Extensive Reorganization of Behavior Accompanies Ontogeny of Aggression in Male Flesh Flies

Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly Sarcophaga crassipalpis, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts (days 1, 2, 3, 4, and 6) upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors (accounting for at least 96% of the total observation time for each age cohort) accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism

Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila.

Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products

Evolution-guided mutagenesis of the cytoplasmic incompatibility proteins: Identifying CifA's complex functional repertoire and new essential regions in CifB.

Wolbachia are the world's most common, maternally-inherited, arthropod endosymbionts. Their worldwide distribution is due, in part, to a selfish drive system termed cytoplasmic incompatibility (CI) that confers a relative fitness advantage to females that transmit Wolbachia to their offspring. CI results in embryonic death when infected males mate with uninfected females but not infected females. Under the Two-by-One genetic model of CI, males expressing the two phage WO proteins CifA and CifB cause CI, and females expressing CifA rescue CI. While each protein is predicted to harbor three functional domains, there is no knowledge on how sites across these Cif domains, rather than in any one particular domain, contribute to CI and rescue. Here, we use evolution-guided, substitution mutagenesis of conserved amino acids across the Cif proteins, coupled with transgenic expression in uninfected Drosophila melanogaster, to determine the functional impacts of conserved residues evolving mostly under purifying selection. We report that amino acids in CifA's N-terminal unannotated region and annotated catalase-related domain are important for both complete CI and rescue, whereas C-terminal residues in CifA's putative domain of unknown function are solely important for CI. Moreover, conserved CifB amino acids in the predicted nucleases, peptidase, and unannotated regions are essential for CI. Taken together, these findings indicate that (i) all CifA amino acids determined to be crucial in rescue are correspondingly crucial in CI, (ii) an additional set of CifA amino acids are uniquely important in CI, and (iii) CifB amino acids across the protein, rather than in one particular domain, are all crucial for CI. We discuss how these findings advance an expanded view of Cif protein evolution and function, inform the mechanistic and biochemical bases of Cif-induced CI/rescue, and continue to substantiate the Two-by-One genetic model of CI

An optimized approach to germ-free rearing in the jewel wasp Nasonia

Development of a Nasonia in vitrogerm-free rearing system in 2012 enabled investigation of Nasonia-microbiota interactions and real-time visualization of parasitoid metamorphosis. However, the use of antibiotics, bleach, and fetal bovine serum introduced artifacts relative to conventional rearing of Nasonia. Here, we optimize the germ-free rearing procedure by using filter sterilization in lieu of antibiotics and by removing residual bleach and fetal bovine serum. Comparison of these methods reveals no influence on larval survival or growth, and a 52% improvement in adult production. Additionally, adult males produced in the new germ-free system are similar in size to conventionally reared males. Experimental implications of these changes are discussed

Extensive Reorganization of Behavior Accompanies Ontogeny of Aggression in Male Flesh Flies

<div><p>Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly <i>Sarcophaga crassipalpis</i>, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts (days 1, 2, 3, 4, and 6) upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors (accounting for at least 96% of the total observation time for each age cohort) accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism.</p></div

Models and Nomenclature for Cytoplasmic Incompatibility : Caution over Premature Conclusions – A Response to Beckmann et al.

Recent studies have identified two genes in bacteriophage WO, cifA and cifB, that contribute to the induction of cytoplasmic incompatibility (CI) [1,2], and one of these two genes, cifA, rescues it [3]. These findings underpin a two-by-one genetic model (Figure 1A) that reflects current understanding of CI genetics and embraces various functional models [3] (Figure 1B). A recent article by Beckmann et al. [4] provides interesting ideas about the mechanism and evolutionary history of the CI genes. Therein, they claim that it is 'clearer than ever that the CI induction and rescue stem from a toxin–antidote (TA) system', and that disputes regarding the operon status of the cif genes are semantic. They also propose a new nomenclature to describe the genes. It is important to test hypotheses and develop nomenclature carefully in the context of current data because misconceptions can sometimes become a narrative for those unfamiliar with the evidence. Here, we present and evaluate three points of criticism of the arguments related to the TA model, the operon hypothesis, and the proposed gene nomenclature. We recommend caution and nuance in interpreting current data (and lack thereof). As we will frequently note, more research will be necessary before a functional narrative should be prescribed for CI

Ontogeny of predominant non-interactive behaviors.

<p>Changes in the proportions of total observation time, with respect to age (in days), occupied by each of the four most predominant non-interactive behaviors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093196#pone-0093196-t001" target="_blank">Table 1</a> for descriptions) in individual, socially naïve male flesh flies: (A) <i>walking</i>, (B) <i>standing</i>, (C) <i>grooming</i>, and (D) <i>upside-down</i>. Flies were paired in a simple observation arena (the same experiment as Fig. 5). Different letters indicate significant differences.</p

Behavioral transition matrix for day 3 age cohort.

<p>The matrix summarizes the frequencies at which each behavior (far left column) is followed by any other behavior (top row). Those transitions occurring more frequently than predicted by chance are indicated in bold. Descriptions of the behaviors (and their abbreviations) are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093196#pone-0093196-t001" target="_blank">Table 1</a>.</p

Lack of dominant individuals within the paired male encounters.

<p>Distributions of the proportions of (A) <i>retreat</i> and (B) high-intensity aggressive behaviors (<i>hold</i> and <i>lunge</i>, combined) performed by individual 1 in the paired male encounters. If the behaviors are exhibited approximately equally by both members of the pairs, the distributions should be centered around 0.5.</p