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Tabanids, commonly known as horseflies and belonging to the family Tabanidae, are blood-feeding arthropods (BFA) found worldwide. They are known for their ability to mechanically and biologically transmit various animal pathogens. Tabanids are potential vectors for diseases such as Francisella tularensis, Anaplasma marginale, Theileria spp., and contributors to lumpy skin diseases. Despite their involvement in common BFA studies, tabanids have not been extensively explored in microbiome research. In this study, the microbiota structure and composition in various organs of four distinct genera of tabanids: Atylotus, Haematopota, Tabanus, and Hybomitra were examined. High-throughput sequencing of the bacterial 16S rRNA gene was performed to gain insights into the microbial communities associated with the different tabanid species. Result display that microbiota composition and diversity, including Firmicutes, Proteobacteria, and Bacteroidetes, varied significantly among the different organs, with the ovaries exhibiting significantly higher diversity. Apart from the Haematopota genus, Tenericutes were enriched in the midgut of other tabanid species, whereas the Malpighian tubules exhibited a higher abundance of Bacteroides. Notably, the ovarian microbiota structure was conserved among the four tabanid species, indicating its potential association with reproductive development. Evaluation of the potential pathogen risk revealed putative pathogens in over 100 genera associated with these tabanid commensal organisms. Twenty genera were annotated as zoonotic agents with a high abundance of Citrobacter and Brucella, highlighting the presence of this important group of zoonotic pathogens. Functional predictions of vector-microbiota interactions indicate that microbiota significantly affects vector biological traits and can influence pathogen transmission via direct interactions or by regulating host immunity and nutrition. For the first time, the distribution characteristics and functions of four genera of horsefly microbiota were analyzed, revealing the presence of multiple potential pathogenic microorganisms. These findings provide valuable insights for future research and the development of symbiotic-based strategies to control insectborne diseases among tabanids. [ABSTRACT FROM AUTHOR]

The abundance of metal pollutants in freshwater habitats poses serious threats to the survival and biodiversity of aquatic organisms and human beings. This study intends for the first time to assess the pernicious influences of heavy metals in Al Marioteya canal freshwater in Egypt, compared to Al Mansoureya canal as a reference site utilizing the creeping water bug (Ilyocoris cimicoides) as an ecotoxicological model. The elemental analysis of the water showed a significantly higher incidence of heavy metals, including cadmium (Cd), cobalt (Co), chromium (Cr), nickel (Ni), and lead (Pb), in addition to the calcium (Ca) element than the World Health Organization's (WHO) permitted levels. The Ca element was measured in the water samples to determine whether exposure to heavy metals-induced oxidative stress engendered Ca deregulation in the midgut tissues of the creeping water bug. Remarkably, increased levels of these heavy metals were linked to an increase in chemical oxygen demand (COD) at the polluted site. Notably, the accumulation of these heavy metals in the midgut tissues resulted in a substantial reduction in antioxidant parameters, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and ascorbate peroxidase (APOX), along with a marked rise in malondialdehyde (MDA), cytochrome P450, and protein carbonyl levels. These results clearly indicate a noticeable disturbance in the antioxidant defense system due to uncontrollable reactive oxygen species (ROS). Notably, the results demonstrated that oxidative stress caused disturbances in Ca levels in the midgut tissue of I. cimicoides from polluted sites. Furthermore, the comet and flow cytometry analyses showed considerable proliferations of comet cells and apoptotic cells in midgut tissues, respectively, exhibiting prominent correlations, with pathophysiological deregulation. Interestingly, histopathological and ultrastructural examinations exposed noticeable anomalies in the midgut, Malpighian tubules, and ovarioles of I. cimicoides, emphasizing our findings. Overall, our findings emphasize the potential use of I. cimicoides as a bioindicator of heavy metal pollution in freshwater to improve sustainable water management in Egypt. [ABSTRACT FROM AUTHOR]

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Voltage-gated ion channels (VGICs) respond to changes in membrane potential (Vm) and typically exhibit fast kinetic properties. They play an important role in signal detection and propagation in excitable tissues. In contrast, the role of VGICs in non-excitable tissues like epithelia is less studied and less clear. Studies in epithelia of vertebrates and invertebrates demonstrate wide expression of VGICs in epithelia of animals. Recently, VGICs have emerged as regulators of ion transport in the Malpighian tubules (MTs) and other osmoregulatory organs of insects. This mini-review aims to concisely summarize which VGICs have been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects to date, and highlight select groups for further study. We have also speculated on the roles VGICs may potentially play in regulating processes connected directly to ion transport in insects (e.g., acid-base balance, desiccation, thermal tolerance). This review is not meant to be exhaustive but should rather serve as a thought-provoking collection of select existing highlights on VGICs, and to emphasize how understudied this mechanism of ion transport regulation is in insect epithelia. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cry1Ab has emerged as a bio‐insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. RESULTS: After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag‐based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up‐regulated and significantly enriched in the serine protease activity pathway, and up‐regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down‐regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein–protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt‐resistance of S. litura larvae under sublethal Cry1Ab exposure. CONCLUSION: Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Halyomorpha halys Stål (Hemiptera: Pentatomidae) is a widespread and very harmful species to many different plant species and can cause lead to great economic damage. The excretory system of H. halys is composed of four Malpighian tubules. Each Malpighian tubule is divided to region according to the external structures: proximal and distal regions. [ABSTRACT FROM AUTHOR]

