A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva

Background The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. Methods A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography–tandem mass spectrometry (LC–MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). Results Up to 264 and 263 proteins were identified by LC–MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. Conclusions The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick–host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits. Graphical Abstract

Exploring the role of gut microbiota in host feeding behavior among breeds in swine

Background The interplay between the gut microbiota and feeding behavior has consequences for host metabolism and health. The present study aimed to explore gut microbiota overall influence on feeding behavior traits and to identify specific microbes associated with the traits in three commercial swine breeds at three growth stages. Feeding behavior measures were obtained from 651 pigs of three breeds (Duroc, Landrace, and Large White) from an average 73 to 163 days of age. Seven feeding behavior traits covered the information of feed intake, feeder occupation time, feeding rate, and the number of visits to the feeder. Rectal swabs were collected from each pig at 73 ± 3, 123 ± 4, and 158 ± 4 days of age. DNA was extracted and subjected to 16 S rRNA gene sequencing. Results Differences in feeding behavior traits among breeds during each period were found. The proportion of phenotypic variances of feeding behavior explained by the gut microbial composition was small to moderate (ranged from 0.09 to 0.31). A total of 21, 10, and 35 amplicon sequence variants were found to be significantly (q-value < 0.05) associated with feeding behavior traits for Duroc, Landrace, and Large White across the three sampling time points. The identified amplicon sequence variants were annotated to five phyla, with Firmicutes being the most abundant. Those amplicon sequence variants were assigned to 28 genera, mainly including Christensenellaceae_R-7_group, Ruminococcaceae_UCG-004, Dorea, Ruminococcaceae_UCG-014, and Marvinbryantia. Conclusions This study demonstrated the importance of the gut microbial composition in interacting with the host feeding behavior and identified multiple archaea and bacteria associated with feeding behavior measures in pigs from either Duroc, Landrace, or Large White breeds at three growth stages. Our study provides insight into the interaction between gut microbiota and feeding behavior and highlights the genetic background and age effects in swine microbial studies.

Impact of a nonnative parasitoid species on intraspecific interference and offspring sex ratio

In an assemblage of multiple predators sharing a single prey species, the combined effects of the component species may scale unpredictably because of emergent interspecific interactions. Prior studies suggest that chaotic but persistent community dynamics are induced by intra-/interspecific interactions between native and nonnative parasitoids competing over a shared host. Here, we test the impact of the nonnative parasitoid Heterospilus prosopidis (Hymenoptera: Braconidae) on the intraspecific interference and offspring sex ratio of the native parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae). We found that the nonnative parasitoid reduced intraspecific interference among native parasitoids and decreased the proportion of female offspring produced by the native parasitoid (predicted under conditions of reduced host availability). At higher host densities, the nonnative parasitoid contributed less to the total proportion of hosts parasitized, as its innate saturating Type II response changed to a dome-shaped Type IV response with increasing density of the native parasitoid, while the native parasitoid retained its increasing Type I response. This inverse host-density-dependent response between the two parasitoids and associated competitive superiority can explain the observed changes in parasitism; at high host densities, the searching efficiency of the native parasitoid increases via host feeding while the nonnative parasitoid experiences egg limitation. These results highlight the importance of the complementary top-down effects of multiple consumers on a single resource.

The Control of Carbon Translocation in a Sea Anemone-Dinoflagellate Symbiosis from New Zealand

