Insecticide resistance of Miami-Dade Culex quinquefasciatus populations and initial field efficacy of a new resistance-breaking adulticide formulation.

Sporadic outbreaks of human cases of West Nile virus (WNV), primarily vectored by Culex quinquefasciatus Say in suburban and urban areas, have been reported since introduction of the virus into Florida in 2001. Miami-Dade County, Florida is part of one of the largest metropolitan areas in the United States, supports Cx. quinquefasciatus year-round, and recently experienced over 60 human cases of WNV during one outbreak. To facilitate more effective integrated vector management and public health protection, we used the Centers for Disease Control and Prevention (CDC) bottle bioassay method to evaluate the susceptibility of adult Cx. quinquefasciatus collected from 29 locations throughout Miami-Dade County to pyrethroid and organophosphate adulticide active ingredients (AIs) used by Miami-Dade County Mosquito Control. We also determined the frequency of the 1014 knockdown resistance (kdr) mutation for Cx. quinquefasciatus from a subset of 17 locations. We detected resistance to two pyrethroid AIs in all tested locations (permethrin: 27 locations, deltamethrin: 28 locations). The 1014F allele was widely distributed throughout all 17 locations sampled; however, 29.4% of these locations lacked 1014F homozygotes even though phenotypic pyrethroid resistance was present. Organophosphate resistance was more variable; 20.7% of the locations tested were susceptible to malathion, and 33.3% of the populations were susceptible to naled. We subsequently conducted a field trial of ReMoa Tri, a recently approved multiple AI adulticide formulation labelled for resistant mosquitoes, against a mixed location field population of Miami-Dade Cx. quinquefasciatus. Average 24-hr mortality was 65.1 ± 7.2% and 48-hr mortality increased to 85.3 ± 9.1%, indicating good control of these resistant Cx. quinquefasciatus. This current study shows that insecticide resistance is common in local Cx. quinquefasciatus but effective options are available to maintain control during active disease transmission in Miami-Dade County.

The L1014F Knockdown Resistance Mutation Is Not a Strong Correlate of Phenotypic Resistance to Pyrethroids in Florida Populations of Culex quinquefasciatus.

Culex quinquefasciatus is an important target for vector control because of its ability to transmit pathogens that cause disease. Most populations are resistant to pyrethroids and often to organophosphates, the two most common classes of active ingredients used by public health agencies. A knockdown resistance (kdr) mutation, resulting in an amino acid change from a leucine to phenylalanine in the voltage gated sodium channel, is one mechanism contributing to the pyrethroid resistant phenotype. Enzymatic resistance has also been shown to play a very important role. Recent studies have shown strong resistance in populations even when kdr is relatively low, which indicates that factors other than kdr may be larger contributors to resistance. In this study, we examined, on a statewide scale (over 70 populations), the strength of the correlation between resistance in the CDC bottle bioassay and the kdr genotypes and allele frequencies. Spearman correlation analysis showed only moderate (-0.51) or weak (-0.29) correlation between the kdr genotype and permethrin or deltamethrin resistance, respectively. The frequency of the kdr allele was an even weaker correlate than genotype. These results indicate that assessing kdr in populations of Culex quinquefasciatus is not a good surrogate for phenotypic resistance testing.

Complete mitogenome sequence of Aedes (Hulecoeteomyia) japonicus japonicus from Hawai'i Island.

We report the first complete mitogenome (Mt) sequence of Aedes japonicus japonicus (Diptera: Culicidae). The sequence was extracted from one adult from the Big Island of Hawai'i Island. The length of the Ae. japonicus japonicus Mt was 16,528bp with 78.1% AT content. Its sequence is most similar to the Mt sequence of Aedes koreicus with 90.81% sequence identity. This is the first full Mt sequence available for this species and provides important genetic resource for studying population genetics and dynamics of this important invasive mosquito species.

Effects of selenomethionine in irradiated human thyroid epithelial cells and tumorigenicity studies.

