Metabarcoding reveals low prevalence of microsporidian infections in castor bean tick (Ixodes ricinus)

Background Microsporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans. Methods In total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI). Results All collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified. Conclusions The lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites. Graphical abstract

Species-Specificity in Thermopreference and CO2-Gated Heat-Seeking in Culex Mosquitoes

Combining thermopreference (Tp) and CO2-gated heat-seeking assays, we studied the thermal preferendum and response to thermal cues in three Culex mosquito species exhibiting differences in native habitat and host preference (e.g., biting cold and/or warm-blooded animals). Results show that these species differ in both Tp and heat-seeking behavior. In particular, we found that Culex territans, which feed primarily on cold-blood hosts, did not respond to heat during heat-seeking assays, regardless of the CO2 concentration, but exhibited an intermediate Tp during resting. In contrast, Cx. quinquefasciatus, which feeds on warm blooded hosts, sought the coolest locations on a thermal gradient and responded only moderately to thermal stimuli when paired with CO2 at higher concentrations. The third species, Cx. tarsalis, which has been shown to feed on a wide range of hosts, responded to heat when paired with high CO2 levels and exhibited a high Tp. This study provides the first insights into the role of heat and CO2 in the host seeking behavior of three disease vectors in the Culex genus and highlights differences in preferred resting temperatures.

Trypanosoma cruzi Parasite Load Modulates the Circadian Activity Pattern of Triatoma infestans

American trypanosomiasis is a disease caused by the flagellate protozoan Trypanosoma cruzi, which is transmitted mainly in endemic areas by blood-sucking triatomine vectors. Triatoma infestans is the most important vector in the southern cone of South America, exhibiting a nocturnal host-seeking behavior. It has been previously documented that the parasite produces changes in some triatomine species, but this is the first time that the behavior of a vector has been evaluated in relation to its parasite load. After comparing the movement events and distance traveled of infected and non-infected T. infestans, we evaluated the change produced by different T. cruzi parasite loads on its circadian locomotor activity. We observed differences between infected and non-infected triatomines, and a significant relation between the parasite load and the increase in locomotor activity of T. infestans, which was accentuated during the photophase. This could have direct implications on the transmission of T. cruzi, as the increased movement and distance traveled could enhance the contact of the vector with the host, while increasing the predation risk for the vector, which could both constitute a risk for vectorial and oral transmission to mammals.

Seasonal Dynamics of Mosquito-Borne Viruses in the Southwestern Florida Everglades, 2016, 2017

Mosquitoes were collected for 12 consecutive months beginning June 2016, from 11 locations in the Florida Everglades, Collier County, and tested for viruses by isolation in Vero cells and subsequent identification. One species complex and 31 species of mosquitoes were identified from 668,809 specimens. Ochlerotatus taeniorhynchus comprised 72.2% of the collection. Other notable species were Anopheles crucians complex, Culex nigripalpus, Cx. erraticus, and Cx. cedecei. Seven species of virus were identified from 110 isolations: Everglades, Gumbo Limbo, Mahogany Hammock, Pahayokee, Shark River, Tensaw, and West Nile viruses. Everglades, West Nile, Tensaw, and Mahogany Hammock viruses were most frequently isolated. Largest numbers of viruses were identified from Cx. cedecei, Cx. nigripalpus, and An. crucians complex. Five species of virus were isolated from Cx. cedecei. Viruses were isolated from mangrove, cypress swamp, hardwood hammock, and sawgrass habitats. West Nile virus was isolated August through October when Cx. nigripalpus was most abundant. Everglades virus was the most frequently isolated virus from nine species of mosquitoes collected from June through August. Tensaw virus was isolated primarily from Anopheles species. Isolations were made in July, August, January, February, and April, suggesting that this virus may be present in host-seeking mosquitoes throughout the year. Mahogany Hammock, Shark River, Gumbo Limbo, and Pahayokee viruses were isolated primarily from Cx. cedecei from June through December. Shotgun metagenomic sequencing was used to document that seven pools of Cx. cedecei were infected with two arboviruses. As communities expand into the Everglades, more humans will become exposed to arboviruses.

Blood meal analysis of Anopheles vectors of simian malaria based on laboratory and field studies

Blood feeding and host-seeking behaviors of a mosquito play an imperative role in determining its vectorial capacity in transmitting pathogens. Unfortunately, limited information is available regarding blood feeding behavior of Anopheles species in Malaysia. Collection of resting Anopheles mosquitoes for blood meal analysis poses a great challenge especially for forest dwelling mosquitoes. Therefore, a laboratory-based study was conducted to evaluate the potential use of mosquitoes caught using human landing catch (HLC) for blood meal analysis, and subsequently to document blood feeding behavior of local Anopheles mosquitoes in Peninsular Malaysia. The laboratory-based experiment from this study revealed that mosquitoes caught using HLC had the potential to be used for blood meal analysis. Besides HLC, mosquitoes were also collected using manual aspirator and Mosquito Magnet. Overall, 47.4% of 321 field-caught Anopheles mosquitoes belonging to six species were positive for vertebrate host DNA in their blood meal. The most frequent blood meal source was human (45.9%) followed by wild boar (27.4%), dog (15.3%) and monkey (7.5%). Interestingly, only Anopheles cracens and Anopheles introlatus (Leucosphyrus Group) fed on monkey. This study further confirmed that members of the Leucosphyrus Group are the predominant vectors for knowlesi malaria transmission in Peninsular Malaysia mainly due to their simio-anthropophagic feeding behavior.