<p>Anthopleura aureoradiata, a common sea anemone of New Zealand's intertidal mudflats and rocky shores, hosts symbiotic dinoflagellates of the genus Symbiodinium. This study investigated the control of photosynthetic carbon translocation in this symbiosis, and in particular the presence and operation of socalled 'host release factor' (HRF). Evidence for HRF exists in a number other algalinvertebrate symbioses, where tissue extracts of the host stimulate carbon release by isolated algal symbionts. However, its identity remains elusive and it has never been studied before in A. aureoradiata. Translocation of photosynthetically-fixed carbon in the intact symbiosis and in the presence of host tissue extract was measured using a 14C label. Zooxanthellae in the intact symbiosis released around 40% of their photosynthetically-fixed carbon to the anemone. Isolated zooxanthellae, however, translocated only 8%, even less than the amount of photosynthate liberated by zooxanthellae in FSW alone (11%). Photosynthetic rates per algal cell were similar in the intact symbiosis and both host homogenate and FSW incubations, meaning that the total amount of photosynthetically-fixed carbon released (in pg C/cell/h) by the zooxanthellae in these different situations reflected the %translocation values. Given the failure of homologous zooxanthellae (i.e. those from A. aureoradiata) to respond to homogenized host tissue, it was tested whether zooxanthellae from other host species (i.e. cultured heterologous algae) responded. Heterologous zooxanthellae representing 5 clades (A-E) of Symbiodinium were incubated in host tissue homogenate and photosynthate release again measured with 14C. The %translocation varied from 12-51% in A. aureoradiata homogenate and 17-67% in FSW, again suggesting a lack of an active HRF in the homogenized tissues of this sea anemone. Photosynthetic rates amongst the different heterologous algae also varied widely with, for instance, freshly isolated zooxanthellae from A. aureoradiata having 6-fold higher photosynthetic rates than cultured algae from the same clade (clade A). The zooxanthellae of A. aureoradiata are known to be N-sufficient in the field, and studies with other species have demonstrated that N-deficient zooxanthellae release more photosynthate in response to HRF than do N-sufficient ones. Therefore, induction of an HRF effect was attempted by starving sea anemones, and hence their zooxanthellae, prior to incubation of freshly isolated zooxanthellae in homogenized tissue. However, even after 8 weeks of starvation, the zooxanthellae showed no signs of N-deficiency (as indicated by the extent to which ammonium enhanced the rate of dark 14C fixation), meaning that the relationship with HRF activity could not be examined. The ability of these temperate zooxanthellae to maintain their Nsufficiency, even after relatively long periods of food deprivation, may indicate a lower reliance on host feeding for nitrogen than is seen in tropical zooxanthellae, or a greater capacity to use internal stores of nitrogen. The lack of photosynthate release by both homologous and heterologous zooxanthellae in host homogenate, as opposed to substantial carbon released in the intact symbiosis, suggests that control of carbon translocation in A. aureoradiata is not related to the activity of an HRF; alternatively, if an HRF is present, its activity is hindered when the symbiosis is disrupted. Further study is needed to determine what is responsible for the control of photosynthate translocation in the A. aureoradiata-Symbiodinium symbiosis.</p>

The Control of Carbon Translocation in a Sea Anemone-Dinoflagellate Symbiosis from New Zealand

<p>Anthopleura aureoradiata, a common sea anemone of New Zealand's intertidal mudflats and rocky shores, hosts symbiotic dinoflagellates of the genus Symbiodinium. This study investigated the control of photosynthetic carbon translocation in this symbiosis, and in particular the presence and operation of socalled 'host release factor' (HRF). Evidence for HRF exists in a number other algalinvertebrate symbioses, where tissue extracts of the host stimulate carbon release by isolated algal symbionts. However, its identity remains elusive and it has never been studied before in A. aureoradiata. Translocation of photosynthetically-fixed carbon in the intact symbiosis and in the presence of host tissue extract was measured using a 14C label. Zooxanthellae in the intact symbiosis released around 40% of their photosynthetically-fixed carbon to the anemone. Isolated zooxanthellae, however, translocated only 8%, even less than the amount of photosynthate liberated by zooxanthellae in FSW alone (11%). Photosynthetic rates per algal cell were similar in the intact symbiosis and both host homogenate and FSW incubations, meaning that the total amount of photosynthetically-fixed carbon released (in pg C/cell/h) by the zooxanthellae in these different situations reflected the %translocation values. Given the failure of homologous zooxanthellae (i.e. those from A. aureoradiata) to respond to homogenized host tissue, it was tested whether zooxanthellae from other host species (i.e. cultured heterologous algae) responded. Heterologous zooxanthellae representing 5 clades (A-E) of Symbiodinium were incubated in host tissue homogenate and photosynthate release again measured with 14C. The %translocation varied from 12-51% in A. aureoradiata homogenate and 17-67% in FSW, again suggesting a lack of an active HRF in the homogenized tissues of this sea anemone. Photosynthetic rates amongst the different heterologous algae also varied widely with, for instance, freshly isolated zooxanthellae from A. aureoradiata having 6-fold higher photosynthetic rates than cultured algae from the same clade (clade A). The zooxanthellae of A. aureoradiata are known to be N-sufficient in the field, and studies with other species have demonstrated that N-deficient zooxanthellae release more photosynthate in response to HRF than do N-sufficient ones. Therefore, induction of an HRF effect was attempted by starving sea anemones, and hence their zooxanthellae, prior to incubation of freshly isolated zooxanthellae in homogenized tissue. However, even after 8 weeks of starvation, the zooxanthellae showed no signs of N-deficiency (as indicated by the extent to which ammonium enhanced the rate of dark 14C fixation), meaning that the relationship with HRF activity could not be examined. The ability of these temperate zooxanthellae to maintain their Nsufficiency, even after relatively long periods of food deprivation, may indicate a lower reliance on host feeding for nitrogen than is seen in tropical zooxanthellae, or a greater capacity to use internal stores of nitrogen. The lack of photosynthate release by both homologous and heterologous zooxanthellae in host homogenate, as opposed to substantial carbon released in the intact symbiosis, suggests that control of carbon translocation in A. aureoradiata is not related to the activity of an HRF; alternatively, if an HRF is present, its activity is hindered when the symbiosis is disrupted. Further study is needed to determine what is responsible for the control of photosynthate translocation in the A. aureoradiata-Symbiodinium symbiosis.</p>

