A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

Correction: Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

[This corrects the article DOI: 10.1371/journal.pone.0292043.].

Morphological and molecular insights into the diversity of Leptoconops biting midges from a heavily infested Mediterranean area.

The genus Leptoconops Skuse (Diptera: Ceratopogonidae) are blood-sucking midges known to pester humans and domestic animals. In certain Mediterranean areas, midges occur in large numbers during summer and limit the use of recreational areas, also raising serious health and social concerns. Despite such impact, the diversity and distribution of Leptoconops in Maremma Regional Park (Tuscany Region, Italy), a heavily infested area, is not well known, and neither molecular nor detailed morphological studies exist. We sampled adult midge females in six areas and used high-resolution digital stereomicroscopy and scanning electron microscopy to identify species and investigate the morphology of structures involved in host searching/recognition (antennae and maxillary palps) and host attack (mouthparts). We also performed energy-dispersive X-ray spectroscopy to characterize the elemental composition of mouthparts. Finally, the cytochrome c oxidase subunit 1 (cox1) gene was amplified and sequenced, to confirm species identification of collected specimens. We identified two species: Leptoconops (L.) irritans Noé and Leptoconops (L.) noei Clastrier & Coluzzi, with the former being more frequently sampled than the latter and closer to sea coast and rivers. The antennal segments appeared slightly more globular in L. noei than in L. irritans. Five types of trichoid, basiconic and chaetic sensilla were found on the antennae, with some differences between the two species. Mouthparts had the labellum visibly larger in L. noei compared with L. irritans. The maxillary palps possessed a pit filled with bulb-shaped sensilla, which appeared denser in L. noei than in L. irritans. Mouthpart cuticle included Calcium (Ca) and Aluminum (Al) at small but significant concentrations (0.3-1.0%) in both species. Our results suggest that the limited but appreciable differences in sensory system between the studied species of Leptoconops and other Ceratopogonidae may reflect different host or habitat preferences, a scenario potentially suggested also by preliminarily data on their distribution in the studied area. The presence of Ca and Al in the cuticle of mouthparts may help host skin drilling during bite activity. Finally, the gene sequences obtained in this study provide a first reference for future investigations on the taxonomy and dispersal patterns of Leptoconops spp. in the Mediterranean area.

Aedes albopictus microbiota: Differences between wild and mass-reared immatures do not suggest negative impacts from a diet based on black soldier fly larvae and fish food.

The "Sterile Insect Technique" (SIT), a promising method to control Aedes albopictus, the Asian tiger mosquito, is gaining increasing interest. Recently, the role of microbiota in mosquito fitness received attention, but the link between microbiota and larval diet in mass rearing programs for SIT remains largely unexplored. We characterized the microbiota of four larval instars, pupae and eggs of non-wild (NW) lab-reared Ae. albopictus fed with a diet based on Black soldier fly (Hermetia illucens) larvae powder and fish food KOI pellets. We compared it with wild (W) field-collected individuals and the bacterial community occurring in rearing water-diet (DIET). A total of 18 bacterial classes with > 0.10% abundance were found overall in the samples, with seven classes being especially abundant. Overall, the microbiota profile significantly differed among NW, W and DIET. Verrucomicrobiae were significantly more abundant in W and DIET, Bacteroidia were more abundant in NW and DIET, and Gammaproteobacteria were only more abundant in W than in DIET. W-eggs microbiota differed from all the other groups. Large differences also appeared at the bacterial genus-level, with the abundance of 14 genera differing among groups. Three ASVs of Acinetobacter, known to have positive effects on tiger mosquitoes, were more abundant in NW than in W, while Serratia, known to have negative or neutral effects on another Aedes species, was less abundant in NW than in W. The bacterial community of W-eggs was the richest in species, while dominance and diversity did not differ among groups. Our data show that the diet based on Black soldier fly powder and fish food KOI influences the microbiota of NW tiger mosquito immature stages, but not in a way that may suggest a negative impact on their quality in SIT programs.

Bacterial gut microbiomes of aculeate brood parasites overlap with their aculeate hosts', but have higher diversity and specialization.

