Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Publishing in the Open Access and Open Science era.

Our research activities would be better served if they were communicated in a manner that is openly accessible to the public and all researchers. The research we share is often limited to representative data included in research papers-science would be much more efficient if all reproducible research data were shared alongside detailed methods and protocols, in the paradigm called Open Science. On the other hand, one primary function of research journals is to select manuscripts of good quality, verify the authenticity of the data and its impact, and deliver to the appropriate audience for critical evaluation and verification. In the current paradigm, where publication in a subset of journals is intimately linked to research evaluation, a hypercompetitive "market" has emerged where authors compete to access a limited number of top-tier journals, leading to high rejection rates. Competition among publishers and scientific journals for market dominance resulted in an increase in both the number of journals and the cost of publishing and accessing scientific papers. Here we summarize the current problems and potential solutions from the development of AI technology discussed in the seminar at the 46th Annual Meeting of the Molecular Biology Society of Japan.

Key Features and Considerations for Using Automated Long-Term Monitoring of Heat-Seeking Behavior of Mosquitoes.

Mosquitoes are vectors of various deadly infectious diseases, including malaria and dengue fever. Because these pathogens are transmitted by mosquito blood-feeding behavior, it is important to understand how mosquitoes are attracted to their hosts and how they feed on blood. The simplest method is to observe their behavior with the naked eye or video recording. Furthermore, various devices have been invented to assay mosquito behavior, such as olfactometers. Although each method has distinctive advantages, all have disadvantages, such as limitations in the number of individuals that can be assayed at one time and in observable durations, objective quantification methods, and others. To solve these problems, we have developed an automated device to quantify the carbon dioxide-activated heat-seeking behavior of Anopheles stephensi and Aedes aegypti with continuous monitoring for up to 1 week. This device, which is detailed in an accompanying protocol, can be applied to search for substances and molecules that affect heat-seeking behavior. It may also be applicable to other hematophagous insects.

Automated Long-Term Monitoring of the Heat-Seeking Behavior of Mosquitoes.

Heat is one of the critical cues female mosquitoes use to find vertebrate hosts, particularly in the final step of attraction (touching down on hosts) before the initiation of blood-sucking. To prevent vector-borne diseases such as malaria and dengue fever, which are transmitted via mosquito blood-sucking, it is essential to understand the dynamics and mechanisms of the heat-seeking behavior of mosquitoes. An automated device to quantify CO2-activated heat-seeking behavior with continuous monitoring for up to a week was established. The device is based on the infrared beam break method; it can simultaneously monitor three independent mosquito behaviors (touchdown on a heated target, feeding, and locomotion activity) by using several pairs of infrared laser sensors. This protocol describes brief instructions for the device's construction, directions for use, and possible issues and solutions.

Molecular action of larvicidal flavonoids on ecdysteroidogenic glutathione S-transferase Noppera-bo in Aedes aegypti.

BACKGROUND: Mosquito control is a crucial global issue for protecting the human community from mosquito-borne diseases. There is an urgent need for the development of selective and safe reagents for mosquito control. Flavonoids, a group of chemical substances with variable phenolic structures, such as daidzein, have been suggested as potential mosquito larvicides with less risk to the environment. However, the mode of mosquito larvicidal action of flavonoids has not been elucidated. RESULTS: Here, we report that several flavonoids, including daidzein, inhibit the activity of glutathione S-transferase Noppera-bo (Nobo), an enzyme used for the biosynthesis of the insect steroid hormone ecdysone, in the yellow fever mosquito Aedes aegypti. The crystal structure of the Nobo protein of Ae. aegypti (AeNobo) complexed with the flavonoids and its molecular dynamics simulation revealed that Glu113 forms a hydrogen bond with the flavonoid inhibitors. Consistent with this observation, substitution of Glu113 with Ala drastically reduced the inhibitory activity of the flavonoids against AeNobo. Among the identified flavonoid-type inhibitors, desmethylglycitein (4',6,7-trihydroxyisoflavone) exhibited the highest inhibitory activity in vitro. Moreover, the inhibitory activities of the flavonoids correlated with the larvicidal activity, as desmethylglycitein suppressed Ae. aegypti larval development more efficiently than daidzein. CONCLUSION: Our study demonstrates the mode of action of flavonoids on the Ae. aegypti Nobo protein at the atomic, enzymatic, and organismal levels.

A simple and affordable method for estimating the fluid volume a mosquito sucks using food dyes.

