Impact of diet and bacterial supplementation regimes on Orius strigicollis microbiota and life history performance.

Given the growing interest in manipulating microbiota to enhance the fitness of mass-reared insects for biological control, this study investigated the impact of an artificial diet on the microbiota composition and performance of Orius strigicollis. We compared the microbiota of O. strigicollis fed on an artificial diet and moth eggs via culturing and 16S rRNA gene amplicon sequencing. Subsequently, we assessed life history traits and immune gene expression of O. strigicollis fed on the artificial diet supplemented with Pantoea dispersa OS1. Results showed that microbial diversity remained largely unaffected by the artificial diet, with similar microbiota compositions in both diet groups. OS1, a minor member of the microbiota but significantly enriched in bugs fed on the artificial diet, improved nymphal survival rates and shifted adult longevity-reproduction life history in females. Additionally, OS1 supplementation elevated the transcription of antimicrobial peptide diptericin. According to population parameters, the group receiving OS1 only during the nymphal stage showed higher population growth potential compared to the group supplemented across all life stages. These findings reveal the resilience of O. strigicollis microbiota under distinct dietary conditions and highlight the potential of using natural symbionts and specific supplementation regimes to improve Orius rearing for future biocontrol programs.

Nanoparticle LDH enhances RNAi efficiency of dsRNA in piercing-sucking pests by promoting dsRNA stability and transport in plants.

Piercing-sucking pests are the most notorious group of pests for global agriculture. RNAi-mediated crop protection by foliar application is a promising approach in field trials. However, the effect of this approach on piercing-sucking pests is far from satisfactory due to the limited uptake and transport of double strand RNA (dsRNA) in plants. Therefore, there is an urgent need for more feasible and biocompatible dsRNA delivery approaches to better control piercing-sucking pests. Here, we report that foliar application of layered double hydroxide (LDH)-loaded dsRNA can effectively disrupt Panonychus citri at multiple developmental stages. MgAl-LDH-dsRNA targeting Chitinase (Chit) gene significantly promoted the RNAi efficiency and then increased the mortality of P. citri nymphs by enhancing dsRNA stability in gut, promoting the adhesion of dsRNA onto leaf surface, facilitating dsRNA internalization into leaf cells, and delivering dsRNA from the stem to the leaf via the vascular system of pomelo plants. Finally, this delivery pathway based on other metal elements such as iron (MgFe-LDH) was also found to significantly improve the protection against P. citri and the nymphs or larvae of Diaphorina citri and Aphis gossypii, two other important piercing-sucking hemipeteran pests, indicating the universality of nanoparticles LDH in promoting the RNAi efficiency and mortality of piercing-sucking pests. Collectively, this study provides insights into the synergistic mechanism for nano-dsRNA systemic translocation in plants, and proposes a potential eco-friendly control strategy for piercing-sucking pests.

The prevalence of pathogens in ticks collected from humans in Belgium, 2021, versus 2017.