The objectives of the present study were to characterize γ-ray, 1 GeV/n proton, and 1 GeV/n iron ion radiation-induced adverse biological effects in terms of toxicity and transformation of HTori-3 human thyroid epithelial cells; to evaluate the ability of L-selenomethionine (SeM) to protect against radiation-induced transformation when present at different times during the assay period; and to evaluate the tumorigenicity of HTori-3 cells derived from anchorage-independent colonies following iron ion radiation exposure. Cell survival was determined by a clonogenic assay, transformation was measured by a soft agar colony formation assay, and the tumorigenic potential of the cells was determined by injecting them subcutaneously into athymic nude mice and monitoring tumor formation. The results demonstrate that exposure of HTori-3 cells to γ-ray, proton, or iron ion radiation resulted in decreased clonogenic survival, which persisted for weeks after the radiation exposure. Treatment with SeM initiated up to 7 days after the radiation exposure conferred significant protection against radiation-induced anchorage-independent growth. HTori-3 cells derived from all evaluated anchorage-independent colonies formed tumors when injected into athymic nude mice, indicating that these cells are tumorigenic and that anchorage-independent colony growth is a reliable surrogate endpoint biomarker for the radiation-induced malignant transformation of HTori-3 cells.

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Multiple Novel Clades of Anopheline Mosquitoes Caught Outdoors in Northern Zambia.

Residual vector populations that do not come in contact with the most frequently utilized indoor-directed interventions present major challenges to global malaria eradication. Many of these residual populations are mosquito species about which little is known. As part of a study to assess the threat of outdoor exposure to malaria mosquitoes within the Southern and Central Africa International Centers of Excellence for Malaria Research, foraging female anophelines were collected outside households in Nchelenge District, northern Zambia. These anophelines proved to be more diverse than had previously been reported in the area. In order to further characterize the anopheline species, sequencing and phylogenetic approaches were utilized. Anopheline mosquitoes were collected from outdoor light traps, morphologically identified, and sent to Johns Hopkins Bloomberg School of Public Health for sequencing. Sanger sequencing from 115 field-derived samples yielded mitochondrial COI sequences, which were aligned with a homologous 488 bp gene segment from known anophelines (n = 140) retrieved from NCBI. Nuclear ITS2 sequences (n = 57) for at least one individual from each unique COI clade were generated and compared against NCBI's nucleotide BLAST database to provide additional evidence for taxonomical identity and structure. Molecular and morphological data were combined for assignment of species or higher taxonomy. Twelve phylogenetic groups were characterized from the COI and ITS2 sequence data, including the primary vector species Anopheles funestus s.s. and An. gambiae s.s. An unexpectedly large proportion of the field collections were identified as An. coustani and An. sp. 6. Six phylogenetic groups remain unidentified to species-level. Outdoor collections of anopheline mosquitoes in areas frequented by people in Nchelenge, northern Zambia, proved to be extremely diverse. Morphological misidentification and underrepresentation of some anopheline species in sequence databases confound efforts to confirm identity of potential malaria vector species. The large number of unidentified anophelines could compromise the malaria vector surveillance and malaria control efforts not only in northern Zambia but other places where surveillance and control are focused on indoor-foraging and resting anophelines. Therefore, it is critical to continue development of methodologies that allow better identification of these populations and revisiting and cleaning current genomic databases.

Mosquito Control Priorities in Florida-Survey Results from Florida Mosquito Control Districts.

Florida lies within a subtropical region where the climate allows diverse mosquito species including invasive species to thrive year-round. As of 2021, there are currently 66 state-approved Florida Mosquito Control Districts, which are major stakeholders for Florida public universities engaged in mosquito research. Florida is one of the few states with extensive organized mosquito control programs. The Florida State Government and Florida Mosquito Control Districts have long histories of collaboration with research institutions. During fall 2020, we carried out a survey to collect baseline data on the current control priorities from Florida Mosquito Control Districts relating to (1) priority control species, (2) common adult and larval control methods, and (3) major research questions to address that will improve their control and surveillance programs. The survey data showed that a total of 17 distinct mosquito species were considered to be priority control targets, with many of these species being understudied. The most common control approaches included truck-mounted ultra-low-volume adulticiding and biopesticide-based larviciding. The districts held interest in diverse research questions, with many prioritizing studies on basic science questions to help develop evidence-based control strategies. Our data highlight the fact that mosquito control approaches and priorities differ greatly between districts and provide an important point of comparison for other regions investing in mosquito control, particularly those with similar ecological settings, and great diversity of potential mosquito vectors, such as in Florida. Our findings highlight a need for greater alignment of research priorities between mosquito control and mosquito research. In particular, we note a need to prioritize filling knowledge gaps relating to understudied mosquito species that have been implicated in arbovirus transmission.

