© 2016. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2016) 219, 2965-2967

INSIDE JEB

Wolbachia makes H. horticola were better at evading M. cf. parasitism’, says Duplouy and stigmaticus infections or became more colleagues, who suspect that bacteria parasite vulnerable to susceptible to the tenacious hyperparasite. may contribute to H. horticola’s hyperparasite First, they had to produce H. horticola- resistance to other parasitic infections infected Granville fritillary caterpillars instead. that they could place in the environment, where half of the caterpillars were 10.1242/jeb.149740 carrying H. horticola that were infected van Nouhuys, S., Kohonen, M. and Duplouy, A. with Wolbachia and the other half were (2016). Wolbachia increases the susceptibility of Wolbachia free. a parasitoid wasp to hyperparasitism. J. Exp. Biol. 219, 2984–2990. Duplouy and colleagues collected Kathryn Knight parasitized fritillary larvae from the Åland Hyposoter horticola Melitaea wasp laying eggs in Islands and waited for the adult H. horticola cinxia eggs. Photo credit: Anne Duplouy. wasps to emerge. Knowing that only 50% Manduca of the parasitic wasps carried Wolbachia, larvae survive Evil parasites are the stuff of horror movies, bursting out of unsuspecting victims in the they then offered clutches of butterfly eggs drowning most ghoulish fashion possible. However, to the H. horticola wasp mothers for them it seems that even parasites are not immune to lay their eggs in. After nurturing the to parasites of their own. Anne Duplouy, butterfly eggs as they developed into Saskya van Nouhuys and Minna Kohonen caterpillars, the team checked that some from the University of Helsinki, Finland, individuals from each clutch were carrying describe how Hyposoter horticola parasitic H. horticola wasp larvae and then wasps, which exclusively target Granville transferred the parasitized caterpillars onto fritillary butterfly caterpillars – on the potted plants, where they spun silky nests Åland Islands off Finland – are susceptible for overwintering; the team finally in turn to Mesochorus cf. stigmaticus transferred the plants and their parasitised caterpillars to Åland Islands to measure the hyperparasitic wasps, which hijack Submerged Manduca sexta pupa. Photo credit: H. horticola larvae as incubators for their impact that Wolbachia infection had on the Steve Lane. own offspring. However, Duplouy had also rate of M. cf. stigmaticus infestation of noticed previously that H. horticola H. horticola larvae. Dragonfly larvae are perfectly equipped populations had lower levels of M. cf. for a life of total immersion in their pond stigmaticus infection where H. horticola Retrieving the caterpillar nests from the homes prior to emergence for their final had high rates of infection by the symbiotic islands several weeks later and metamorphosis. However, other species Wolbachia bacteria (doi: 10.1371/journal. comparing the number of butterfly nests may be less well prepared for soggy starts pone.0134843). Explaining that Wolbachia that had been targeted by the M. cf. in life. Art Woods from the University of infections are often beneficial to their hosts stigmaticus hyperparasites, the team Montana, USA, explains that although the in order to maintain their welcome, found that 74% of the H. horticola larvae and pupae of other insects spend Duplouy wondered whether the bacteria larvae that carried Wolbachia were some portion of their lives buried in dry also boosted the resistance of H. horticola infested by M. cf. stigmaticus larvae, in soil, their conditions can change rapidly to M. stigmaticus hyperparasitic lodgers – contrast to the Wolbachia-free H. when the weather turns. Whether it’s the consequently reducing M. cf. stigmaticus horticola, which only suffered a 40% onset of the monsoon or a rapid thaw, dry infection rates when Wolbachia infection M. stigmaticus infestation rate. So, soil in river beds and desiccated flood rates are high. However, there was an Wolbachia infections seem to make H. plains can flood quickly, submerging alternative explanation for low levels of horticola larvae more vulnerable to larvae as the soil saturates. ‘Anoxia can be M. cf. stigmaticus infection: if Wolbachia parasitic infection, which poses the highly stressful, even in highly reduced the resistance of H. horticola to question of why H. horticola maintains anoxia-tolerant individuals’, says Woods, M. cf. stigmaticus, this would allow the close relationship with symbiotic who was intrigued by how well Manduca M. stigmaticus to kill off Wolbachia- bacteria that increase its vulnerability in sexta moth pupae survive drowning. infected H. horticola to keep their numbers some circumstances. ‘In order to persist down when M. cf. stigmaticus infection in the host population, [Wolbachia] Graduate student Steven Lane submerged rates were high. must have a positive effect on the pupae for up to a fortnight and plucked the fitness of infected parasitic wasps that insects from the water on alternate days Intrigued by both possibilities, the team could outweigh the costly burden of (from day 1 to 13) to find out whether investigated whether Wolbachia-infected susceptibility to widespread they survived and, if so, how long it took