Innate immunity is the immediate and exclusive defense mechanism in invertebrates. It acts against foreign agents and pathogens. In insects, this defense mechanism involves a combination of humoral and cellular responses, orchestrated by specific recognition receptors and the activation of various signaling pathways. Remarkably, Toll and Toll‐related proteins play a pivotal role in insect immunity by inducing the production of antimicrobial peptides (AMPs) in response to pathogen attacks. Toll receptors, which are type I transmembrane proteins, significantly affect insect development and the innate immune response, primarily via the nuclear factor kappa‐B signaling pathway. The Malpighian tubules of insects play a crucial role in waste elimination, hemostasis maintenance, and immune defense. The Toll signaling pathway in Malpighian tubules is mainly involved in the production of AMPs. This review provides valuable insights into the mechanisms of the Toll signaling pathway and the morphological and physiological attributes of Malpighian tubules. Moreover, it discusses the classification and functionalities of AMPs synthesized in Malpighian tubules in response to pathogens, thereby contributing to our understanding of innate immunity in insects. [ABSTRACT FROM AUTHOR]

Oestrid flies (Diptera: Oestridae) are obligate parasites of mammals during their larval stage and show anatomical adaptations for the infestation of host tissues. Unlike the species that parasitize domestic mammals, those oestrid species that infest wild mammal hosts remain poorly known. With the use of x‐ray micro‐computed tomography, we describe for the first time the anatomy of the digestive and excretory systems of the second and third larval instars of Pharyngomyia picta (Meigen), a parasite of cervids that, like other species within the subfamily Oestrinae, causes nasopharyngeal myiasis. Both larval instars of P. picta show a pair of remarkably large salivary glands arranged in a characteristic 'glandular band', a convoluted and thickly uniform midgut and a greatly enlarged distal region of the anterior pair of Malpighian tubules. These anatomical features also have been described in other species within the subfamily Oestrinae, whereas they differ from the observations in other oestrid subfamilies. We discuss the potential functional significance of the anatomy of the digestive and excretory systems of Oestrinae larvae as specific adaptations to parasitize the nasopharyngeal cavities of mammal hosts. [ABSTRACT FROM AUTHOR]

Triatoma infestans (Klug) is an insect recognized as not only an important vector of South American trypanosomiasis (Chagas disease) but also a model of specific cellular morphofunctional organization and epigenetic characteristics. The purpose of the present review is to highlight certain cellular processes that are particularly unveiled in T. infestans, such as the following: (1) somatic polyploidy involving nuclear and cell fusions that generate giant nuclei; (2) diversification of nuclear phenotypes in the Malpighian tubules during insect development; (3) heterochromatin compartmentalization into large bodies with specific spatial distribution and presumed mobility in the cell nuclei; (4) chromatin remodeling and co-occurrence of necrosis and apoptosis in the Malpighian tubules under stress conditions; (5) epigenetic markers; and (6) response of heterochromatin to valproic acid, an epidrug that inhibits histone deacetylases and induces DNA demethylation in other cell systems. These cellular processes and epigenetic characteristics emphasize the role of T. infestans as an attractive model for cellular research. A limitation of these studies is the availability of insect supply by accredited insectaries. For studies that require the injection of drugs, the operator's dexterity to perform insect manipulation is necessary, especially if young nymphs are used. For studies involving in vitro cultivation of insect organs, the culture medium should be carefully selected to avoid inconsistent results. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are integral membrane proteins that can transport water and small molecules across cell membranes in organisms. In a previous report, we identified an AQP (SlAQP1) gene of the cotton leafworm, Spodoptera littoralis. In the current study, we identify and characterize two more SlAQP genes and their developmental and tissues expression. Predicted amino acid sequence and phylogenetic analysis revealed that SlAQP2 is a glycerol channel belonging to the insect specific Eglp subfamily, whereas SlAQP3 is a water specific channel belonging to the Prip subfamily. SlAQP2 expression was detected mainly in the first four larval instars and adult males, whereas SlAQP3 was detected in all developmental stages. SlAQP2 gene expression was mainly detected in larval midgut and Malpighian tubules and adult male testes, whereas SlAQP3 was detected in all the tissues tested. RNAi mediated knockdown of each gene separately resulted in deleterious effects including larval and pupal mortality, deformed pupae and adults and prolonged development. Results were confirmed using qRT-PCR, which revealed downregulation of both genes after injection of larvae with gene specific dsRNA. Our results confirm the presence of Prip and Eglp AQPs in S. littoralis and suggest that Eglp has a role in male reproductive ability.