Despite growing interest in gut microbiomes of aculeate Hymenoptera, research so far focused on social bees, wasps, and ants, whereas non-social taxa and their brood parasites have not received much attention. Brood parasitism, however, allows to distinguish between microbiome components horizontally transmitted by spill-over from the host with such inherited through vertical transmission by mothers. Here, we studied the bacterial gut microbiome of adults in seven aculeate species in four brood parasite-host systems: two bee-mutillid (host-parasitoid) systems, one halictid bee-cuckoo bee system, and one wasp-chrysidid cuckoo wasp system. We addressed the following questions: (1) Do closely related species possess a more similar gut microbiome? (2) Do brood parasites share components of the microbiome with their host? (3) Do brood parasites have different diversity and specialization of microbiome communities compared with the hosts? Our results indicate that the bacterial gut microbiome of the studied taxa was species-specific, yet with a limited effect of host phylogenetic relatedness and a major contribution of shared microbes between hosts and parasites. However, contrasting patterns emerged between bee-parasite systems and the wasp-parasite system. We conclude that the gut microbiome in adult brood parasites is largely affected by their host-parasite relationships and the similarity of trophic food sources between hosts and parasites.

Host-trailing satellite flight behaviour is associated with greater investment in peripheral visual sensory system in miltogrammine flies.

Insect sensory systems are the subjects of different selective pressures that shape their morphology. In many species of the flesh fly subfamily Miltogramminae (Diptera: Sarcophagidae) that are kleptoparasitic on bees and wasps, females perch on objects close to the host nests and, once a returning host is detected, they follow it in flight at a fixed distance behind until reaching the nest. We hypothesized that such satellite (SAT) flight behaviour, which implies a finely coordinated trailing flight, is associated with an improved visual system, compared to species adopting other, non-satellite (NON-SAT) strategies. After looking at body size and common ancestry, we found that SAT species have a greater number of ommatidia and a greater eye surface area when compared to NON-SAT species. Ommatidium area is only affected by body size, suggesting that selection changes disproportionately (relative to body size variation) the number of ommatidia and as a consequence the eye area, instead of ommatidium size. SAT species also tend to have larger ocelli, but their role in host-finding was less clear. This suggests that SAT species may have a higher visual acuity by increasing ommatidia number, as well as better stability during flight and motion perception through larger ocelli. Interestingly, antennal length was significantly reduced in SAT species, and ommatidia number negatively correlated with antennal length. While this finding does not imply a selection pressure of improved antennal sensory system in species adopting NON-SAT strategies, it suggests an inverse resource (i.e. a single imaginal disc) allocation between eyes and antennae in this fly subfamily.

A sting affair: A global quantitative exploration of bee, wasp and ant hosts of velvet ants.

The vast majority of species of velvet ants (Hymenoptera: Aculeata: Mutillidae) are ectoparasitoids of immature stages of other aculeate Hymenoptera (bees, wasps and ants). Due to their cryptic, furtive behaviour at the host nesting sites, however, even basic information on their biology, like host use diversity, is still unknown for entire subfamilies, and the known information, scattered in over two centuries of published studies, is potentially hiding tendencies to host specialization across velvet ant lineages. In this review, based on 305 host associations spanning 132 species in 49 genera and 10 main lineages (tribes/subfamilies), we explored patterns of host use in velvet ants. Overall, 15 families and 29 subfamilies of Aculeata are listed as hosts of mutillids, with a strong predominance of Apoidea (bees and apoid wasps: 19 subfamilies and 82.3% of host records). A series of bipartite networks, multivariate analyses and calculations of different indices suggested possible patterns of specialization. Host taxonomic spectrum (number of subfamilies) of velvet ants was very variable and explained by variation in the number of host records. Instead, we found a great variation of network-based host specialization degree and host taxonomic distinctness that did not depend on the number of host records. Differences in host use patterns seemed apparent across mutillid tribes/subfamilies, among genera within several tribes/subfamilies, and to lesser extent within genera. Taxonomic host use variation seemed not dependent on phylogeny. Instead, it was likely driven by the exploitation of hosts with different ecological traits (nest type, larval diet and sociality). Thus, taxonomically more generalist lineages may use hosts that essentially share the same ecological profile. Interestingly, closely related mutillid lineages often show contrasting combinations of host ecological traits, particularly sociality and larval diet, with a more common preference for ground-nesting hosts across most lineages. This review may serve as a basis to test hypotheses for host use evolution in this fascinating family of parasitoids.