Expression, activity, and consequences of biochemical inhibition of α- and ß-glucosidases in different life stages of Culex quinquefasciatus.

Mosquitoes have a wide range of digestive enzymes that enable them to utilize requisite blood and sugar meals for survival and reproduction. Sugar meals, typically derived from plant sources, are critical to maintain energy in both male and female mosquitoes, whereas blood meals are taken only by females to complete oogenesis. Enzymes involved in sugar digestion have been the subject of study for decades but have been limited to a relatively narrow range of mosquito species. The southern house mosquito, Culex quinquefasciatus, is of public health importance and seldom considered in these types of studies outside of topics related to Bacillus sphaericus, a biocontrol agent that requires interaction with a specific gut-associated α-glucosidase. Here we sought to describe the nature of α-glucosidases and unexplored ß-glucosidases that may aid Cx. quinquefasciatus larvae in acquiring nutrients from cellulosic sources in their aquatic habitats. Consistent with our hypothesis, we found both α- and ß-glucosidase activity in larvae. Interestingly, ß-glucosidase activity all but disappeared at the pupal stage and remained low in adults, while α-glucosidase activity remained in the pupal stage and then exceeded larval activity by approximately 1.5-fold. The expression patterns of the putative α- and ß-glucosidase genes chosen did not consistently align with observed enzyme activities. When the α-glucosidase inhibitor acarbose was administered to adults, mortality was seen especially in males but also in females after two days of exposure and key energetic storage molecules, glycogen and lipids, were significantly lower than controls. In contrast, administering the ß-glucosidase inhibitor conduritol ß-epoxide to larvae did not produce mortality even at the highest soluble concentration. Here we provide insights into the importance of α- and ß-glucosidases on the survival of Cx. quinquefasciatus in their three mobile life stages.

Using environmental factors to predict Rhipicephalus sanguineus s.l. (Acari: Ixodidae) mortality.

BACKGROUND: Rhipicephalus sanguineus s.l. (Latreille, 1806) can establish indoor populations, which increases the risk of pathogen transmission to humans and companion dogs. Rhipicephalus sanguineus s.l. ticks spend most of their life cycle off the host, which subjects developmental timescale to abiotic factors. Previous studies showed that both temperature and relative humidity (RH) influenced Rhipicephalus sanguineus s.l. survival time across all life stages. However, quantified relationships between environmental factors and Rhipicephalus sanguineus s.l. mortality is not currently available. Here, three Rhipicephalus sanguineus s.l. strains were evaluated for mortality under 20 combinations of five temperatures and four RHs. The data obtained were analyzed to quantify the relationship between environmental factors and Rhipicephalus sanguineus s.l. RESULTS: Mortality probabilities did not show a consistent pattern between the three tick strains. Temperature, RH, and their interaction influenced Rhipicephalus sanguineus s.l. mortality probabilities across all life stages, with mortality probability generally increasing with temperature but decreasing with RH. With 50% and lower RH, larvae cannot survive for more than 1 week. However, mortality probabilities in all strains and stages were more sensitive to temperature than to RH. CONCLUSION: This study identified the predictive relationship between environmental factors and Rhipicephalus sanguineus s.l. survival, which enables estimations of tick survival time under varied residential situations, allows parameterization of population models, and provides guidance for pest control professionals to develop efficient management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evidence of Permethrin Resistance and Fipronil Tolerance in Rhipicephalus sanguineus s.l. (Acari: Ixodidae) Populations From Florida and California.