BACKGROUND: Ticks carry a variety of microorganisms, some of which are pathogenic to humans. The human risk of tick-borne diseases depends on, among others, the prevalence of pathogens in ticks biting humans. To follow-up on this prevalence over time, a Belgian study from 2017 was repeated in 2021. METHODS: During the tick season 2021, citizens were invited to have ticks removed from their skin, send them and fill in a short questionnaire on an existing citizen science platform for the notification of tick bites (TekenNet). Ticks were morphologically identified to species and life stage level and screened using multiplex qPCR targeting, among others, Borrelia burgdorferi (sensu lato), Anaplasma phagocytophilum, Borrelia miyamotoi, Neoehrlichia mikurensis, Babesia spp., Rickettsia helvetica and tick-borne encephalitis virus (TBEV). The same methodology as in 2017 was used. RESULTS: In 2021, the same tick species as in 2017 were identified in similar proportions; of 1094 ticks, 98.7% were Ixodes ricinus, 0.8% Ixodes hexagonus and 0.5% Dermacentor reticulatus. A total of 928 nymphs and adults could be screened for the presence of pathogens. Borrelia burgdorferi (s.l.) was detected in 9.9% (95% CI 8.2-12.0%), which is significantly lower than the prevalence of 13.9% (95% CI 12.2-15.7%) in 2017 (P = 0.004). The prevalences of A. phagocytophilum (4.7%; 95% CI 3.5-6.3%) and R. helvetica (13.3%; 95% CI 11.2-15.6%) in 2021 were significantly higher compared to 2017 (1.8%; 95% CI 1.3-2.7% and 6.8%; 95% CI 5.6-8.2% respectively) (P < 0.001 for both). For the other pathogens tested, no statistical differences compared to 2017 were found, with prevalences ranging between 1.5 and 2.9% in 2021. Rickettsia raoultii was again found in D. reticulatus ticks (n = 3/5 in 2021). Similar to 2017, no TBEV was detected in the ticks. Co-infections were found in 5.1% of ticks. When combining co-infection occurrence in 2017 and 2021, a positive correlation was observed between B. burgdorferi (s.l.) and N. mikurensis and B. burgdorferi (s.l.) and B. miyamotoi (P < 0.001 for both). CONCLUSIONS: Although the 2021 prevalences fell within expectations, differences were found compared to 2017. Further research to understand the explanations behind these differences is needed.

Geographical, Seasonal, and Growth-Related Dynamics of Gut Microbiota in a Grapevine Pest, Apolygus spinolae (Heteroptera: Miridae).

A number of insects are associated with gut symbiotic microorganisms, wherein symbiotic partners play pivotal metabolic roles for each other such as nutrient supplementation, diet degradation, and pesticide detoxification. Despite the ecological and evolutionary importance of gut microbial communities in insects, their diversity and dynamics remain unclear in many species. The green plant bug Apolygus spinolae, a notorious grapevine pest in Japan, damages grape shoots and severely reduces grape berry yield and quality. The plant bug possesses a simple tubular gut housing ~ 104 bacteria. Here, we investigated geographic, seasonal, and growth-related dynamics of gut microbiota by high-throughput sequencing in 82 individuals (11 nymphs and 71 adults) from five locations in Hokkaido, Japan. In plant bugs, gut microbiota changed dynamically depending on region, season, and developmental stage. Among the gut bacteria, Serratia was consistently and abundantly detected and was significantly affected by seasonal changes. In addition, Caballeronia, known as a specific symbiont in some stinkbug species, was abundantly detected, especially in insects collected in late summer despite A. spinolae complete lack of midgut crypts known as symbiotic organ harboring Caballeronia in other stinkbug species. Considering their prevalence among host bug populations, it is possible these gut microorganisms play a pivotal role in the adaptation of the green plant bug to grapevine fields, although further confirmation through rearing experiments is needed.

The potential risk of human exposure to tick borne infection by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Babesia microti in selected recreational areas of the Poprad Landscape Park in southern Poland.

INTRODUCTION AND OBJECTIVE: Ticks (Acari:Ixodida) are dangerous ectoparasites and, at the same time, vectors and/or resevoirs of many pathogens, among others Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti. These ethiological agents of Lyme borreliosis, anaplasmosis and babesiosis are transferred to humans mainly by ticks during feeding. The aim of this study was to estimate the potential risk of human exposure to tick borne infection of B. burgdorferi s.l., A. phagocytophilum and B. microti in selected areas of Poprad Landscape Park in southern Poland [PLP]. MATERIAL AND METHODS: Ixodes ricinus ticks were collected from vegetation by the flagging method. Under a stereoscopic microscope, specimens were determined to the species and developmental stage. In total, DNA was isolated from 363 ticks. To detect B. burgdorferi s.l,.two pairs of primers specific to the flagelline gene were used. In turn, to detect A. phagocytophilum and B. microti, two pairs of primers specific to the 16S rRNA gene fragment and 18S rRNA gene fragment were used, respectively. The amplification products were separated electrophoretically in 2% ethidium bromide stained agarose gels, and visualized under ultra violet light. RESULTS: Generally, pathogens were observed in 19.6% of ticks. Borrelia burgdorferi sensu lato was detected in 11.8% of studied ticks. In turn, A. phagocytophlium and B. microti were presented, respectively, in 0.3% and 7.4% of examined I. ricinus. CONCLUSIONS: The study indicated a potentially high risk of human exposure to infection with tick-borne pathogens, mainly B. burgdorferi s.l. and B. microti, in the areas of PLP. In turn, the presence of A. phagocytophilum in lower percentage was shown in the studied ticks.

