Protocol for bloodmeal identification in ticks using retrotransposon-targeted real time PCR

Tick-borne infections are a global public health burden. Our ability to develop effective control measures relies on understanding the natural transmission cycles as well as the mode of exposure to humans. Our current knowledge of the relative importance of the hosts upon which tick vectors feed is based on indirect measurements, such as infestation indices. Bloodmeal identification can determine the host upon which a questing tick had fed in the previous life stage and is an unbiased method for incriminating reservoir hosts. Although bloodmeal identification has been utilized for mosquito ecology since the 1950s, the extended life-cycle of ticks and complete intracellular digestion of the bloodmeal make such analyses for ticks more complicated. We have recently developed a highly sensitive assay for identification of bloodmeals in ticks based upon real-time PCR amplification of mammalian retrotransposons. Although other PCR-based methods for bloodmeal analysis in ticks have been published previously, they have an inadequate success rate with field-collected ticks and have not been adopted for general use. We describe our methods in detail in order to make this assay widely accessible for use in other laboratories.

Novel insights on obligate symbiont lifestyle and adaptation to chemosynthetic environment as revealed by the giant tubeworm genome

The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulphur metabolism, detoxification, anti-oxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establishes that the trophosome is a multi-functional organ marked by intracellular digestion of endosymbionts, storage of excretory products and haematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbour highly expressed genes involved with cell cycle, programmed cell death, and immunity indicating a high cell turnover and defence mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whilst simultaneously provides new insights into the development, whole organism functions and evolution in the giant tubeworm.

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

Macrophages are the first encounters of invading bacteria and are responsible for engulfing and digesting pathogens through phagocytosis leading to initiation of the innate inflammatory response. Intracellular digestion occurs through a close relationship between phagocytic/endocytic and lysosomal pathways, in which proteolytic enzymes, such as cathepsins, are involved. The presence of cathepsins in the endo-lysosomal compartment permits direct interaction with and killing of bacteria, and may contribute to processing of bacterial antigens for presentation, an event necessary for the induction of antibacterial adaptive immune response. Therefore, it is not surprising that bacteria can control the expression and proteolytic activity of cathepsins, including their inhibitors – cystatins, to favor their own intracellular survival in macrophages. In this review, we summarize recent developments in defining the role of cathepsins in bacteria-macrophage interaction and describe important strategies engaged by bacteria to manipulate cathepsin expression and activity in macrophages. Particularly, we focus on specific bacterial species due to their clinical relevance to humans and animal health, i.e., Mycobacterium, Mycoplasma, Staphylococcus, Streptococcus, Salmonella, Shigella, Francisella, Chlamydia, Listeria, Brucella, Helicobacter, Neisseria, and other genera.

Morphological characteristics of respiratory and digestive organs of Roman snail (Helix pomatia L., 1758)

The aim of this work was to study the structure of lung and hepatopancreas of Roman snail (Helix of pomatia of L., 1758). The study found that the lung occupies the lower turn of shell and presented by a saccate cavity, in the wall of that there are a kidney and heart with a pericardium, and also a rectum and ureter pass. An external surface of lungs covered by a shell and covered by an epidermis. An internal surface is covered by a flat ciliated epithelium and forms numerous folds in which pulmonary vessels and lacunae are accommodated. The branches of pulmonary vein have a thick muscular wall, that consists of circular and longitudinal muscular layers. An internal surface of lungs covered by the layer of mucus. Inhalation and exhalation are carried out due to reduction and relaxation of muscles of dorsal wall of the body that is named a “diaphragm”. Gas exchange occurs through the hemolymphatic capillaries of the lung wall. Respiratory motions take place not rhythmically, but through the different intervals of time depending on a requirement in oxygen. The frequency of pneumostome closing and opening is typically one time in a minute. At subzero humidity of atmospheric air of pneumostome closed by a mantle, and also one (or a few) epiphragms. The hepatopancreas (“liver” or liver gland) is in the upper rotation of the sink and formed by two parts: right and left, from which two liver ducts enter into the stomach respectively. The liver gland consists of many acinuss, surrounded by connecting tissue, that contains small number of muscular fibres. Calcium cells have a pyramidal form and usually do not reach the lumen of the acinus. Cytoplasm of calcium cells contains inclusions: grains of phosphoricacid lime and drops of fat. The digestive cells of the hepatopencreas are more elongated, often clavicular. Сytoplasm of digestive cells is loose and vacuolated and contain inclusions of yellow-green color. Enzyme cells on histopreparations are difficult to distinguish from digestive ones. They contain transparent vacuoles with a large round inclusion of yellow-green color, which consists of a cluster of several grains of different sizes. Hepatopancreas performs the following functions: secretory (enzyme cells), absorption and intracellular digestion (digestive cells), preservation of nutrients and calcium (calcium cells), and also excretory function.