Rhipicephalus sanguineus s.l. (Latreille), is a vector of multiple disease-causing pathogens to humans and dogs. Permethrin and fipronil are two acaricides used to manage R. sanguineus s.l. infestations. Homeowners have reported treatment failures in managing brown dog ticks using permethrin and fipronil based products. Previous studies demonstrated that high permethrin resistance in some R. sanguineus s.l. populations was due to metabolic detoxification and target site insensitivity. In this study, three R. sanguineus s.l. strains, one from a laboratory colony (NC) and two colonies originally collected from Florida (FL) and California (CA), were evaluated for resistance expression against permethrin and fipronil. Metabolic detoxification mechanisms were evaluated in the FL strain using three synergists, while a polymerase chain reaction assay was used to detect a resistance mutation in all strains. The NC strain was susceptible to both permethrin and fipronil, while both the FL and CA strains exhibited high resistance to permethrin and tolerance to fipronil. The synergist tests and PCR results indicated that the FL strain utilized both metabolic resistance and target site insensitivity against permethrin, while the CA strain was documented to have the target-site insensitivity resistant allele. This study confirmed permethrin resistance in both California and Florida populations and its persistence in Florida populations, although its susceptibility can potentially be increased by adding a synergist. Fipronil resistance was not detected suggesting this acaricide may provide suitable tick control.

Variable Effects of Temperature and Relative Humidity on Rhipicephalus sanguineus s.l. (Acari: Ixodidae) Development.

Rhipicephalus sanguineus s.l. (Latreille, 1806) can establish populations in residences and may lead to severe domestic and peridomestic infestations. Detection in the early infestation stage is challenging because of their small body size and the lack of visibility when ticks stay in sheltered refugia. The residents may believe that the infestation has been eliminated when no ticks are observed until ticks reappear when seeking hosts. Thus, it is necessary to improve our understanding of tick phenology to achieve more effective infestation management. In this study, the relationships between environmental conditions and tick development were explored in laboratory and using linear and nonlinear models. Three R. sanguineus s.l. strains, from one colony of the temperate lineage and two of the tropical lineage, were evaluated for the development of all life stages and conversion efficiency index (CEI) under five temperatures and four relative humidities (RHs). The development times differed between the three tick strains across stages and were primarily dependent on temperature. The CEIs had little variance explained by temperature, RH, or strains. Compared with the linear and exponential models with temperature as the only variable, the Brière-1 model was the best approximating model for most of the developmental rates. The developmental temperature thresholds for R. sanguineus s.l. development estimated by the Brière-1 model varied inconsistently across strains and life stages. We developed a more predictive relationship between environmental factors and R. sanguineus s.l. development, which can be utilized to predict tick development using temperature and develop appropriate control strategies.

The effects of habitat type and pathogen infection on tick host-seeking behaviour.

Tick-borne pathogens pose a significant risk to livestock, wildlife and public health. Host-seeking behaviours may depend on a combination of infection status and environmental factors. Here, we assessed the effects of habitat type and pathogen infection on host-seeking behaviour (questing) in the lone star tick, Amblyomma americanum. Ticks were collected using a tick drag from two different habitat types: xeric hammock and successional hardwood forests. Using a standardized assay, we recorded the likelihood of questing for each tick, the average height quested and total time spent questing and then tested each tick for the presence of Rickettsia spp. and Ehrlichia spp. using conventional polymerase chain reaction. We did not detect Ehrlichia in any ticks, although 30% tested positive for Rickettsia amblyommatis, a member of the Rickettsia spotted fever group. Ticks infected with R. amblyommatis spent less time questing compared to uninfected ticks, with infected ticks spending 85 s on average questing and uninfected ticks spending 112 s. Additionally, ticks collected from xeric hammock habitats spent over twice as long questing compared to ticks from successional hardwood forests. Ticks from xeric hammock spent 151 s on average questing while ticks from successional hardwood forest spent only 58 s during a 10-min observation period. These results demonstrate that habitat type and infection status can influence tick host-seeking behaviours, which can play a pivotal role in disease dynamics.

Combining DL-Methionine and Bacillus thuringiensis Subspecies israelensis: Prospects for a Mosquito Larvicide.

