Tenebrio molitor as a model system to study Staphylococcus spp virulence and horizontal gene transfer.

Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

Evolutionary origin and function of NOX4-art, an arthropod specific NADPH oxidase.

BACKGROUND: NADPH oxidases (NOX) are ROS producing enzymes that perform essential roles in cell physiology, including cell signaling and antimicrobial defense. This gene family is present in most eukaryotes, suggesting a common ancestor. To date, only a limited number of phylogenetic studies of metazoan NOXes have been performed, with few arthropod genes. In arthropods, only NOX5 and DUOX genes have been found and a gene called NOXm was found in mosquitoes but its origin and function has not been examined. In this study, we analyzed the evolution of this gene family in arthropods. A thorough search of genomes and transcriptomes was performed enabling us to browse most branches of arthropod phylogeny. RESULTS: We have found that the subfamilies NOX5 and DUOX are present in all arthropod groups. We also show that a NOX gene, closely related to NOX4 and previously found only in mosquitoes (NOXm), can also be found in other taxonomic groups, leading us to rename it as NOX4-art. Although the accessory protein p22-phox, essential for NOX1-4 activation, was not found in any of the arthropods studied, NOX4-art of Aedes aegypti encodes an active protein that produces H2O2. Although NOX4-art has been lost in a number of arthropod lineages, it has all the domains and many signature residues and motifs necessary for ROS production and, when silenced, H2O2 production is considerably diminished in A. aegypti cells. CONCLUSIONS: Combining all bioinformatic analyses and laboratory work we have reached interesting conclusions regarding arthropod NOX gene family evolution. NOX5 and DUOX are present in all arthropod lineages but it seems that a NOX2-like gene was lost in the ancestral lineage leading to Ecdysozoa. The NOX4-art gene originated from a NOX4-like ancestor and is functional. Although no p22-phox was observed in arthropods, there was no evidence of neo-functionalization and this gene probably produces H2O2 as in other metazoan NOX4 genes. Although functional and present in the genomes of many species, NOX4-art was lost in a number of arthropod lineages.

Morphophysiological study of digestive system litter-feeding termite Cornitermes cumulans (Kollar, 1832).

Termites are the major decomposers of lignocellulosic biomass on Earth and are commonly considered as biological reactor models for lignocellulose degradation. Despite their biotechnological potential, few studies have focused on the morphophysiological aspects of the termite digestive system. We therefore analyze the morphology, ultrastructure and gut luminal pH of the digestive system in workers of the litter-feeding termite Cornitermes cumulans (Blattodea: Termitidae). Their digestive system is composed of salivary glands and an alimentary canal with a pH ranging from neutral to alkaline. The salivary glands have an acinar structure and present cells with secretory characteristics. The alimentary canal is differentiated into the foregut, midgut, mixed segment and hindgut, which comprises the ileum (p1), enteric valve (p2), paunch (p3), colon (p4) and rectum (p5) segments. The foregut has a well-developed chewing system. The midgut possesses a tubular peritrophic membrane and two cell types: digestive cells with secretory and absorptive features and several regenerative cells in mitosis, both cell types being organized into regenerative crypts. The mixed segment exhibits cells rich in glycogen granules. Hindgut p1, p4 and p5 segments have flattened cells with a few apical invaginations related to mitochondria and a thick cuticular lining. Conversely, the hindgut p3 segment contains large cuboid cells with extensive apical invaginations associated with numerous mitochondria. These new insights into the morphophysiology of the digestive system of C. cumulans reveal that it mobilizes lignocellulose components as a nutritional source by means of a highly compartmentalized organization with specialized segments and complex microenvironments.

Unravelling the genome of the brackish water malaria vector Anopheles aquasalis.