A Mutation in the SH2 domain of STAT2 prolongs tyrosine phosphorylation of STAT1 and promotes type I IFN-induced apoptosis.

Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.

Broad-spectrum antibiotic or G-CSF as potential countermeasures for impaired control of bacterial infection associated with an SPE exposure during spaceflight.

A major risk for astronauts during prolonged space flight is infection as a result of the combined effects of microgravity, situational and confinement stress, alterations in food intake, altered circadian rhythm, and radiation that can significantly impair the immune system and the body's defense systems. We previously reported a massive increase in morbidity with a decrease in the ability to control a bacterial challenge when mice were maintained under hindlimb suspension (HS) conditions and exposed to solar particle event (SPE)-like radiation. HS and SPE-like radiation treatment alone resulted in a borderline significant increase in morbidity. Therefore, development and testing of countermeasures that can be used during extended space missions in the setting of exposure to SPE radiation becomes a serious need. In the present study, we investigated the efficacy of enrofloxacin (an orally bioavailable antibiotic) and Granulocyte colony stimulating factor (G-CSF) (Neulasta) on enhancing resistance to Pseudomonas aeruginosa infection in mice subjected to HS and SPE-like radiation. The results revealed that treatment with enrofloxacin or G-CSF enhanced bacterial clearance and significantly decreased morbidity and mortality in challenged mice exposed to suspension and radiation. These results establish that antibiotics, such as enrofloxacin, and G-CSF could be effective countermeasures to decrease the risk of bacterial infections after exposure to SPE radiation during extended space flight, thereby reducing both the risk to the crew and the danger of mission failure.

Potential Beneficial Effects of Si-Wu-Tang on White Blood Cell Numbers and the Gastrointestinal Tract of γ-Ray Irradiated Mice.

Si-Wu-Tang (SWT) is a decoction consisting of a mixture of ingredients of Rehmanniae Radix, Angelica Radix, Chuanxiong Rhizoma and Paeoniae Radix. As a traditional Chinese herbal decoction, SWT has been widely used for the treatment of diseases characterized as blood and/or energy deficit. The present study was performed to evaluate the effects of SWT on the different populations of circulating white blood cells (WBCs) and gastrointestinal changes in γ-ray irradiated mice. Female mice were treated daily with orally administered SWT seven days before irradiation, until one day before irradiation or until one day before sample collection. WBC counts were determined from peripheral blood samples taken from the mice at different times post-irradiation. Hematoxylin and eosin (H&E) staining, as well as immunohistochemical analysis of fibrinogen, were utilized to evaluate the effects of SWT in the intestines of mice after radiation exposure. The results of the present studies demonstrate that SWT has protective effects against radiation damage to circulating WBCs, specifically to lymphocytes, and to the gastrointestinal tract of the irradiated animals.

Effect of Gender on the Radiation Sensitivity of Murine Blood Cells.

Space travel beyond the Earth's protective magnetosphere risks exposing astronauts to ionizing radiation, such as that generated during a solar particle event (SPE). Ionizing radiation has well documented effects on blood cells and it is generally assumed that these effects contribute to the hematopoietic syndrome (HS), observed in animals and humans, following exposure to total body irradiation (TBI). The purpose of the current study was to assess the role of gender on the effects of gamma radiation on blood cells. C3H/HeN mice were irradiated with a 137Cs gamma source. Radiation had similar effects on white blood cells (WBCs), lymphocytes, and granulocytes in male and female C3H/HeN mice, while red blood cell (RBC) counts and hematocrit values remained stable following radiation exposure. Non-irradiated male mice had 13% higher platelet counts, compared with their female counterparts, and showed enhanced recovery of platelets on day 16 following radiation exposure. Hence, gender differences influence the response of platelets to TBI exposure.