Detection and prevalence of Anaplasma phagocytophilum in Ixodes ricinus ticks in eastern Poland.

INTRODUCTION: Anaplasma phagocytophilum are tick-borne bacteria affecting both human and animal health. The aim of the study was to examine the prevalence of A. phagocytophilum in questing I. ricinus ticks collected in Lublin Province, eastern Poland. MATERIAL AND METHODS: Ticks were collected by the flagging method. Total DNA from ticks was extracted by boiling in ammonium hydroxide. Detection of A. phagocytophilum was performed by amplifying a fragment of the 16S rDNA gene. RESULTS: Overall, 626 I. ricinus ticks were tested for the presence of A. phagocytophilum DNA. The prevalence of the pathogenic bacteria was 1.28%. The occurrence of A. phagocytophilum among adults was 1.8%, whereas none of the collected I. ricinus nymphs were infected. CONCLUSIONS: The study revealed the presence of A. phagocytophilum in I. ricinus in eastern Poland, which constitutes a potential health risk for residents, tourists, forestry, and agricultural workers.

Candidatus Midichloria mitochondrii can be vertically transmitted in Hyalomma anatolicum.

In this study, a tick intracellular symbiont, Candidatus Midichloria mitochondrii, was detected in Hyalomma anatolicum from Xinjiang, China. Morphological identification and cytochrome oxidase subunit I sequence alignment were used for molecular identification of the tick species. PCR detection further revealed the presence of endosymbiont C. M. mitochondrii in the tick. Specific primers were designed for Groel and 16S rRNA genes of C. M. mitochondrii for PCR amplification and phylogenetic analysis. To further investigate the vertical transmission characteristics of C. M. mitochondrii, specific primers were designed based on the FabⅠ gene fragment to detect C. M. mitochondrii in different developmental stages and organs of the tick using qPCR. Of the 336 tick specimens collected from the field, 266 samples were identified as H. anatolicum on the basis of morphological characteristics. The gene fragment alignment results of COI confirmed that these ticks were H. anatolicum. The phylogenetic analysis showed that Groel gene of C. M. mitochondrii clustered with Midichloria strains detected in Ixodes ricinus ticks from Italy and Ixodes holocyclus ticks from Australia, with 100% sequence similarity. Furthermore, the 16S rRNA gene of C. M. mitochondrii clusters with the strains isolated from Hyalomma rufipes ticks in Italy, exhibiting the highest degree of homology. qPCR results showed that C. M. mitochondrii was present at all developmental stages of H. anatolicum, with the highest relative abundance in eggs, and lower relative abundance in nymphs and unfed males. With female tick blood feeding, the relative abundance of C. M. mitochondrii increased, and a particularly high relative abundance was detected in the ovaries of engorged female ticks. This study provides information for studying the survival adaptability of H. anatolicum, and provides data for further investigation of the mechanisms regulating tick endosymbionts in ticks, enriching the reference materials for comprehensive prevention and control of tick-borne diseases.

Host-pathogen associations inferred from bloodmeal analyses of Ixodes scapularis ticks in a low biodiversity setting.

