The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Tympanal ears mediate male-male competition, courtship and mating success in Bicyclus anynana butterflies.

The presence of intra-specific acoustic communication in diurnal butterflies is not well established. Here, we examined the function of the tympanal ear (Vogel's organ, VO) in the seasonally polyphenic butterfly Bicyclus anynana in the context of sexual signalling. We investigated how the VO and the flanking enlarged veins, which are suggested sound resonance chambers, scale with wing size across sexes and seasonal forms, and how disruptions to the VO alter courtship behaviour and mating outcomes. We found that males have VOs similar in size to females despite having smaller wings, and dry season (DS) male cubital and anal veins do not scale with the wing size. This suggests that the VO plays an important role in males and that cubital and anal veins in DS males may be tuned to amplify specific sound frequencies. Behavioural assays performed with deafened and hearing males of different seasonal forms, in pair and triad settings, showed that deafened DS males, but not wet season males, experienced lower mating success relative to their hearing counterparts. Our study documents a novel function for the wing tympanal membrane in mediating courtship and mating outcomes in diurnal butterflies.

The yellow gene regulates behavioural plasticity by repressing male courtship in Bicyclus anynana butterflies.

Seasonal plasticity in male courtship in Bicyclus anynana butterflies is due to variation in levels of the steroid hormone 20E (20-hydroxyecdysone) during pupation. Wet season (WS) males have high levels of 20E and become active courters. Dry season (DS) males have lower levels of 20E and reduced courtship rates. However, WS courtship rates can be achieved if DS male pupae are injected with 20E at 30% of pupation. Here, we investigated the genes involved in male courtship plasticity and examined whether 20E plays an organizational role in the pupal brain that later influences the sexual behaviour of adults. We show that DS pupal brains have a sevenfold upregulation of the yellow gene relative to the WS brains, and that knocking out yellow leads to increased male courtship. We find that injecting 20E into DS pupa reduced yellow expression although not significantly. Our results show that yellow is a repressor of the neural circuity for male courtship behaviour in B. anynana. 20E levels experienced during pupation could play an organizational role during pupal brain development by regulating yellow expression, however, other factors might also be involved. Our findings are in striking contrast to Drosophila where yellow is required for male courtship.

Dissections of Larval, Pupal and Adult Butterfly Brains for Immunostaining and Molecular Analysis.

Butterflies possess impressive cognitive abilities, and investigations into the neural mechanisms underlying these abilities are increasingly being conducted. Exploring butterfly neurobiology may require the isolation of larval, pupal, and/or adult brains for further molecular and histological experiments. This procedure has been largely described in the fruit fly, but a detailed description of butterfly brain dissections is still lacking. Here, we provide a detailed written and video protocol for the removal of Bicyclus anynana adult, pupal, and larval brains. This species is gradually becoming a popular model because it uses a large set of sensory modalities, displays plastic and hormonally controlled courtship behaviour, and learns visual mate preference and olfactory preferences that can be passed on to its offspring. The extracted brain can be used for downstream analyses, such as immunostaining, DNA or RNA extraction, and the procedure can be easily adapted to other lepidopteran species and life stages.

Early-exposure to new sex pheromone blends alters mate preference in female butterflies and in their offspring.

While the diversity of sex pheromone communication systems across insects is well documented, the mechanisms that lead to such diversity are not well understood. Sex pheromones constitute a species-specific system of sexual communication that reinforces interspecific reproductive isolation. When odor blends evolve, the efficacy of male-female communication becomes compromised, unless preference for novel blends also evolves. We explore odor learning as a possible mechanism leading to changes in sex pheromone preferences in the butterfly Bicyclus anynana. Our experiments reveal mating patterns suggesting that mating bias for new blends can develop following a short learning experience, and that this maternal experience impacts the mating outcome of offspring without further exposure. We propose that odor learning can be a key factor in the evolution of sex pheromone blend recognition and in chemosensory speciation.