Advances in Understanding Vector Behavioural Traits after Infection

Vector behavioural traits, such as fitness, host-seeking, and host-feeding, are key determinants of vectorial capacity, pathogen transmission, and epidemiology of the vector-borne disease. Several studies have shown that infection with pathogens can alter these behavioural traits of the arthropod vector. Here, we review relevant publications to assess how pathogens modulate the behaviour of mosquitoes and ticks, major vectors for human diseases. The research has shown that infection with pathogens alter the mosquito’s flight activity, mating, fecundity, host-seeking, blood-feeding, and adaptations to insecticide bed nets, and similarly modify the tick’s locomotion, questing heights, vertical and horizontal walks, tendency to overcome obstacles, and host-seeking ability. Although some of these behavioural changes may theoretically increase transmission potential of the pathogens, their effect on the disease epidemiology remains to be verified. This study will not only help in understanding virus–vector interactions but will also benefit in establishing role of these behavioural changes in improved epidemiological models and in devising new vector management strategies.

Heterogeneity of Rift Valley fever virus transmission potential across livestock hosts, quantified through a model-based analysis of host viral load and vector infection

Rift Valley fever (RVF) is a viral, vector-borne, zoonotic disease. The relative contributions of livestock species to RVFV transmission has not been previously quantified. To estimate their potential to transmit the virus over the course of their infection, we 1) fitted a within-host model to viral RNA and infectious virus measures, obtained daily from infected lambs, calves, and young goats, 2) estimated the relationship between vertebrate host infectious titers and probability to infect mosquitoes, and 3) estimated the net infectiousness of each host species over the duration of their infectious periods, taking into account different survival outcomes for lambs. Our results indicate that the efficiency of viral replication, along with the lifespan of infectious particles, could be sources of heterogeneity between hosts. For similar infectious titers, we found that infection rates in Aedes spp. vectors were significantly higher than in Culex spp. vectors. Consequently, for Aedes infections, we estimated the net infectiousness of lambs to be 2.93 (median) and 3.65 times higher than that of calves and goats, respectively. Among lambs, individuals which eventually died from the infection were 1.93 times more infectious than lambs recovering. Beyond infectiousness, the relative contributions of host species to transmission depend on local ecological factors, including relative abundances and vector host-feeding preferences. Quantifying these contributions will ultimately help design efficient, targeted, surveillance and vaccination strategies.

Synchrony between daily rhythms of malaria parasites and hosts is driven by an essential amino acid

Background: Rapid asexual replication of blood stage malaria parasites is responsible for the severity of disease symptoms and fuels the production of transmission forms. Here, we demonstrate that a Plasmodium chabaudi’s schedule for asexual replication can be orchestrated by isoleucine, a metabolite provided to the parasite in a periodic manner due to the host’s rhythmic intake of food. Methods: We infect female C57BL/6 and Per1/2-null mice which have a disrupted canonical (transcription translation feedback loop, TTFL) clock with 1×105 red blood cells containing P. chabaudi (DK genotype). We perturb the timing of rhythms in asexual replication and host feeding-fasting cycles to identify nutrients with rhythms that match all combinations of host and parasite rhythms. We then test whether perturbing the availability of the best candidate nutrient in vitro changes the schedule for asexual development. Results: Our large-scale metabolomics experiment and follow up experiments reveal that only one metabolite - the amino acid isoleucine – fits criteria for a time-of-day cue used by parasites to set the schedule for replication. The response to isoleucine is a parasite strategy rather than solely the consequences of a constraint imposed by host rhythms, because unlike when parasites are deprived of other essential nutrients, they suffer no apparent costs from isoleucine withdrawal. Conclusions: Overall, our data suggest parasites can use the daily rhythmicity of blood-isoleucine concentration to synchronise asexual development with the availability of isoleucine, and potentially other resources, that arrive in the blood in a periodic manner due to the host’s daily feeding-fasting cycle. Identifying both how and why parasites keep time opens avenues for interventions; interfering with the parasite’s time-keeping mechanism may stall replication, increasing the efficacy of drugs and immune responses, and could also prevent parasites from entering dormancy to tolerate drugs.