two categories according to their role in the fig tree’s ecology and their potential impact on its reproduction. He called the categories “early fig interlopers” and “fallen fig fauna”. 

All the insects identified have representatives in both categories except for ten wasp species belonging to three families that are not fig wasps but that bear some resemblance to them. All ten are early fig interlopers that oviposit in figs and the larvae of which compete directly with those of fig wasps for food and space inside the fig or simply feed on them, leaving the fig when they reach adulthood.

In the article, Palmieri describes the modus operandi of several early fig interlopers in detail. One is Lissocephala, a genus of flies that lay eggs in the ostiole at the same time as the original female wasp is entering the fig. The fly larvae migrate to the interior of the fig and feed exclusively on yeast and bacteria brought inside by the pollinating wasp. The flies finish their development inside the fig and leave by the exit hole previously chewed in the fig wall by male wasps.

Butterflies and moths are the most aggressive group of insects in terms of the damage to figs. They lay eggs in the fig wall. In the C phase, their larvae bore through the fig wall and feed indiscriminately on fig pulp, wasps and seeds. The larvae destroy the hanging fig and crawl out to pupate in cocoons attached to branches of the tree. 

In the case of fallen fig fauna, Palmieri explained, the category comprises various organisms that feed on the fleshy parts or seeds of ripe figs not consumed by fruit-eating vertebrates. They take advantage of the window of opportunity created by the figs that fall under the parent tree in the F phase.

Fallen fig fauna, which includes some ants, butterflies and bugs, consists mainly of beetles that feed on fruit remains. Beetles take advantage of the fig development cycle in various ways. Some colonize figs on the tree in the early C phase. Their larvae grow inside the figs and stay there when the ripe fruit falls to the ground. They then migrate to the soil, where they dig holes and pupate in cocoons.

“These examples give us just a glimpse of a far greater complexity of interactions. In addition to the evolutionary implications of pollination mutualism, an additional factor relating to the success of the 750-odd fig species is probably the highly diversified fauna of insects associated with fig trees, such as nonpollinating wasp species. Pressure from these parasitic wasps will have been, and continues to be, a key driver of fig species diversification,” Palmieri said. 

The article “The role of non-fig-wasp insects on fig tree biology, with a proposal of the F phase (fallen figs)” (https://doi.org/10.1016/j.actao.2017.10.006) by Luciano Palmieri and Rodrigo Augusto Santinelo Pereira is published at: sciencedirect.com/science/article/pii/S1146609X17300395. 

Ovipositor differences

Some 650 species of fig wasp belonging to various families have been described, representing less than half the estimated number of fig wasp species, including pollinators and parasitic nonpollinators.

“The latter are opportunists. They lay eggs from outside using a structure called an ovipositor to pierce the fig wall and insert their eggs into flowers or galls,” said Larissa Galante Elias, a researcher affiliated with the Insect-Plant Interaction Laboratory at FFCLRP-USP.

In another article published in the special issue of Acta Oecologica, Elias and colleagues analyze morphological differences in the ovipositors of several pollinating and nonpollinating wasp species. The study was supervised by Santinelo Pereira and supported by FAPESP. 

“Over the course of millions of years of evolution, the ovipositor adapted in order to perform different functions. My research interest is understanding how wasps are able to do such complex and differentiated things with their ovipositors, such as laying eggs outside the fig wall and injecting them accurately inside a flower or placing eggs in galls or even inside the fig wall. In present-day wasps, we see the ovipositor performing all these functions,” Elias said. 

First author of the article alongside other Brazilian researchers and colleagues from France and China, Elias analyzed variations in ovipositor morphology in 24 fig wasp species belonging to nine different genera.

“The ovipositor is a structure found in all wasp species, but it’s slightly different in each one. It’s very thin and long, and can be up to three times longer than the wasp’s body,” Elias said. 

Pollinating fig wasps lay eggs when the flowers are young. The flowers of a fig tree are the hundreds of filaments inside the fig, which has a convex base. Each flower containing a larva will change into a gall. 

Parasitic wasps lay eggs at the same time as pollinating wasps or a little later, when the larvae are developing in galls. “They may parasitize hundreds of galls, but they lay only one egg in each gall. Their larvae feed on the larvae that are already there,” Elias explained.

Her study included ancestral state reconstruction analysis, which is the basis for interpreting the evolution of a range of morphological, ecological and behavioral characters in a given group of organisms.

“The ovipositor has tooth-like structures at its extremity. I realized that the morphology of these teeth varies a great deal and decided to investigate whether the structures varied between different wasp species depending on the stage of fig development in which they lay eggs, such as when the fig is young or when galls have formed,” Elias said. 

Samples were taken from 24 species belonging to all the main clades (group that have evolved from a common ancestor) of agaonids, including representatives of all described genera of nonpollinating wasps in the family. There were species from Brazil, Australia, China, Laos, Senegal, Indonesia, Cameroon, India and the Solomon Islands. Some were collected in the field, while others were obtained from the collection of Jean-Yves Rasplus, research director of the Center for Biology and Population Management (CBGP) at France’s National Agricultural Research Institute (INRA). 

Using a stereomicroscope, Elias performed a series of measurements of the bodies and ovipositors of 10-20 individuals from each species and analyzed characteristics relating to ovipositor teeth to investigate their potential role in drilling and anchoring the ovipositor while allowing it to move through the fig substrate. 

“I found that the distance between ovipositor teeth correlated with what the wasp was doing. The teeth were more spread out or closer together,” Elias said. 

The wasps studied by Elias belong to different ecological groups. Wasps that insert eggs by piercing the fig wall with their ovipositor when the fig is young and depositing the eggs in flowers on the inside are called gallers because egg deposition stimulates the development of galls. “We discovered that in the case of gallers, the ovipositor teeth are closer together,” she noted. 

Another group comprises wasps that use their ovipositors to insert eggs into galls through the fig wall. These are known as parasitoids because they parasitize the galls. “Their ovipositors have irregular teeth and are further apart,” Elias said. 

“Ancestral state reconstruction analysis suggested that the common ancestor of agaonids had an ovipositor adapted to laying eggs in young flowers,” she explained. In other words, the ovipositor adapted over millions of years to insert eggs into galls, and this adaptation was a driver of diversification for the group.

“It’s interesting to have this new method of identifying the type of fig wasp by analyzing the ovipositor because this means it’s no longer necessary to analyze the entire fig development cycle in order to see which wasp is doing what in the context of these interactions,” she said. 

The article “Ovipositor morphology correlates with life history evolution in agaonid fig wasps” (doi: https://doi.org/10.1016/j.actao.2017.10.007) by Larissa Galante Elias, Finn Kjellberg, Fernando Henrique Antoniolli Farache, Eduardo A.B. Almeida, Jean-Yves Rasplus, Astrid Cruaud, Yan-Qiong Peng, Da-Rong Yang and Rodrigo Augusto Santinelo Pereira is published at: sciencedirect.com/science/article/pii/S1146609X17300358. 

The special volume with four articles by Brazilian researchers is available at sciencedirect.com/journal/acta-oecologica/vol/90/suppl/C.