Tick-borne pathogen emergence is dependent on the abundance and distribution of competent hosts in the environment. Ixodes scapularis ticks are generalist feeders, and their pathogen infection prevalence depends on their relative feeding on local competent and non-competent hosts. The ability to determine what host a larval life stage tick fed on can help predict infection prevalence, emergence, and spread of certain tick-borne pathogens and the risks posed to public health. Here, we use a newly developed genomic target-based technique to detect the source of larval bloodmeals by sampling questing nymphs from Block Island, RI, a small island with a depauperate mammalian community. We used previously designed specific assays to target all known hosts on this island and analyzed ticks for four human pathogenic tick-borne pathogens. We determined the highest proportion of larvae fed on avian species (42.34%), white-footed mice (36.94%), and white-tailed deer (20.72%) and occasionally fed on feral cats, rats, and voles, which are in low abundance on Block Island. Additionally, larvae that had fed on white-footed mice were significantly more likely to be infected with Borrelia burgdorferi and Babesia microti, while larvae that had fed on white-footed mice or white-tailed deer were significantly more likely to be infected with, respectively, mouse- and deer-associated genotypes of Anaplasma phagocytophilum. The ability to detect a nymph's larval host allows for a better understanding of tick feeding behavior, host distribution, pathogen prevalence, and zoonotic risks to humans, which can contribute to better tick management strategies. IMPORTANCE: Tick-borne diseases, such as Lyme disease, babesiosis, and anaplasmosis, pose significant public health burdens. Tick bloodmeal analysis provides a noninvasive sampling method to evaluate tick-host associations and combined with a zoonotic pathogen assay, can generate crucial insights into the epidemiology and transmission of tick-borne diseases by identifying potential key maintenance hosts. We investigated the bloodmeals of questing Ixodes scapularis nymphs. We found that avian hosts, white-footed mice, and white-tailed deer fed the majority of larval ticks and differentially contributed to the prevalence of multiple tick-borne pathogens and pathogen genotypes in a low biodiversity island setting. Unraveling the intricate network of host-vector-pathogen interactions will contribute to improving wildlife management and conservation efforts, to developing targeted surveillance, and vector and host control efforts, ultimately reducing the incidence of tick-borne diseases and improving public health.

Prevalence of tick-borne bacterial pathogens in Germany-has the situation changed after a decade?

Introduction: Tick-borne pathogens, such as Borreliella spp., Rickettsia spp., and Anaplasma spp., are frequently detected in Germany. They circulate between animals and tick vectors and can cause mild to severe diseases in humans. Knowledge about distribution and prevalence of these pathogens over time is important for risk assessment of human and animal health. Methods: Ixodes ricinus nymphs were collected at different locations in 2009/2010 and 2019 in Germany and analyzed for tick-borne pathogens by real-time PCR and sequencing. Results: Borreliella spp. were detected with a prevalence of 11.96% in 2009/2010 and 13.10% in 2019 with B. afzelii and B. garinii as dominant species. Borrelia miyamotoi was detected in seven ticks and in coinfection with B. afzelii or B. garinii. Rickettsia spp. showed a prevalence of 8.82% in 2009/2010 and 1.68% in 2019 with the exclusive detection of R. helvetica. The prevalence of Anaplasma spp. was 1.00% in 2009/2010 and 7.01% in 2019. A. phagocytophilum was detected in seven tick samples. None of the nymphs were positive for C. burnetii. Discussion: Here, observed changes in prevalence were not significant after a decade but require longitudinal observations including parameters like host species and density, climatic factors to improve our understanding of tick-borne diseases.

Modeling platform to assess the effectiveness of single and integrated Ixodes scapularis tick control methods.