Abstract Background Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. Methods This study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA. Results Malpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed. Conclusions Our data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity. Graphical Abstract

The high adaptability of insects to food sources has contributed to their ranking among the most abundant and diverse species on Earth. However, the molecular mechanisms underlying the rapid adaptation of insects to different foods remain unclear. We explored the changes in gene expression and metabolic composition of the Malpighian tubules as an important metabolic excretion and detoxification organ in silkworms (Bombyx mori) fed mulberry leaf and artificial diets. A total of 2436 differentially expressed genes (DEGs) and 245 differential metabolites were identified between groups, with the majority of DEGs associated with metabolic detoxification, transmembrane transport, and mitochondrial function. Detoxification enzymes, such as cytochrome P450 (CYP), glutathione-S-transferase (GST), and UDP-glycosyltransferase, and ABC and SLC transporters of endogenous and exogenous solutes were more abundant in the artificial diet group. Enzyme activity assays confirmed increased CYP and GST activity in the Malpighian tubules of the artificial diet-fed group. Metabolome analysis showed increased contents of secondary metabolites, terpenoids, flavonoids, alkaloids, organic acids, lipids, and food additives in the artificial diet group. Our findings highlight the important role of the Malpighian tubules in adaptation to different foods and provide guidance for further optimization of artificial diets to improve silkworm breeding. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are integral membrane proteins that can transport water and small molecules across cell membranes in organisms. In a previous report, we identified an AQP (SlAQP1) gene of the cotton leafworm, Spodoptera littoralis. In the current study, we identify and characterize two more SlAQP genes and their developmental and tissues expression. Predicted amino acid sequence and phylogenetic analysis revealed that SlAQP2 is a glycerol channel belonging to the insect specific Eglp subfamily, whereas SlAQP3 is a water specific channel belonging to the Prip subfamily. SlAQP2 expression was detected mainly in the first four larval instars and adult males, whereas SlAQP3 was detected in all developmental stages. SlAQP2 gene expression was mainly detected in larval midgut and Malpighian tubules and adult male testes, whereas SlAQP3 was detected in all the tissues tested. RNAi mediated knockdown of each gene separately resulted in deleterious effects including larval and pupal mortality, deformed pupae and adults and prolonged development. Results were confirmed using qRT-PCR, which revealed downregulation of both genes after injection of larvae with gene specific dsRNA. Our results confirm the presence of Prip and Eglp AQPs in S. littoralis and suggest that Eglp has a role in male reproductive ability. [ABSTRACT FROM AUTHOR]

Tissue-specific gene promoters are desired as they provide the specificity needed for control of gene expression in transgenic animals. Here we describe a relatively rapid two-component transient expression assay that was used to identify a gene promoter active in the larval salivary glands of the green blow fly, Lucilia sericata. Sterile L. sericata maggots are widely used for wound debridement. A larval salivary gland gene promoter could be used to make maggots that secrete factors for enhanced wound therapy. Embryos from a line that carry a tetracycline transactivator (tTA)-activated red fluorescent protein gene were injected with plasmid DNA with the tTA gene driven by a constitutive or tissue-specific gene promoter. The hatched larvae were reared on diet and then examined for red fluorescence. A promoter from the LsCG30371 gene was active in the larval salivary glands. The tissue-specificity of the promoter was subsequently confirmed with stable transgenic lines that carried the LsCG3 0371-tTA gene. The relatively rapid transient expression assay could potentially be used to determine the tissue-specificity of other gene promoters. Further, the stable LsCG3 0371-tTA lines could be used to make sterile maggots that secrete factors from the salivary glands for enhanced wound healing. [Display omitted] • Tissue-specific gene promoters offer fine control of gene expression. • A tissue-specific gene promoter was identified using a transient expression assay. • The tissue-specificity of the promoter was confirmed with transgenic lines. [ABSTRACT FROM AUTHOR]