The effect of flood on seasonal dynamics of Haemaphysalis (Acari: Ixodidae) tick vectors in Western Ghats forest area of Kerala, South India

Aim: Climate and weather conditions play a crucial role in the dynamics and distribution of ticks and tick-borne diseases. In this study, we explored the influence of a heavy rainfall (flood) occurrence on the seasonal activity and density of host-seeking Haemaphysalis tick vectors in Wayanad district, Kerala, India. Methodology: Wayanad district in Kerala state was selected as the study area. Ticks were collected from December 2017 to May 2019, monthly for five consecutive days by dragging method. Tick density was analyzed with climate data obtained from the meteorological station. Results: The total number of ticks collected post-flood decreased to 59% in Kurichiyad (site 1) and 63% in Muthanga (site 2), and the seasonal nymphal peak density was shifted. A seasonal peak of tick activity was normally observed from December to February. This peak occurrence was missing after flood in the study areas created with waterlogging and vegetation overgrowth. Interpretation: The present study revealed the effect of flood events in the study sites with significant differences in the abundance of five Haemaphysalis tick species during pre and post-flood periods and forest and wildlife habitats. This difference in the changing climatic conditions and increasing annual flood seasons in the Western Ghats may shift this region's ticks questing activity and tick-borne disease ecology.

Daily Variation in Sampled Densities of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) Nymphs at a Single Site—Implications for Assessing Acarological Risk

The public health challenge posed by tick-borne disease (TBD) has increased efforts to characterize the spatial and temporal distribution of ticks and associated pathogens to better focus tick control strategies and personal protection measures. We describe variability in nymphal Ixodes scapularis Say and Amblyomma americanum (L.) density derived from daily drag sampling at a single location in New Jersey over 4 yr and explore how observed differences in daily collections might affect the estimation of acarological risk. We found significant variability in the density of host-seeking nymphs that could suggest substantially different rates of human-tick encounters depending on sampling date, habitat, and ambient weather conditions. The spatial and temporal variability in the distribution of 2 sympatric tick species with different host preferences and questing strategies, suggests that to produce results that are comparable among sites across the area sampled, surveillance efforts may be limited to shorter collection seasons, fewer sites or less sampling effort (fewer plots or fewer visits) per site, and a geographic scope that minimizes the potential temporal and spatial biases indicated here. Our results illustrate that evaluation of models of tick distribution or relative acarological risk based on surveillance data requires a full description of the diversity of habitats sampled and the conditions under which sampling is performed. The array of factors that affect tick host-seeking and that could bias interpretation of sampling results emphasizes the need to standardize sampling protocols and for more caution when interpreting tick sampling data collected over large temporal and spatial scales.

Mosquito Host-Seeking Diel Rhythm and Chemosensory Gene Expression is Affected by Age and Plasmodium Stages

Malaria parasites can affect vector-related behaviours, increasing transmission success. Using Anopheles gambiae and Plasmodium falciparum, we consider the effect of interaction between infection stage and vector age on diel locomotion in response to human odour and the expression of antennal chemosensory genes. We demonstrate age-dependent behavioural diel compartmentalisation by uninfected females. Infection disrupts overall and diel activity patterns compared with age-matched controls. Mosquitoes carrying transmissible sporozoites are more active, shifting activity periods to coincide with human host availability, in response to human odour. Older, uninfected females reduce activity during their peak host-seeking period in response to human odour. Age- and infection stage-specific changes in odour-mediated locomotion coincide with altered transcript abundance of select chemosensory genes providing a possible molecular mechanism regulating the behaviour. Our results support the hypothesis that vector-related behaviours of female mosquitoes are altered by infection stage and further modulated by the age of the vector, and have important implications for malaria transmission and disease dynamics.

Human skin triglycerides prevent bed bug (Cimex lectularius L.) arrestment

Bed bugs (Cimex lectularius) have proliferated globally and have become one of the most challenging pests to control indoors. They are nocturnal and use multiple sensory cues to detect and orient towards their human hosts. After feeding, usually on a sleeping human, they return to a shelter on or around the sleeping surface, but not directly on the host. We hypothesized that although human skin odors attract hungry bed bugs, human skin compounds may also prevent arrestment on hosts. We used arrestment assays to test human skin swabs, extracts from human skin swabs, and pure compounds identified from human skin swabs. When given a choice, bed bugs preferred to arrest on substrates not previously conditioned by humans. These responses were consistent among laboratory-reared and apartment-collected bed bugs. The compounds responsible for this behavior were found to be extractable in hexane, and bed bugs responded to such extracts in a dose-dependent manner. Bioassay-guided fractionation paired with thin-layer chromatography, GC–MS, and LC–MS analyses suggested that triglycerides (TAGs), common compounds found on human skin, were preventing arrestment on shelters. Bed bugs universally avoided sheltering in TAG-treated shelters, which was independent of the number of carbons or the number of double bonds in the TAG. These results provide strong evidence that the complex of human skin compounds serve as multifunctional semiochemicals for bed bugs, with some odorants attracting host-seeking stages, and others (TAGs and possibly other compounds) preventing bed bug arrestment. Host chemistry, environmental conditions and the physiological state of bed bugs likely influence the dual nature behavioral responses of bed bugs to human skin compounds.