BACKGROUND: Lyme disease continues to expand in Canada and the USA and no single intervention is likely to curb the epidemic. METHODS: We propose a platform to quantitatively assess the effectiveness of a subset of Ixodes scapularis tick management approaches. The platform allows us to assess the impact of different control treatments, conducted either individually (single interventions) or in combination (combined efforts), with varying timings and durations. Interventions include three low environmental toxicity measures in differing combinations, namely reductions in white-tailed deer (Odocoileus virginianus) populations, broadcast area-application of the entomopathogenic fungus Metarhizium anisopliae, and fipronil-based rodent-targeted bait boxes. To assess the impact of these control efforts, we calibrated a process-based mathematical model to data collected from residential properties in the town of Redding, southwestern Connecticut, where an integrated tick management program to reduce I.xodes scapularis nymphs was conducted from 2013 through 2016. We estimated parameters mechanistically for each of the three treatments, simulated multiple combinations and timings of interventions, and computed the resulting percent reduction of the nymphal peak and of the area under the phenology curve. RESULTS: Simulation outputs suggest that the three-treatment combination and the bait boxes-deer reduction combination had the overall highest impacts on suppressing I. scapularis nymphs. All (single or combined) interventions were more efficacious when implemented for a higher number of years. When implemented for at least 4 years, most interventions (except the single application of the entomopathogenic fungus) were predicted to strongly reduce the nymphal peak compared with the no intervention scenario. Finally, we determined the optimal period to apply the entomopathogenic fungus in residential yards, depending on the number of applications. CONCLUSIONS: Computer simulation is a powerful tool to identify the optimal deployment of individual and combined tick management approaches, which can synergistically contribute to short-to-long-term, costeffective, and sustainable control of tick-borne diseases in integrated tick management (ITM) interventions.

Born suckers: the cibarial pump of Philaenus spumarius scales across ontogeny to ensure functional equivalence.

A select group of hemipterans within the suborder Auchenorrhyncha are the only animals that feed exclusively on xylem sap - a nutritionally poor liquid that exists under negative pressure within a plant's xylem vessels. To consume it, xylem-feeding bugs have evolved enlarged cibarial pumps capable of generating enormous negative pressures. A previous study examining the allometry of this feeding model suggested that small xylem feeders pay relatively higher energetic costs while feeding, favouring the evolution of larger-bodied species. However, this interspecific analysis only considered adult xylem-feeding insects and neglected the considerable intraspecific change in size that occurs across the insect's development. Here, we examine the changes in cibarial pump morphology and function that occur during the development of Philaenus spumarius, the common meadow spittlebug. We show that the cibarial pump scales largely as expected from isometry and that the maximum negative pressure is mass independent, indicating that size has no effect on the xylem-feeding capacity of juvenile spittlebugs. We conclude that a first instar nymph with a body mass 2% of the adult can still feed at the >1 MPa tension present in a plant's xylem vessels without a substantial energetic disadvantage.

Acaricidal potential of essential oils on Rhipicephalus linnaei: Alternatives and prospects.

The acaricidal potential of various essential oils (EOs) has been evaluated based on their benefits in tick control. This study aimed to investigate the tick-killing activity of Pogostemon cablin "patchouli," Cymbopogon martinii "palmarosa," and Cymbopogon flexuosus "lemongrass" EOs on Rhipicephalus linnaei. Engorged females were collected from domiciled and non-domiciled dogs from Jataí city, Goiás state, to obtain larvae and nymphs in a controlled environment. Two commercial EOs brands were used in this study in different EOs concentrations (2.5, 5, 10, and 20 mg/mL), and was tested by immersion of larvae and nymphs. In the in vitro evaluation of EOs toxicity against R. linnaei larvae, 100 % mortality was achieved with 10 mg/mL of P. cablin oil, whereas mortality rates greater than 98 % were observed with 20 mg/mL of C. martinii and C. flexuosus. In nymphs, high sensitivity was observed, with 100 % mortality achieved using 5 mg/mL of P. cablin and 20 mg/mL of C. martinii and C. flexuosus. The EO of P. cablin "patchouli" demonstrated in vitro toxicity at a lower concentration than the other oils in the two development stages of R. linnaei, and was considered the most efficient and with verified acaricidal activity. Oils of C. martinii "palmarosa" and C. flexuosus "lemongrass" achieved mortality greater than 95 % in larvae and nymphs only at the highest concentration. Therefore, the acaricidal effects of the tested EOs are promising, especially of patchouli oil, which promoted high mortality at a low concentration (LC90 of 2.21 mg/mL).

No evidence of Bartonella infections in host-seeking Ixodes scapularis and Ixodes pacificus ticks in the United States.