Inside JEB highlights the key developments in Journal of Experimental Biology. Written by science journalists, the short reports give the inside viewof the science in JEB. Journal of Experimental Biology

2965 INSIDE JEB Journal of Experimental Biology (2016) 219, 2965-2967 them to recover from total immersion. mechanisms underlying patterns of gas lengthy sections of the three calcium Impressively, all of the pupae that were exchange in insects’. channel subunits, fed them to removed from the water after submersion P. tetraurelia for up to 72 h and then for 5 days survived; however, the partially 10.1242/jeb.149591 transferred the cells to a solution that drowned pupae took 7 days longer to Woods, H. A. and Lane, S. J. (2016). Metabolic triggers swimming in reverse to assess recovery from drowning by insect pupae. J. Exp. emerge as adults than pupae that had Biol. 219, 3126–3136. how losing channel components affected remained dry, and extending the swimming performance. Comparing immersion by 2 days proved fatal. Kathryn Knight Paramecium cells that had received the ‘Survival times of pupal M. sexta in RNAi diet with those that had not, the immersion (anoxia) are impressive but team found that cells that had lost one not unprecedented’, says Woods, adding CaV channels send channel subunit could only reverse for that other species that suffer flooding Paramecium into reverse ∼3–5 s and cells that had lost all three are capable of surviving similar periods. barely reversed at all (∼2 s); however, And when Lane measured the amount of cells that not been fed RNAi swam lactic acid produced by the submerged backward for ∼23 s. The CaV insects, the pupae that had been channel proteins found in the cilia submerged for longest had the largest were responsible for Paramecium’s amount and it took 2 days to dissipate; about-turn. the insects had switched from aerobic to anaerobic respiration while Paramecium with 10 µm-long cilia from Grass However, the team needed further submerged. Calendar, 1985. Picture credit: Judith Van convincing that the CaV1a–c channel Houten. subunits provided the essential calcium Recording the pupae’s respiration patterns current, so they turned their attention to a as they recovered, it was clear to Lane that The tissues and organs of most animal mutant form of P. tetraurelia – known as they initially opened their spiracles for an bodies are lined by ceaselessly beating ‘Pawn’–which, just like its chess piece – extended period to release large amounts microscopic hairs from the structures that namesake, is unable to reverse. As there waft cerebrospinal fluid around the brain to of CO2. However, once the CO2 levels were two possible explanations for the had fallen sufficiently, individual lung-cleansing cilia, and many single-cell Pawn mutants’ inability to reverse – either organisms, such as Paramecium,use spiracles began to close, possibly because they lacked CaV channels in their cilia or the pH of the body tissues had increased similar structures for propulsion in the they had the channels, but the proteins pursuit of food and to avoid difficult as the CO2 seeped out. And then the CO2 were inactive for some reason – Lodh and emission pattern switched again, rising conditions. Junji Yano and Judith Van Yano added a tag to the end of the Pawn Houten from the University of Vermont, and falling every 0.8–2.2 min, suggesting CaV1c gene and allowed the cells to grow either that the pupa was opening and USA, explain that the relentlessly beating before collecting the cilia. Then they closing its spiracles rapidly or that the motion of Paramecium cilia is driven by tested for evidence of the tagged channel insect was pumping its abdomen while calcium currents passing through protein protein in the cilia, but there was none, channels within the microscopic structures holding its spiracles open to expel the suggesting Pawn mutants lack CaV that switch the direction in which the cilia accumulated CO2. It was also clear that channels in their cilia. the recovering insect’s metabolic rate was beat to send the cell into reverse. The true 50–75% higher than that of pupae that had identity of the proteins that comprise these Lodh and Yano then inserted normal copies not been submerged. As the spiracle channels had remained elusive for almost of thegenesthat are known to be defective in ’ opening patterns that produce intermittent half a century until Van Houten s team Pawn cells (known as PWA and PWB)and breathing patterns in insects in other identified components of the calcium tested the modified cells; not only had they circumstances are regulated by the channel in the cilia of Paramecium regained the ability to reverse but also the tetraurelia in 2013. But there was still no interplay between CO2 and O2 levels in essential CaV channel proteins were present the spiracles and adjacent tissues, direct evidence that these channels were the in the cilia. In addition, the normal PwB Woods explains that only one scenario – origin of the essential calcium current, so protein appears to be associated with the Yano, Sukanya Lodh and Van Houten set where spiracle opening and closing are CaV1c protein in the endoplasmic triggered by the acidity of the about directly testing the role of the channel reticulum, where proteins are synthesised, surrounding tissue – seemed likely to in Paramecium swimming behaviour. suggesting that PwB may be involved in produce the conditions that could trigger transporting CaV1c to the cilia, where the some, but not all, aspects of the RNA interference (RNAi) – where protein contributes to production of the recovering pupae’s unusual high- segments of RNA that match the sequence calcium current that is essential for the frequency breathing pattern. of a target gene are fed to organisms to change in direction of P. tetraurelia’s prevent production of the protein that is beating cilia. So, the pupae of species that occasionally encoded by the gene – is a powerful suffer flooding and are at risk of drowning technique that allows scientists to assess 10.1242/jeb.149724 are capable of recovering after several the physiological role of specific genes. Lodh, S., Yano, J., Valentine, M. S. and Van days of submersion, and Woods says, As Van Houten’s team had previously Houten, J. L. (2016). Voltage-gated calcium ‘Pupae of Manduca would make a good sequenced three calcium channel alpha 1 channels of Paramecium cilia. J. Exp. Biol. 219, 3028–3038. model system for further studies linking subunits (CaV1a, 1b, 1c), Lodh and Yano immersion, anoxia tolerance and the produced RNA fragments containing Kathryn Knight Journal of Experimental Biology