Effects of Solar Particle Event-Like Proton Radiation and/or Simulated Microgravity on Circulating Mouse Blood Cells.

Astronauts traveling in space missions outside of low Earth orbit will be exposed for longer times to a microgravity environment. In addition, the increased travel time involved in exploration class missions will result in an increased risk of exposure to significant doses of solar particle event (SPE) radiation. Both conditions could significantly affect the number of circulating blood cells. Therefore, it is critical to determine the combined effects of exposure to both microgravity and SPE radiation. The purpose of the present study was to assess these risks by evaluating the effects of SPE-like proton radiation and/or microgravity, as simulated with the hindlimb unloading (HU) system, on circulating blood cells using mouse as a model system. The results indicate that exposure to HU alone caused minimal or no significant changes in mouse circulating blood cell numbers. The exposure of mice to SPE-like proton radiation with or without HU treatment caused a significant decrease in the number of circulating lymphocytes, granulocytes and platelets. The reduced numbers of circulating lymphocytes, granulocytes, and platelets, resulting from the SPE-like proton radiation exposure, with or without HU treatment, in mice suggest that astronauts participating in exploration class missions may be at greater risk of developing infections and thrombotic diseases; thus, countermeasures may be necessary for these biological endpoints.

Pharmacologic suppression of JAK1/2 by JAK1/2 inhibitor AZD1480 potently inhibits IL-6-induced experimental prostate cancer metastases formation.

Metastatic prostate cancer is lethal and lacks effective strategies for prevention or treatment, requiring novel therapeutic approaches. Interleukin-6 (IL-6) is a cytokine that has been linked with prostate cancer pathogenesis by multiple studies. However, the direct functional roles of IL-6 in prostate cancer growth and progression have been unclear. In the present study, we show that IL-6 is produced in distant metastases of clinical prostate cancers. IL-6-activated signaling pathways in prostate cancer cells induced a robust 7-fold increase in metastases formation in nude mice. We further show that IL-6 promoted migratory prostate cancer cell phenotype, including increased prostate cancer cell migration, microtubule reorganization, and heterotypic adhesion of prostate cancer cells to endothelial cells. IL-6-driven metastasis was predominantly mediated by Stat3 and to lesser extent by ERK1/2. Most importantly, pharmacologic inhibition of Jak1/2 by AZD1480 suppressed IL-6-induced signaling, migratory prostate cancer cell phenotypes, and metastatic dissemination of prostate cancer in vivo in nude mice. In conclusion, we demonstrate that the cytokine IL-6 directly promotes prostate cancer metastasis in vitro and in vivo via Jak-Stat3 signaling pathway, and that IL-6-driven metastasis can be effectively suppressed by pharmacologic targeting of Jak1/2 using Jak1/2 inhibitor AZD1480. Our results therefore provide a strong rationale for further development of Jak1/2 inhibitors as therapy for metastatic prostate cancer.

Leukocyte activity is altered in a ground based murine model of microgravity and proton radiation exposure.

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.

Comparison of Two Methods for the Determination of the Effects of Ionizing Radiation on Blood Cell Counts in Mice.

A reliable technique is needed to determine the effect of ionizing radiation on white blood cell (WBC) counts. Facilities that utilize automated methods can provide this service. However, utilizing external facilities can introduce additional variables, such as differences between time of sample collection and time of sample processing, which may affect the results. The purpose of the present study was to determine whether an automated method at an external facility can accurately determine radiation-induced changes in total WBC, lymphocyte and granulocyte counts when samples are analyzed at periods of time up to 24 hours after collection and stored either at room temperature or at 4°C. To accomplish this, we compared automated blood cell counts determined at an external facility with our manual blood cell counts processed immediately after sample collection or 24 h after sample collection and stored either at room temperature or 4°C from mice exposed to 2 Gy proton or 2 Gy gamma radiation. Our results show a close correlation and good agreement between the two methods, indicating that neither a delay of 24 hours in sample processing nor storage temperature affected white blood cell counts. Analysis of the effects of radiation on blood cell counts by either manual or automated cell counts revealed a statistically significant decrease in lymphocyte and granulocyte counts at different days post-irradiation, with no statistically significant difference between the methods employed; therefore both manual and automated blood cell counts are reliable methods to determine the effects of ionizing radiation in blood cells.