BACKGROUND: Blood-sucking by mosquitoes is an inevitable behavior when pathogens are transmitted among humans. Adenine nucleotides such as ATP are known as phagostimulants for mosquitoes and are widely used to induce and enhance the blood-sucking activity in an artificial manner. Although using ATP solution is convenient to introduce a variety of substances (for example chemicals and pathogens) into the mosquito body via sucking, establishing an easy and cost-effective method to quantify the amount of solution ingested has yet to be reported. RESULTS: A set of commercial food dyes (green, blue, yellow, and red) was employed in this study. Each dye was added to ATP solution used to colorize the abdomen of Ae. aegypti female mosquitoes after ingestion. The intake of food dyes did not show any toxicity to the mosquitoes, affecting neither ATP-sucking behavior nor survival of the mosquitoes. We observed that quantifying the color intensity of green dye in the mosquito abdomen by spectral analysis, as well as distinguishing the size of the colored abdomen using the naked eye, allowed the estimation of the amount of ingested solution. Using this method, capsaicin, a pungent component of chili peppers, was identified as an aversive tastant that can discourage mosquitoes from sucking the ATP solution. CONCLUSIONS: Employing commercially available, non-toxic food dyes converted ATP-driven membrane feeding into an easy-to-use method to estimate the amount of solution ingested by mosquitoes. This method can be further applied for a variety of experiments such as introducing a certain quantity of chemical compounds or microbes into the mosquito body.

The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses.

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.

A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability.

During neural development, regulation of microtubule stability is essential for proper morphogenesis of neurons. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was revealed to be involved in diverse cellular processes. However, there is little evidence that STRIPAK components regulate microtubule dynamics, especially in vivo. Here, we show that one of the core STRIPAK components, Strip, is required for microtubule organization during neuronal morphogenesis. Knockdown of Strip causes a decrease in the level of acetylated α-tubulin in Drosophila S2 cells, suggesting that Strip influences the stability of microtubules. We also found that Strip physically and genetically interacts with tubulin folding cofactor D (TBCD), an essential regulator of α- and ß-tubulin heterodimers. Furthermore, we demonstrate the genetic interaction between strip and Down syndrome cell adhesion molecule (Dscam), a cell surface molecule that is known to work with TBCD. Thus, we propose that Strip regulates neuronal morphogenesis by affecting microtubule stability.

Linking cell surface receptors to microtubules: tubulin folding cofactor D mediates Dscam functions during neuronal morphogenesis.

Formation of functional neural networks requires the coordination of cell surface receptors and downstream signaling cascades, which eventually leads to dynamic remodeling of the cytoskeleton. Although a number of guidance receptors affecting actin cytoskeleton remodeling have been identified, it is relatively unknown how microtubule dynamics are regulated by guidance receptors. We used Drosophila olfactory projection neurons to study the molecular mechanisms of neuronal morphogenesis. Dendrites of each projection neuron target a single glomerulus of ∼50 glomeruli in the antennal lobe, and the axons show stereotypical pattern of terminal arborization. In the course of genetic analysis of the dachsous mutant allele (ds(UAO71)), we identified a mutation in the tubulin folding cofactor D gene (TBCD) as a background mutation. TBCD is one of five tubulin-folding cofactors required for the formation of α- and ß-tubulin heterodimers. Single-cell clones of projection neurons homozygous for the TBCD mutation displayed disruption of microtubules, resulting in ectopic arborization of dendrites, and axon degeneration. Interestingly, overexpression of TBCD also resulted in microtubule disruption and ectopic dendrite arborization, suggesting that an optimum level of TBCD is crucial for in vivo neuronal morphogenesis. We further found that TBCD physically interacts with the intracellular domain of Down syndrome cell adhesion molecule (Dscam), which is important for neural development and has been implicated in Down syndrome. Genetic analyses revealed that TBCD cooperates with Dscam in vivo. Our study may offer new insights into the molecular mechanism underlying the altered neural networks in cognitive disabilities of Down syndrome.

Drosophila Strip serves as a platform for early endosome organization during axon elongation.

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. Here we show that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis.

Development of olfactory projection neuron dendrites that contribute to wiring specificity of the Drosophila olfactory circuit.

The antennal lobe (AL) of Drosophila is the first olfactory processing center in which olfactory input and output are spatially organized into distinct channels via glomeruli to form a discrete neural map. In each glomerulus, the axons of a single type of olfactory receptor neurons (ORNs) synapse with the dendrites of a single type of projection neurons (PNs). The AL is an ideal place to study how the wiring specificity between specific types of ORNs and PNs is established during development. During the past two decades, the involvement of diverse molecules in the specification and patterning of ORNs and PNs has been reported. Furthermore, local interneurons-another component of glomeruli-have been recently catalogued and their functions have been gradually dissected. Although there is accumulating knowledge about the involvement of these three cell types in the wiring specificity of the olfactory system, in this review, we focus especially on the development of PN dendrites.