BACKGROUND: Bartonella spp. infect a variety of vertebrates throughout the world, with generally high prevalence. Several Bartonella spp. are known to cause diverse clinical manifestations in humans and have been recognized as emerging pathogens. These bacteria are mainly transmitted by blood-sucking arthropods, such as fleas and lice. The role of ticks in the transmission of Bartonella spp. is unclear. METHODS: A recently developed quadruplex polymerase chain reaction (PCR) amplicon next-generation sequencing approach that targets Bartonella-specific fragments on gltA, ssrA, rpoB, and groEL was applied to test host-seeking Ixodes scapularis ticks (n = 1641; consisting of 886 nymphs and 755 adults) collected in 23 states of the eastern half of the United States and Ixodes pacificus ticks (n = 966; all nymphs) collected in California in the western United States for the presence of Bartonella DNA. These species were selected because they are common human biters and serve as vectors of pathogens causing the greatest number of vector-borne diseases in the United States. RESULTS: No Bartonella DNA was detected in any of the ticks tested by any target. CONCLUSIONS: Owing to the lack of Bartonella detection in a large number of host-seeking Ixodes spp. ticks tested across a broad geographical region, our results strongly suggest that I. scapularis and I. pacificus are unlikely to contribute more than minimally, if at all, to the transmission of Bartonella spp.

The effect of fluralaner treatment of small mammals on the endemic cycle of Borrelia burgdorferi in a natural environment.

Among approaches aimed at reducing Lyme disease risk in the environment, those targeting reservoirs of Borrelia burgdorferi Johnson are promising because they have the potential to reduce both the density of questing Ixodes scapularis Say (Acari: Ixodidea) ticks and the prevalence of B. burgdorferi in the tick population. In this 4-yr field study, we treated a population of wild small mammals with 2 densities of fluralaner baits and investigated the effect of the treatment on 3 parameters of the endemic cycle of B. burgdorferi: (i) the prevalence of infected Peromyscus mice (PIM), (ii) the density of questing nymphs (DON), and (iii) the prevalence of infected questing nymphs (NIP). We demonstrated that fluralaner baiting is effective at reducing tick infestation of Peromyscus mice, the main reservoir of B. burgdorferi in central and northeastern North America, in the laboratory and the field. Results from this study showed a significant decrease in B. burgdorferi infection in mice (odds ratio: 0.37 [CI95: 0.17 to 0.83]). A reduction in the DON between 45.4% [CI95: 22.4 to 61.6] and 62.7% [CI95: 45.9 to 74.2] occurred in treated area when compared with control areas. No significant effect was reported on the NIP. These results confirm the hypothesis that fluralaner baits have an effect on B. burgdorferi endemic cycle, with the potential to reduce the density of B. burgdorferi-infected ticks in the environment. Further studies performed in various habitats and public health intervention contexts are needed to refine and operationalize this approach for reducing Lyme disease risk in the environment.

Life history data of four populations of Triatoma mexicana (Hemiptera: Reduviidae) from Central Mexico.

Triatomine bugs are vectors for the Trypanosoma cruzi Chagas parasites, the etiological agent for Chagas disease. This study evaluated 6 epidemiologically significant behaviors (development time, number of blood meals required for molting to the next instar, mortality rate, aggressiveness, feeding duration, and defecation delay) across 4 populations of Triatoma mexicana Herrich-Schaeffer (Heteroptera: Reduviidae), a major T. cruzi vector in Central Mexico. We collected triatomines from areas characterized by high (HP), medium (MP), medium-high (MHP), and low (LP) prevalence of human T. cruzi infection. The MHP population had the shortest development time, <290 days. Both the HP and MP populations required the most blood meals to molt to the next instar, with a median of 13. Mortality rates varied across all populations, ranging from 44% to 52%. All of the tested populations showed aggressive behavior during feeding. All populations shared similar feeding durations, with most exceeding 13 min and increasing with each instar. Quick defecation, during feeding, immediately after or less than 1 min after feeding, was observed in most nymphs (78%-90%) from the MP and MHP populations and adults (74%-92%) from HP, MP, and MHP populations. Though most parameters suggest a low potential for T. mexicana to transmit T. cruzi, unique feeding and defecation behaviors in 3 populations (excluding the LP group) could elevate their epidemiological importance. These population-specific differences may contribute to the varying prevalence rates of T. cruzi infection in areas where T. mexicana is found.