Transgenerational inheritance of learned preferences for novel host plant odors in Bicyclus anynana butterflies.

Many phytophagous insects have strong preferences for their host plants, which they recognize via odors, making it unclear how novel host preferences develop in the course of insect diversification. Insects may learn to prefer new host plants via exposure to their odors and pass this learned preference to their offspring. We tested this hypothesis by examining larval odor preferences before and after feeding them with leaves coated with control and novel odors and by examining odor preferences again in their offspring. Larvae of the parental generation developed a preference for two of these odors over their development. These odor preferences were also transmitted to the next generation. Offspring of butterflies fed on these new odors chose these odors more often than offspring of butterflies fed on control leaves. In addition, offspring of butterflies fed on banana odors had a significant naïve preference for the banana odors in contrast to the naïve preference for control leaves shown by individuals of the parental generation. Thus, butterflies can learn to prefer novel host plant odors via exposure to them during larval development and transmit these learned preferences to their offspring. This ability potentially facilitates shifts in host plant use over the course of insect diversification.

Haze smoke impacts survival and development of butterflies.

The Southeast Asian transboundary haze contains a mixture of gases and particles from forest fires and negatively impacts people's health and local economies. However, the effect of the haze on organisms other than humans has not yet been sufficiently studied. Insects are important members of food webs and environmental disturbances that affect insects may impact whole ecosystems. Here we studied how haze directly and indirectly affects the survival, growth, and development of insects by rearing Bicyclus anynana butterflies under artificially generated smoke as well as reared in clean air but fed on plants previously exposed to smoke. Direct haze exposure significantly increased the mortality of caterpillars, increased larval development time, and decreased pupal weight, while indirect haze exposure, via ingestion of haze-exposed food plants, also affected development time and pupal weight. No smoke particles were found in the tracheae of subjects from the smoke treatment suggesting that the increase in development time and mortality of B. anynana under smoke conditions might be due to toxic smoke gases and toxic food, rather than particulate matter. These results document significant deleterious effect of haze smoke to the development, adult size, and survival of insects, key players in food-webs.

Phenotypic plasticity in sex pheromone production in Bicyclus anynana butterflies.

Phenotypic plasticity refers to the environmental control of phenotypes. Cues experienced during development (developmental plasticity) or during adulthood (acclimatization) can both affect adult phenotypes. Phenotypic plasticity has been described in many traits but examples of developmental plasticity in physiological traits, in particular, remain scarce. We examined developmental plasticity and acclimatization in pheromone production in the butterfly Bicyclus anynana in response to rearing temperature. B. anynana lives in the African tropics where warm rearing temperatures of the wet season produce active males that court and females that choose, whereas cooler temperatures of the dry season lead to choosy less active males and courting females. We hypothesized that if male pheromone production is costly, it should be reduced in the dry season form. After describing the ultrastructure of pheromone producing cells, we showed that dry season males produced significantly less sex pheromones than wet season males, partly due to acclimatization and partly due to developmental plasticity. Variation in levels of one of the compounds is associated with differential regulation of a pheromone biosynthetic enzyme gene. This plasticity might be an adaptation to minimize pheromone production costs during the stressful dry season.

Does sex-biased dispersal account for the lack of geographic and host-associated differentiation in introduced populations of an aphid parasitoid?

Host recognition and use in female parasitoids strongly relies on host fidelity, a plastic behavior which can significantly restrict the host preferences of parasitoids, thus reducing the gene flow between parasitoid populations attacking different insect hosts. However, the effect of migrant males on the genetic differentiation of populations has been frequently ignored in parasitoids, despite its known impact on gene flow between populations. Hence, we studied the extent of gene flow mediated by female and male parasitoids by assessing sibship relationships among parasitoids within and between populations, and its impact on geographic and host-associated differentiation in the aphid parasitoid Aphidius ervi. We report evidences of a high gene flow among parasitoid populations on different aphid hosts and geographic locations. The high gene flow among parasitoid populations was found to be largely male mediated, suggested by significant differences in the distribution of full-sib and paternal half-sib dyads of parasitoid populations.

