Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13-33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Rhamnolipids stabilize quorum sensing mediated cooperation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main models to study social behaviors in bacteria since it synthesizes several exoproducts, including exoproteases and siderophores and release them to the environment. Exoproteases and siderophores are public goods that can be utilized by the individuals that produce them but also by non-producers, that are considered social cheaters. Molecularly exoprotease cheaters are mutants in regulatory genes such as lasR, and are commonly isolated from chronic infections and selected in the laboratory upon serial cultivation in media with protein as a sole carbon source. Despite that the production of exoproteases is exploitable, cooperators have also ways to restrict the growth and selection of social cheaters, for instance by producing toxic metabolites like pyocyanin. In this work, using bacterial competitions, serial cultivation and growth assays, we demonstrated that rhamnolipids which production is regulated by quorum sensing, selectively affect the growth of lasR mutants and are able to restrict social cheating, hence contributing to the maintenance of cooperation in Pseudomonas aeruginosa populations.

Pyocyanin Restricts Social Cheating in Pseudomonas aeruginosa.

Quorum sensing (QS) in Pseudomonas aeruginosa coordinates the expression of virulence factors, such as exoproteases and siderophores, that are public goods utilized by the whole population of bacteria, regardless of whether they invested or not in their production. These public goods can be used by QS defective mutants for growth, and since these mutants do not contribute to public goods production, they are considered social cheaters. Pyocyanin is a phenazine that is a toxic, QS-controlled metabolite produced by P. aeruginosa. It is a redox-active compound and promotes the generation of reactive oxygen species; it also possesses antibacterial properties and increases fitness in competition with other bacterial species. Since QS-deficient individuals are less able to tolerate oxidative stress, we hypothesized that the pyocyanin produced by the wild-type population could promote selection of functional QS systems in this bacterium. Here, we demonstrate, using competition experiments and mathematical models, that, indeed, pyocyanin increases the fitness of the cooperative QS-proficient individuals and restricts the appearance of social cheaters. In addition, we also show that pyocyanin is able to select QS in other bacteria such as Acinetobacter baumannii.

Effects of Trypanosoma cruzi on the phenoloxidase and prophenoloxidase activity in the vector Meccus pallidipennis (Hemiptera: Reduviidae).

BACKGROUND: Triatomine insects are vectors of Trypanosoma cruzi, the causal agent of Chagas disease. The insect-parasite interaction has been studied in relation to the transmission and prevalence of this disease. For most triatomines, however, several crucial aspects of the insect immune response are still unknown. For example, only for Rhodnius prolixus and Triatoma infestans has the activity of phenoloxidase (PO) and its zymogen prophenoloxidase (proPO) been reported in relation to the hemolymph and anterior midgut (AM). The aim of this study was to gain insight into the immune response to T. cruzi infection of an important triatomine in Mexico, Meccus pallidipennis. METHODS: Parasites were quantified in the rectal contents of infected M. pallidipennis groups. We examined some key factors in disease transmission, including the systemic (hemolymph) and local (gut) immune response. RESULTS: Parasites were present in the rectal contents at 4 days post-infection (pi) and reached their maximum density on day 7 pi. At 7 and 9 days pi mainly metacyclic trypomastigotes occurred. Compared to the control, the infected insects exhibited diminished PO activity in the hemolymph on days 9, 16 and 20 pi, and in the AM only on day 9. Additionally, infected insects displayed lower proPO activity in the hemolymph on day 1, but greater activity in the AM on day 28. CONCLUSIONS: The parasite strain originating from M. pallidipennis rapidly colonized the rectum of nymphs of this triatomine and developed high numbers of metacyclic trypomastigotes. Neither the changes of concentrations of PO and proPO in the hemolymph nor in the AM correlated with the changes in the population of T. cruzi.

Bacterial symbionts in human blood-feeding arthropods: Patterns, general mechanisms and effects of global ecological changes.

Due to their high impact on public health, human blood-feeding arthropods are one of the most relevant animal groups. Bacterial symbionts have been long known to play a role in the metabolism, and reproduction of these arthropod vectors. Nowadays, we have a more complete picture of their functions, acknowledging the wide influence of bacterial symbionts on processes ranging from the immune response of the arthropod host to the possible establishment of pathogens and parasites. One or two primary symbiont species have been found to co-evolve along with their host in each taxon (being ticks an exception), leading to various kinds of symbiosis, mostly mutualistic in nature. Moreover, several secondary symbiont species are shared by all arthropod groups. With respect to gut microbiota, several bacterial symbionts genera are hosted in common, indicating that these bacterial groups are prone to invade several hematophagous arthropod species feeding on humans. The main mechanisms underlying bacterium-arthropod symbiosis are discussed, highlighting that even primary symbionts elicit an immune response from the host. Bacterial groups in the gut microbiota play a key role in immune homeostasis, and in some cases symbiont bacteria could be competing directly or indirectly with pathogens and parasites. Finally, the effects climate change, great human migrations, and the increasingly frequent interactions of wild and domestic animal species are analyzed, along with their implications on microbiota alteration and their possible impacts on public health and the control of pathogens and parasites harbored in arthropod vectors of human parasites and pathogens.

The effects of food shortage during larval development on adult body size, body mass, physiology and developmental time in a tropical damselfly.

Few studies have looked jointly at the effects of larval stressors on life history and physiology across metamorphosis, especially in tropical insects. Here we investigated how the variation of food availability during the larval stage of the tropical and territorial American rubyspot damselfly (Hetaerina americana) affects adult body size and body mass, and two physiological indicators of condition--phenoloxidase activity (an indicator of immune ability) and protein concentration. We also investigated whether larval developmental time is prolonged when food is scarce, an expected situation for tropical species whose larval time is less constrained, compared to temperate species. Second instar larvae were collected from their natural environments and reared in one of two diet regimes: (i) "rich" provided with five Artemia salina prey every day, and (ii) "poor" provided with two A. salina prey every day. In order to compare how distinct our treatments were from natural conditions, a second set of last-instar larvae were also collected and allowed to emerge. Only body size and phenoloxidase increased in the rich regime, possibly to prioritize investment on sexually selected traits (which increase mating opportunities), and immune ability, given pathogen pressure. The sexes did not differ in body size in relation to food regimes but they did differ in body mass and protein concentration; this can be explained on the basis of the energetically demanding territorial activities by males (for the case of body mass), and female allocation to egg production (for the case of protein). Finally, animals delayed larval development when food was scarce, which is coherent for tropical environments. These findings provide key insights in the role of food availability in a tropical species.