A morpho-phylogenetic update on ixodid ticks infesting cattle and buffalos in Vietnam, with three new species to the fauna and a checklist of all species indigenous to the country.

BACKGROUND: Southeast Asia is regarded as a hotspot for the diversity of ixodid ticks. In this geographical region, Vietnam extends through both temperate and tropical climate zones and therefore has a broad range of tick habitats. However, molecular-phylogenetic studies on ixodid tick species have not been reported from this country. METHODS: In this study, 1788 ixodid ticks were collected from cattle, buffalos and a dog at 10 locations in three provinces of northern Vietnam. Tick species were identified morphologically, and representative specimens were molecularly analyzed based on the cytochrome c oxidase subunit I (cox1) and 16S rRNA genes. Fifty-nine tick species that are indigenous in Vietnam were also reviewed in the context of their typical hosts in the region. RESULTS: Most ticks removed from cattle and buffalos were identified as Rhipicephalus microplus, including all developmental stages. Larvae and nymphs were found between January and July but adults until December. Further species identified from cattle were Rhipicephalus linnaei, Rhipicephalus haemaphysaloides, Amblyomma integrum and Haemaphysalis cornigera. Interestingly, the latter three species were represented only by adults, collected in one province: Son La. The dog was infested with nymphs and adults of R. linnaei in July. Phylogenetically, R. microplus from Vietnam belonged to clade A of this species, and R. haemaphysaloides clustered separately from ticks identified under this name in China, Taiwan and Pakistan. Amblyomma integrum from Vietnam belonged to the phylogenetic group of haplotypes of an Amblyomma sp. reported from Myanmar. The separate clustering of H. cornigera from Haemaphysalis shimoga received moderate support. CONCLUSIONS: Three tick species (R. linnaei, A. integrum and H. cornigera) are reported here for the first time in Vietnam, thus increasing the number of indigenous tick species to 62. Clade A of R. microplus and at least R. linnaei from the group of Rhipicephalus sanguineus sensu lato occur in the country. There is multiple phylogenetic evidence that different species might exist among the ticks that are reported under the name R. haemaphysaloides in South and East Asia. This is the first report of A. integrum in Southeastern Asia.

Ixodes ricinus as potential vector for Usutu virus.

Usutu virus (USUV) is an emerging flavivirus that is maintained in an enzootic cycle with mosquitoes as vectors and birds as amplifying hosts. In Europe, the virus has caused mass mortality of wild birds, mainly among Common Blackbird (Turdus merula) populations. While mosquitoes are the primary vectors for USUV, Common Blackbirds and other avian species are exposed to other arthropod ectoparasites, such as ticks. It is unknown, however, if ticks can maintain and transmit USUV. We addressed this question using in vitro and in vivo experiments and field collected data. USUV replicated in IRE/CTVM19 Ixodes ricinus tick cells and in injected ticks. Moreover, I. ricinus nymphs acquired the virus via artificial membrane blood-feeding and maintained the virus for at least 70 days. Transstadial transmission of USUV from nymphs to adults was confirmed in 4.9% of the ticks. USUV disseminated from the midgut to the haemocoel, and was transmitted via the saliva of the tick during artificial membrane blood-feeding. We further explored the role of ticks by monitoring USUV in questing ticks and in ticks feeding on wild birds in the Netherlands between 2016 and 2019. In total, 622 wild birds and the Ixodes ticks they carried were tested for USUV RNA. Of these birds, 48 (7.7%) carried USUV-positive ticks. The presence of negative-sense USUV RNA in ticks, as confirmed via small RNA-sequencing, showed active virus replication. In contrast, we did not detect USUV in 15,381 questing ticks collected in 2017 and 2019. We conclude that I. ricinus can be infected with USUV and can transstadially and horizontally transmit USUV. However, in comparison to mosquito-borne transmission, the role of I. ricinus ticks in the epidemiology of USUV is expected to be minor.