Malaria is a severe public health problem in several developing tropical and subtropical countries. Anopheles aquasalis is the primary coastal malaria vector in Central and South America and the Caribbean Islands, and it has the peculiar feature of living in water with large changes in salinity. Recent research has recognised An. aquasalis as an important model for studying the interactions of murine and human Plasmodium parasites. This study presents the complete genome of An. aquasalis and offers insights into its evolution and physiology. The genome is similar in size and gene content to other Neotropical anophelines, with 162 Mb and 12,446 protein-coding genes. There are 1387 single-copy orthologs at the Diptera level (eg. An. gambiae, An. darlingi and Drosophila melanogaster). An. aquasalis diverged from An. darlingi, the primary malaria vector in inland South America, nearly 20 million years ago. Proteins related to ion transport and metabolism belong to the most abundant gene families with 660 genes. We identified gene families relevant to osmosis control (e.g., aquaporins, vacuolar-ATPases, Na+/K+-ATPases, and carbonic anhydrases). Evolutionary analysis suggests that all osmotic regulation genes are under strong purifying selection. We also observed low copy number variation in insecticide resistance and immunity-related genes for all known classical pathways. The data provided by this study offers candidate genes for further studies of parasite-vector interactions and for studies on how anophelines of brackish water deal with the high fluctuation in water salinity. We also established data and insights supporting An. aquasalis as an emerging Neotropical malaria vector model for genetic and molecular studies.

Morphological Characterization of the Antennal Sensilla of the Afrotropical Sand Fly, Phlebotomus duboscqi (Diptera: Psychodidae).

We investigated by scanning electron microscopy the morphology, distribution, and abundance of antennal sensilla of females Phlebotomus duboscqi sand fly, an important vector of zoonotic cutaneous leishmaniasis at Afrotropical region. Thirteen well-differentiated sensilla were identified, among six types of cuticular sensilla. The probable function of these sensillary types is discussed in relation to their external structure and distribution. Five sensillary types were classified as olfactory sensilla, as they have specific morphological characters of sensilla with this function. Number and distribution of sensilla significantly differed between antennal segments. The results of the present work, besides corroborating in the expansion of the morphological and ultrastructural knowledge of P. duboscqi, can foment future electrophysiological studies for the development of volatile semiochemicals, to be used as attractants in traps for monitoring and selective vector control of this sand fly.

Aedes aegypti Infection With Trypanosomatid Strigomonas culicis Alters Midgut Redox Metabolism and Reduces Mosquito Reproductive Fitness.

Aedes aegypti mosquitoes transmit arboviruses of important global health impact, and their intestinal microbiota can influence vector competence by stimulating the innate immune system. Midgut epithelial cells also produce toxic reactive oxygen species (ROS) by dual oxidases (DUOXs) that are essential players in insect immunity. Strigomonas culicis is a monoxenous trypanosomatid that naturally inhabits mosquitoes; it hosts an endosymbiotic bacterium that completes essential biosynthetic pathways of the parasite and influences its oxidative metabolism. Our group previously showed that S. culicis hydrogen peroxide (H2O2)-resistant (WTR) strain is more infectious to A. aegypti mosquitoes than the wild-type (WT) strain. Here, we investigated the influence of both strains on the midgut oxidative environment and the effect of infection on mosquito fitness and immunity. WT stimulated the production of superoxide by mitochondrial metabolism of midgut epithelial cells after 4 days post-infection, while WTR exacerbated H2O2 production mediated by increased DUOX activity and impairment of antioxidant system. The infection with both strains also disrupted the fecundity and fertility of the females, with a greater impact on reproductive fitness of WTR-infected mosquitoes. The presence of these parasites induced specific transcriptional modulation of immune-related genes, such as attacin and defensin A during WTR infection (11.8- and 6.4-fold, respectively) and defensin C in WT infection (7.1-fold). Thus, we propose that A. aegypti oxidative response starts in early infection time and does not affect the survival of the H2O2-resistant strain, which has a more efficient antioxidant system. Our data provide new biological aspects of A. aegypti-S. culicis relationship that can be used later in alternative vector control strategies.

How are arbovirus vectors able to tolerate infection?