Mosquito larvicides can reduce mosquito populations at the source, potentially decreasing biting rates and pathogen transmission. However, there is a growing need for mosquito larvicides that are environmentally sustainable. Bacillus thuringiensis subspecies israelensis (BTI) is a naturally occurring bacterium commonly used as a larvicide to manage mosquito populations. Methionine is an essential amino acid that has demonstrated toxic properties against larval mosquitoes in laboratory experiments, while having minimal effects on non-target organisms. The goal of this study was to evaluate the potential for a novel combination larvicide by testing for compatibility between these two active ingredients. We began by determining the lethal concentration values (LCs) of BTI and DL-methionine against Anopheles quadrimaculatus Say and Aedes aegypti Linnaeus (Diptera: Culicidae) larvae. These bioassays were conducted in glass jars and mortality was observed 48 h post-treatment. We found that while DL-methionine was more toxic to An. quadrimaculatus than Ae. aegypti, the opposite was true for BTI. Then, we used these LCs to conduct bioassays with a combination of BTI and DL-methionine to determine the relationship between the two active ingredients when used against An. quadrimaculatus and Ae. aegypti larvae. The findings of this study demonstrate that BTI and DL-methionine have the potential to be complementary due to their additive properties at higher concentrations and effect levels when tested against An. quadrimaculatus. However, an antagonistic relationship was detected at the concentrations tested with Ae. aegypti. These results are encouraging and imply that a DL-methionine or BTI/DL-methionine combination larvicide could be used in management of Anopheles species.

Delivery of gene-specific dsRNA by microinjection and feeding induces RNAi response in Sri Lanka weevil, Myllocerus undecimpustulatus undatus Marshall.

BACKGROUND: RNA interference (RNAi) is widely used in entomological research for functional analysis of genes and is being considered as a new tool for insect pest management. Sri Lanka weevil (SLW) is a highly polyphagous pest of agronomically important plants, but currently only a few control methods are available for this insect. RESULTS: In the present study, we evaluated the stability of candidate reference genes ß-ACT, α-TUB, EF1-α, RPL12 and GAPDH, and identified EF1-α as the most reliable for gene expression normalization. Four target genes involved in different cellular processes, including Prosα2, RPS13, Snf7 and V-ATPase A were selected to evaluate whether RNAi response in SLW adults can be triggered by microinjection and oral feeding of their double-stranded RNAs (dsRNAs). Three days after injection of the dsRNAs for the target genes, their transcript levels were significantly reduced (up to 91.4%) when compared to the control. Additionally, weevils fed with the target dsRNAs showed significant decreases in gene transcript levels and significant mortality was observed in insects treated with Prosα2 and Snf7 dsRNAs (78.6 to 92.7%). CONCLUSION: Our data demonstrate that microinjection and feeding of dsRNA produce a strong RNAi response in SLW, indicating that RNAi-based strategies could be explored to develop a selective and environmentally safe control method against SLW. © 2019 Society of Chemical Industry.

CRISPR/Cas9 mediated knockout of the abdominal-A homeotic gene in fall armyworm moth (Spodoptera frugiperda).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is an important pest of maize in the Americas and has recently been introduced into Africa. Fall armyworm populations have developed resistance to control strategies that depend on insecticides and transgenic plants expressing Bacillus thuringiensis toxins. The study of various resistance mechanisms at the molecular level and the development novel control strategies have been hampered by a lack of functional genomic tools such as gene editing in this pest. In the current study, we explored the possibility of using the CRISPR/Cas9 system to modify the genome of FAW. We first identified and characterized the abdominal-A (Sfabd-A) gene of FAW. Sfabd-A single guide RNA (sgRNA) and Cas9 protein were then injected into 244 embryos of FAW. Sixty-two embryos injected with Sfabd-A sgRNA hatched. Of these hatched embryos, twelve developed into larvae that displayed typical aba-A mutant phenotypes such as fused segments. Of the twelve mutant larvae, three and five eventually developed into female and male moths, respectively. Most mutant moths were sterile, and one female produced a few unviable eggs when it was outcrossed to a wild-type male. Genotyping of 20 unhatched Sfabd-A sgRNA-injected embryos and 42 moths that developed from Sfabd-A sgRNA-injected embryos showed that 100% of the unhatched embryos and 50% of the moths contained indel mutations at the Sfabd-A genomic locus near the guide RNA target site. These results suggest that the CRISPR/Cas9 system is highly efficient in editing FAW genome. Importantly, this gene editing technology can be used to validate gene function to facilitate an understanding of the resistance mechanism and lead to the development of novel pest management approaches.