Neurobehavioral, neurotransmitter and redox modifications in Nauphoeta cinerea under mixed heavy metal (silver and mercury) exposure.

Heavy metals are encountered in nature, and are used in several human endeavors, including in dental fillings. It is well known that the safety of metals depends on their chemical form, as well as the dose and route through which biological systems are exposed to them. Here, we used the Nauphoeta cinerea model to examine the mechanism by which salts of the heavy metals used in dental fillings - silver and mercury - exert their neurotoxicity. Nymphs exposed to heavy metals presented with reduced motor and exploratory abilities as they spent more time immobile, especially in the periphery of a novel object, and covered less distance compared with control nymphs. Exposure to AgNO3 and HgCl2 also exacerbated levels of oxidative stress markers (MDA & ROS) and the neurotransmitter regulators - AChE and MAO, while reducing antioxidant activity markers, both in biochemical (thiol & GST) and RT-qPCR (TRX, GST, SOD, Catalase) examinations, in neural tissues of the cockroach. The observed disruptions in neurolocomotor control, synaptic transmission and redox balance explain how heavy metal salts may predispose organisms to neurological disorders.

Dual Guardians of Immunity: FoRab10 and FoRab29 in Frankliniella occidentalis Confer Resistance to Tomato Spotted Wilt Orthotospovirus.

Rab GTPase is critical for autophagy processes and is implicated in insect immunity against viruses. In this study, we aimed to investigate the role of FoRabs in the autophagic regulation of antiviral defense against tomato spotted wilt orthotospovirus (TSWV) in Frankliniella occidentalis. Transcriptome analysis revealed the downregulation of FoRabs in viruliferous nymph and adults of F. occidentalis in response to TSWV infection. Manipulation of autophagy levels with 3-MA and Rapa treatments resulted in a 5- to 15-fold increase and a 38-64% decrease in viral titers, respectively. Additionally, interference with FoRab10 in nymphs and FoRab29 in adults led to a 20-90% downregulation of autophagy-related genes, a decrease in ATG8-II (an autophagy marker protein), and an increase in the TSWV titers by 1.5- to 2.5-fold and 1.3- to 2.0-fold, respectively. In addition, the leaf disk and the living plant methods revealed increased transmission rates of 20.8-41.6 and 68.3-88.3%, respectively. In conclusion, FoRab10 and FoRab29 play a role in the autophagic regulation of the antiviral defense in F. occidentalis nymphs and adults against TSWV, respectively. These findings offer insights into the intricate immune mechanisms functional in F. occidentalis against TSWV, suggesting potential targeted strategies for F. occidentalis and TSWV management.

Functional redundancy of the three insulin receptors of cockroaches.

Gene duplication is a fundamental evolutionary process which provides opportunities to acquire new gene functions. In the case of the insulin receptors (InRs) in cockroaches and close-related insects, two successive duplications determined the occurrence of three InR genes: InR2, InR1 and InR3, the last two forming a sister cluster to InR2. The biological role of each of the gene duplicates and whether they resulted from neofunctionalization or subfunctionalization is still unclear. The analysis of the sequences from different lineages did not detect positive selection as driving the divergence of InR1 and InR3, discarding neofunctionalization, and suggesting that there is no functional divergence between both gene copies. Using the cockroach Blattella germanica as a model, we have determined that BgInR2 is the gene with the highest expression levels in all the tissues analyzed, both in adult females and males, as well as in nymphs and embryos. BgInR3 is second in expression levels while BgInR1 is expressed at lower levels and only in some tissues. The selective depletion by RNAi of each of the three InRs, analyzed in terms of phenotype and fat body transcriptomic profiles, resulted in essentially redundant effects, with a magnitude approximately proportional to the level of expression of the respective InR. Therefore, the results indicate that the InR duplicates likely experienced a subfunctionalization process, by which the three InRs maintained similar functions but contributing to those functions proportionally to their expression levels.