One of the defining features of mosquito vectors of arboviruses such as Dengue and Zika is their ability to tolerate high levels of virus proliferation without suffering significant pathology. This adaptation is central to vector competence and disease spread. The molecular mechanisms, pathways, cellular and metabolic adaptations responsible for mosquito disease tolerance are still largely unknown and may represent effective ways to control mosquito populations and prevent arboviral diseases. In this review article, we describe the key link between disease tolerance and pathogen transmission, and how vector control methods may benefit by focusing efforts on dissecting the mechanisms underlying mosquito tolerance of arboviral infections. We briefly review recent work investigating tolerance mechanisms in other insects, describe the state of the art regarding the mechanisms of disease tolerance in mosquitos, and highlight the emerging role of gut microbiota in mosquito immunity and disease tolerance.

Ultrastructural Analysis of Mouthparts of Adult Horn Fly (Diptera: Muscidae) From the Brazilian Midwest Region.

The ultrastructure of the mouthparts of Haematobia irritans (L.) was investigated by scanning electron microscopy. The morphological characteristics of the maxillary palps, labium (prementum and postmentum), labrum, hypopharynx, haustellum, and labellar lobes are described, as well as of the sensilla evidenced on all the surface of the mouthparts, and the set of different positions assumed by the mouth apparatus of this fly. Based on their morphology, 12 well-differentiated sensilla were identified, among three types of cuticular sensilla: trichoidea, coeloconica, and campaniformia. A slight sexual dimorphism in the sensilla patterns found in the mouthparts of H. irritans was evidenced. These observations are discussed with reference to the current literature on the functional morphology of sense organs of Insecta. These results could facilitate the recognition of the chemosensory sensilla by electrophysiological techniques, and foment future taxonomic and phylogenetic studies to better elucidate the evolution of Diptera, Muscomorpha.

Ultrastructure of the Antennae and Sensilla of Nyssomyia intermedia (Diptera: Psychodidae), Vector of American Cutaneous Leishmaniasis.

The antennal sensilla and the antenna of females Nyssomyia intermedia, one of the main vectors of American cutaneous leishmaniasis, were studied by scanning electron microscopy. The main goal was to characterize the quantity, typology, and topography of the sensilla with particular attention to the olfactory types. The insects were captured in the city of Corte de Pedra, State of Bahia, Brazil, by CDC-type light traps and raised in a laboratory as a new colony. Fourteen well-differentiated sensilla were identified, among six cuticular types: trichoidea, campaniformia, squamiformia, basiconica, chaetica, and coeloconica. Of these, six sensilla were classified as olfactory sensilla due to their specific morphological features. Smaller noninnervated pilosities of microtrichiae type were also evidenced by covering all antennal segments. The antennal segments differ in shapes and sizes, and the amount and distribution of types and subtypes of sensilla. This study may foment future taxonomic and phylogenetic analysis for a better evolutionary understanding of the sand flies. Besides, it may assist the targeting of future electrophysiological studies by Single Sensillum Recording, and aim to develop alternative measures of monitoring and control of this vector.

The relationship between oxidant levels and gut physiology in a litter-feeding termite.

The termite gut is an efficient decomposer of polyphenol-rich diets, such as lignocellulosic biomasses, and it has been proposed that non-enzymatic oxidative mechanisms could be involved with the digestive process in these animals. However, oxidant levels are completely unknown in termites, as well as protective mechanisms against oxidative damage to the termite gut and its microbiota. As the first step in investigating the role oxidants plays in termite gut physiology, this work presents oxidant levels, antioxidant enzymatic defenses, cell renewal and microbiota abundance along the litter-feeding termite Cornitermes cumulans gut compartments (foregut, midgut, mixed segment and hindgut p1, p3, p4, and p5 segments) and salivary glands. The results show variable levels of oxidants along the C. cumulans gut, the production of antioxidant enzymes, gut cell renewal as potential defenses against oxidative injuries and the profile of microbiota distribution (being predominantly inverse to oxidant levels). In this fashion, the oxidative challenges imposed by polyphenol-rich diet seem to be circumvented by the C. cumulans gut, ensuring efficiency of the digestive process together with preservation of tissue homoeostasis and microbiota growth. These results present new insights into the physicochemical properties of the gut in a litter-feeding termite, expanding our view in relation to termites' digestive physiology.