Rapid and persistent RNAi response in western corn rootworm adults.

RNA interference (RNAi) has proven effective for controlling pest insects such as western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Previous studies have shown that WCR adults display a robust RNAi response to orally-administered double-stranded RNA (dsRNA). However, it is unclear how quickly the response occurs after ingestion or how long RNAi effect lasts after WCR stop ingesting diet containing dsRNA. In the current study, WCR adult females were provided with diet treated with dsRNAs of Laccase 2 and Argonaute 2, two nonessential genes, for 8 days. RNAi response in WCR females commenced as early as 10 h after exposure to dsRNA and lasted up to 40 days after exposure to dsRNA ended. Our results show that dsRNA-mediated RNAi response in WCR females is rapid and long-lasting. These findings suggest that even a short-term ingestion of transgenic plants expressing dsRNA by WCR may have a sustained impact on this insect.

Multidisciplinary Approaches to Chest Wall Recurrences of Breast Cancer.

The management of postmastectomy chest wall recurrences of breast cancer has long challenged clinicians. A tissue diagnosis combined with proper imaging and staging of patients to ensure the disease is localized are the first steps in management. Multimodal therapy offers patients the best chances of cure. In properly selected patients, complete surgical resection to negative margins, including full-thickness chest wall resection when required, followed by reconstruction that is well planned, can provide local control with very low surgical mortality and acceptable morbidity. Radiation therapy provides additional local control, while systemic therapy is an adjunct that prolongs survival in many cases. Multidisciplinary care combined with careful patient selection are the keys to successful chest wall resection for locally recurrent breast cancer after mastectomy.

Distinct fitness costs associated with the knockdown of RNAi pathway genes in western corn rootworm adults.

RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.

Management of Breast Cancer Therapy-Related Sexual Dysfunction.

Sexual dysfunction is a prevalent concern among breast cancer survivors, and is often multifactorial in nature. Causes can be both physiologic and psychological; chemotherapy, endocrine therapy, and disfigurement from breast cancer surgery can all play a role. Sexual dysfunction can have a profound impact on quality of life and relationships and can also impact adherence to endocrine therapy. However, it can be a difficult topic for both patients and providers to bring up, and as a result, it is often inadequately addressed. Here, we will review the mechanisms of sexual dysfunction that occurs after breast cancer, offer some practical guidance on how to discuss this effectively with patients, and review the data and our recommendations for optimizing management.

Manure Preferences and Postemergence Learning of Two Filth Fly Parasitoids, Spalangia cameroni and Muscidifurax raptor (Hymenoptera: Pteromalidae).

The efficiency of host-seeking behavior is crucial to the reproductive performance of female parasitoids. Initially, parasitoids may use chemical information garnered from the microhabitat in which they emerge to locate hosts. Spalangia cameroni and Muscidifurax raptor are commercially available parasitoids of filth flies. Postemergence exposure to a specific manure may provide a way to increase parasitism in specific microhabitats found at livestock facilities upon release. In this study, female parasitoids of both species were exposed to equine manure, bovine manure, or clean pupae. Females from each emergence exposure were tested in a two-choice arena (house fly hosts in bovine manure versus clean pupae, equine manure versus clean pupae, and equine manure versus bovine manure) for progeny production. There was a detectable but small effect of postemergence exposure on S. cameroni, but it was not sufficient to reverse innate preferences. Females consistently produced more progeny in hosts found in any manure over clean pupae, and in equine manure over bovine manure. The effect of postemergence exposure on M. raptor was also detectable but small. Females produced equal numbers of progeny in bovine manure versus clean pupae, as opposed to preferring to oviposit in clean pupae as with all other treatments. Preferences by M. raptor were overall less marked than for S. cameroni; indeed most of the variability observed for this species did not result from the treatment design. Residual host mortality was also detectably altered by exposure in both species, but the effect was small. Thus, postemergence exposure did not consistently and effectively manipulate these parasitoids to producing progeny in different exposure manures, suggesting that microhabitat preferences are largely determined by other factors.