New elements in the C-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption.

C-type natriuretic peptide (CNP) and its cognate receptor, natriuretic peptide receptor (NPR) B, have been shown to promote cGMP production in granulosa/cumulus cells. Once transferred to the oocyte through the gap junctions, the cGMP inhibits oocyte meiotic resumption. CNP has been shown to bind another natriuretic receptor, NPR-C. NPR-C is known to interact with and degrade bound CNP, and has been reported to possess signaling functions. Therefore, NPR-C could participate in the control of oocyte maturation during swine in vitro maturation (IVM). Here, we examine the effect of CNP signaling on meiotic resumption, the amount of cGMP and gap junctional communication (GJC) regulation during swine IVM. The results show an inhibitory effect of CNP in inhibiting oocyte meiotic resumption in follicle-stimulating hormone (FSH)-stimulated IVM. We also found that an NPR-C-specific agonist (cANP([4-23])) is likely to play a role in maintaining meiotic arrest during porcine IVM when in the presence of a suboptimal dose of CNP. Moreover, we show that, even if CNP can increase intracellular concentration of cGMP in cumulus-oocyte complexes, cANP((4-23)) had no impact on cGMP concentration, suggesting a potential cGMP-independent signaling pathway related to NPR-C activation. These data support a potential involvement of cANP((4-23)) through NPR-C in inhibiting oocyte meiotic resumption while in the presence of a suboptimal dose of CNP. The regulation of GJC was not altered by CNP, cANP((4-23)), or the combination of CNP and cANP((4-23)), supporting their potential contribution in sending signals to the oocytes. These findings offer promising insights in to new elements of the signaling pathways that may be involved in inhibiting resumption of meiosis during FSH-stimulated swine IVM.

Modulatory effects of the serotonergic and histaminergic systems on reaction to light in the crustacean Gammarus pulex.

Serotonin modulates reaction to light in many animals. In the freshwater amphipod Gammarus pulex, exogenous administration of serotonin induces a transient reversal of photic behaviour from strong photophobia to photophily. We have elucidated further the neuromodulation of photic behaviour in G. pulex by using in vivo pharmacology and behavioural testing. Using several mammalian 5-HT receptor antagonists and agonists, we provide evidence for a role of serotonin receptors in the 5-HT-dependent regulation of G. pulex photic behaviour, possibly involving 5-HTR2 subtype. Serotonin-induced photophily was blocked by the 5-HT receptor antagonists, mianserin, cyproheptadine, and ritanserin, but not by ketanserin and methiothepin. In addition, two agonists at 5-HT receptor, mCPP and α-methylserotonin, partially mimicked 5-HT-induced photophily. The natural photophobia of G. pulex was also decreased by exogenous administration of the histamine receptor antagonist cimetidine, suggesting that photic behaviour may be controlled by more complex interactions between serotonergic and histaminergic systems.

Symbiont infection affects aphid defensive behaviours.

Aphids harbour both an obligate bacterial symbiont, Buchnera aphidicola, and a wide range of facultative ones. Facultative symbionts can modify morphological, developmental and physiological host traits that favour their spread within aphid populations. We experimentally investigated the idea that symbionts may also modify aphid behavioural traits to enhance their transmission. Aphids exhibit many behavioural defences against enemies. Despite their benefits, these behaviours have some associated costs leading to reduction in aphid reproduction. Some aphid individuals harbour a facultative symbiont Hamiltonella defensa that provides protection against parasitoids. By analysing aphid behaviours in the presence of parasitoids, we showed that aphids infected with H. defensa exhibited reduced aggressiveness and escape reactions compared with uninfected aphids. The aphid and the symbiont have both benefited from these behavioural changes: both partners reduced the fitness decrements associated with the behavioural defences. Such symbiont-induced changes of behavioural defences may have consequences for coevolutionary processes between host organisms and their enemies.