Whole-Genome Resequencing of Twenty Branchiostoma belcheri Individuals Provides a Brand-New Variant Dataset for Branchiostoma

As the extant representatives of the basal chordate lineage, amphioxi (including the genera Branchiostoma, Asymmetron and Epigonichthys) play important roles in tracing the state of chordate ancestry. Previous studies have reported that members of the Branchiostoma species have similar morphological phenotypic characteristics, but in contrast, there are high levels of genetic polymorphisms in the populations. Here, we resequenced 20 Branchiostomabelcheri genomes to an average depth of approximately 12.5X using the Illumina HiSeq 2000 platform. In this study, over 52 million variations (~12% of the total genome) were detected in the B. belcheri population, and an average of 12.8 million variations (~3% of the total genome) were detected in each individual, confirming that Branchiostoma is one of the most genetically diverse species sequenced to date. Demographic inference analysis highlighted the role of historical global temperature in the long-term population dynamics of Branchiostoma, and revealed a population expansion at the Greenlandian stage of the current geological epoch. We detected 594 Single nucleotide polymorphism and 148 Indels in the Branchiostoma mitochondrial genome, and further analyzed their genetic mutations. A recent study found that the epithelial cells of the digestive tract in Branchiostoma can directly phagocytize food particles and convert them into absorbable nontoxic nutrients using powerful digestive and immune gene groups. In this study, we predicted all potential mutations in intracellular digestion-associated genes. The results showed that most “probably damaging” mutations were related to rare variants (MAF < 0.05) involved in strengthening or weakening the intracellular digestive capacity of Branchiostoma. Due to the extremely high number of polymorphisms in the Branchiostoma genome, our analysis with a depth of approximately 12.5X can only be considered a preliminary analysis. However, the novel variant dataset provided here is a valuable resource for further investigation of phagocytic intracellular digestion in Branchiostoma and determination of the phenotypic and genotypic features of Branchiostoma.

Dinoflagellate symbionts escape vomocytosis by host cell immune suppression

Emergence of the symbiotic lifestyle fostered the immense diversity of all ecosystems on Earth, but symbiosis plays a particularly remarkable role in marine ecosystems. Photosynthetic dinoflagellate endosymbionts power reef ecosystems by transferring vital nutrients to their coral hosts. The mechanisms driving this symbiosis, specifically those which allow hosts to discriminate between beneficial symbionts and pathogens, are not well understood. Here, we uncover that host immune suppression is key for dinoflagellate endosymbionts to avoid elimination by the host using a comparative, model systems approach. Unexpectedly, we find that the clearance of non-symbiotic microalgae occurs by non-lytic expulsion (vomocytosis) and not intracellular digestion, the canonical mechanism used by professional immune cells to destroy foreign invaders. We provide evidence that suppression of TLR signalling by targeting the conserved MyD88 adapter protein has been co-opted for this endosymbiotic lifestyle, suggesting that this is an evolutionarily ancient mechanism exploited to facilitate symbiotic associations ranging from coral endosymbiosis to the microbiome of vertebrate guts.

Ultrastructure of the midgut epithelium in three species of Macrobiotidae (Tardigrada: Eutardigrada: Parachela)

Three species of Macrobiotidae, Macrobiotus polonicus, Macrobiotus diversus and Macrobiotus pallarii, were selected for analysis of the fine structure of the midgut epithelium. They are gonochoric and carnivorous species that live in wet terrestrial and freshwater environments. The ultrastructure of the midgut epithelium of the investigated Macrobiotidae species was analysed in both males and females. Their digestive system is composed of fore- and hindguts that are covered by a cuticle, and the middle region, termed the midgut. It is lined with a simple epithelium that is formed by digestive cells that have a distinct brush border. Crescent-shaped cells that form an anterior ring in the border between the fore- and midgut were detected. The ultrastructure of the intestinal epithelium of the examined species differs slightly depending on sex. The digestive cells of the posterior segment of the intestine contain numerous lipid droplets, which are the reserve material. We concluded that the digestive cells of the Macrobiotidae midgut are responsible for its intracellular digestion owing to endocytosis. They also participate in the extracellular digestion owing to merocrine secretion (exocytosis). However, the midgut is not the main organ that accumulates reserve material. Additionally, the midgut epithelium does not participate in oogenesis.

Autophagy in Pulmonary Innate Immunity

Autophagy is a major intracellular digestion system that delivers cytoplasmic components for degradation and recycling. In this capacity, autophagy plays an important role in maintaining cellular homeostasis by mediating the degradation of cellular macromolecules and dysfunctional organelles and regeneration of nutrients for cell growth. Autophagy is important in innate immunity, as it is responsible for the clearance of various pathogens. Deficiency of intracellular autophagy can result in exaggerated activation of the inflammasome. The latter is an innate immune complex that senses diverse pathogen-associated or danger-associated molecular patterns and activates the expression of inflammatory cytokines. In autophagy-deficient cells, accumulation of damaged organelles, misfolded proteins, and reactive oxygen species contribute to inflammasome activation. The lung is continuously exposed to pathogens from the environment, rendering it vulnerable to infection. The lung innate immune cells act as a crucial initial barrier against the continuous threat from pathogens. In this review, we will summarize recent findings on the regulation of autophagy and its inter­action with innate immunity, focusing on the lung.

Evolution of self-limited cell division of symbionts

In mutualism between unicellular hosts and their endosymbionts, symbiont's cell division is often synchronized with its host's, ensuring the permanent relationship between endosymbionts and their hosts. The evolution of synchronized cell division thus has been considered to be an essential step in the evolutionary transition from symbionts to organelles. However, if symbionts would accelerate their cell division without regard for the synchronization with the host, they would proliferate more efficiently. Thus, it is paradoxical that symbionts evolve to limit their own division for synchronized cell division. Here, we theoretically explore the condition for the evolution of self-limited cell division of symbionts, by assuming that symbionts control their division rate and that hosts control symbionts' death rate by intracellular digestion and nutrient supply. Our analysis shows that symbionts can evolve to limit their own cell division. Such evolution occurs if not only symbiont's but also host's benefit through symbiosis is large. Moreover, the coevolution of hosts and symbionts leads to either permanent symbiosis where symbionts proliferate to keep pace with their host, or the arms race between symbionts that behave as lytic parasites and hosts that resist them by rapid digestion.