Analysis of white blood cell counts in mice after gamma- or proton-radiation exposure.

In the coming decades human space exploration is expected to move beyond low-Earth orbit. This transition involves increasing mission time and therefore an increased risk of radiation exposure from solar particle event (SPE) radiation. Acute radiation effects after exposure to SPE radiation are of prime importance due to potential mission-threatening consequences. The major objective of this study was to characterize the dose-response relationship for proton and γ radiation delivered at doses up to 2 Gy at high (0.5 Gy/min) and low (0.5 Gy/h) dose rates using white blood cell (WBC) counts as a biological end point. The results demonstrate a dose-dependent decrease in WBC counts in mice exposed to high- and low-dose-rate proton and γ radiation, suggesting that astronauts exposed to SPE-like radiation may experience a significant decrease in circulating leukocytes.

Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2.

Type I IFNs (IFN-alpha/beta) are pleitropic cytokines widely used in the treatment of certain malignancies, hepatitis B and C, and multiple sclerosis. IFN resistance is a challenging clinical problem to overcome. Hence, understanding the molecular mechanism by which IFN immunotherapy ceases to be effective is of translational importance. In this study, we report that continuous IFN-alpha stimulation of the human Jurkat variant H123 led to resistance to type I IFN-induced apoptosis due to a loss of signal transducers and activators of transcription 2 (STAT2) expression. The apoptotic effects of IFN-alpha were hampered as STAT2-deficient cells were defective in activating the mitochondrial-dependent death pathway and ISGF3-mediated gene activation. Reconstitution of STAT2 restored the apoptotic effects of IFN-alpha as measured by the loss of mitochondrial membrane potential, cytochrome c release from mitochondria, caspase activation, and ultimately cell death. Nuclear localization of STAT2 was a critical event as retention of tyrosine-phosphorylated STAT2 in the cytosol was not sufficient to activate apoptosis. Furthermore, silencing STAT2 gene expression in Saos2 and A375S.2 tumor cell lines significantly reduced the apoptotic capacity of IFN-alpha. Altogether, we show that STAT2 is a critical mediator in the activation of type I IFN-induced apoptosis. More importantly, defects in the expression or nuclear localization of STAT2 could lessen the efficacy of type I IFN immunotherapy.

IFNalpha and IFNlambda differ in their antiproliferative effects and duration of JAK/STAT signaling activity.

Interferon (IFN)lambda, also known as IL-28A, IL-28B or IL-29, is a new type III IFN, which like type I IFN(alpha/beta), activates common elements of the JAK/STAT signaling pathway and exhibits antiproliferative activity. Currently, IFNalpha is used in the treatment of certain forms of cancer, but its antitumor effects are limited and associated with high toxicity. In this study, we determined whether IFNlambda induced the same level of cell growth inhibition relative to IFNalpha. To this effect HaCaT cells, which are typically growth inhibited by IFNalpha, underwent apoptosis in response to IFNlambda. Next, in contrast to IFNalpha stimulation, IFNlambda prolonged the duration of activated STAT1 and STAT2. Furthermore, the kinetics of IFN-stimulated genes was different as IFNlambda induced a delayed but stronger induction of IFN-responsive genes. Components of the JAK/STAT pathway remained essential for the antiproliferative effects of IFNalpha and IFNlambda. IFNlambda-induced persistence of STAT activation required de novo protein synthesis and was in part due to a delay in STAT2 inactivation. Thus our data demonstrate that the duration of IFNlambda signaling is different from that of IFNalpha, and that IFNlambda could be a suitable cytokine to evaluate for cancer therapy.