2966 INSIDE JEB Journal of Experimental Biology (2016) 219, 2965-2967

Testosterone regulates scarlet plumage in red-backed fairy-wrens

As the days begin to lengthen and winter of red-backed fairy-wrens altered were unable to increase the levels of the recedes for yet another year, many birds after they received a testosterone hormone in their blood. dust off their most glamorous plumage implant. to attract a mate. Males often opt for So, it seems that testosterone is the ostentatious displays while females tend Initially, the males and females both hormone that regulates the production of to the dowdier end of the spectrum. But looked drab; however, soon after the birds the carotenoid pigments that produce the what factors regulate the dramatic received the implant, the females moulted males’ scarlet plumage; however, the differences in avian regalia between and began sprouting shorter male-like hormone was unable to elicit production of males and females? Willow Lindsay feathers. The females also produced a the jet-black melanin pigment that from the University of Gothenburg, vibrant patch of red feathers on their produces their distinctive dark feathers. It Sweden, explains that the hormones that backs, similar to the bright red streak on seems that the female fairy-wrens still regulate sexual differences were thought the males’ backs. In addition, the feathers maintain the essential hormone mechanism to play a key role in defining plumage on the crown, belly and breast of the that allows the males to put on their gaudy patterns, but the precise role of females turned a shade of pinky-orange, display, although the females are unable to individual hormones was less clear. As which Lindsay said was: ‘Never produce sufficient testosterone to trigger testosterone starts flowing in spring, previously documented on a red-backed the masculine transformation. male red-backed fairy-wrens (Malurus fairy-wren’. Only one female went on to 10.1242/jeb.149609 melanocephalus) shed their dreary produce black feathers like those of the brown feathers in favour of a vivid males, although the beaks of all of the Lindsay, W. R., Barron, D. G., Webster, M. S. and Schwabl, H. (2016). Testosterone activates sexual scarlet and glossy black coat, while the females became darker. And when dimorphism including male-typical carotenoid but females retain their dingy brown and Lindsay and Barron injected some not melanin plumage pigmentation in a female bird. white plumage. So, Lindsay and Douglas females with gonadotropin-releasing J. Exp. Biol. 219, 3091–3099. Barron headed south to Queensland, hormone, in a bid to naturally raise the Kathryn Knight Australia, to find out how the plumage birds’ testosterone levels, the females [email protected] Journal of Experimental Biology

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