Background: Canine heartworm is a widespread and potentially fatal mosquito-borne disease caused by infections with the parasitic nematode, Dirofilaria immitis. We have previously shown that systemic activation of the Toll immune pathway via silencing of the negative regulator Cactus in Aedes aegypti blocks parasite development in the Malpighian tubules (MT), the mosquito renal organ. However, it was not established whether the MT were directly responding to Toll activation or were alternatively responding to upregulated proteins or other changes to the hemolymph driven by other tissues. Distinguishing these possibilities is crucial for developing more precise strategies to block D. immitis while potentially avoiding the fitness cost to the mosquito associated with Cactus silencing. Methods: This study defines the transcriptional response of the MT and changes to the hemolymph proteome of Ae. aegypti after systemic Toll activation via intra-thoracic injection of double-stranded Cactus (dsCactus) RNA. Results: Malpighian tubules significantly increased expression of the Toll pathway target genes that significantly overlapped expression changes occurring in whole mosquitoes. A significant overlap between the transcriptional response of the MT and proteins upregulated in the hemolymph was also observed. Conclusions: Our data show that MT are capable of RNA interference-mediated gene silencing and directly respond to dsCactus treatment by upregulating targets of the canonical Toll pathway. Although not definitive, the strong correspondence between the MT transcriptional response and the hemolymph proteomic responses provides evidence that the MT may contribute to mosquito humoral immunity. [ABSTRACT FROM AUTHOR]

Fipronil has been widely used in agriculture and forestry in Brazil to control several pests. However, this insecticide may be hazardous to non-target organisms, including stingless bees, which are essential pollinators of crops and natural environments. Here, we investigated the effect of 24-h acute oral exposure to LC50 of fipronil on the Malpighian tubules of the stingless bee Partamona helleri (Friese). Insecticide exposure decreases the respiration rate of forager bees, and the Malpighian tubules are severely affected, as shown by the epithelial architecture disorganization, loss of cytoplasmic content, degradation of the brush border, and nuclear pyknosis. In addition, fipronil ingestion increases the number of Malpighian cells positive for peroxidase, LC3, cleaved caspase-3, and JNK. However, Notch and ERK1/2-positive cells decrease in the exposed bees. These changes in the signaling proteins indicate an increase in oxidative stress, autophagy and apoptosis, and impairment of cell recovery. Overall, our results demonstrate the toxicological effects of fipronil on a stingless bee, which compromises the physiology of this important pollinator. [ABSTRACT FROM AUTHOR]

The pioneering studies carried out on heat shock-induced synthesis of specific proteins in the early 1970s did not identify any Hsp60 family protein in Drosophila. By the early 1980s, although the members of Hsp60 family of heat shock proteins (Hsp) were identified in a wide range of eukaryotes as homologs of the bacterial GroEL, none was known in Drosophila. The existence of the Hsp60 family protein was serendipitously revealed in Drosophila in my laboratory in 1989. Contrary to the earlier reports that all tissues in flies display the canonical heat shock response, the larval Malpighian tubules (MT) did not show induction of any of the major Hsps but synthesis of a putative Hsp60 family protein was found to be the most abundant in this tissue. A few years later, we identified this MT-specific heat shock-induced protein to indeed be a member of the Hsp60/chaperonin family. The Drosophila genome sequence projects subsequently revealed four putative Hsp60 gene sequences in the D. melanogaster genome. The present historical perspective chronicles contributions from my and other laboratories that unraveled several aspects of intriguing biology of the multiple Hsp60 genes in D. melanogaster, and highlights challenging questions awaiting future studies. [ABSTRACT FROM AUTHOR]

Diuretic hormones (DHs) activate corresponding G protein-coupled receptors (GPCRs), mediating the water and ion homeostasis in arthropods. There are two different DHs known to be expressed in insects, calcitonin (CT)-like DH31 and corticotropin-releasing factor (CRF)-like DH44. In this study, we identified and characterized a DH44 and five GPCR variants, DH44-R1 and DH44-R2a/b/c/d, in Drosophila suzukii (spotted-wing drosophila), causing detrimental damage to fresh and soft-skinned fruits. Among the five DH44 receptors, DH44-R1 was the longest GPCR and most strongly responded to DH44, and the other DH44-R2 splicing variants were relatively shorter and over 90 % similar to each other. Some DH44-Rs including DH44-R1 utilized both cAMP and Ca 2+ as second messengers. Interestingly, DH44-R1 was dominantly expressed in the brain, whereas DH44-R2 variants were dominant in the digestive organs, particularly the Malpighian tubules (MTs) by their gene expressions. The results suggest that DH44 may have multiple physiological functions, including the regulation of the sleep-wake cycle and diuretic activity. Injection of DH44 stimulated fluid secretion in adults, and the rate of the excretion increased in a dose-dependent manner. Moreover, when the flies were injected with a mixture of DH31 and DH44, a high mortality rate was observed. Here, we demonstrate the gene structures, expressions, characterization of DH44 and five GPCRs, their second messengers, and the effects of DH peptides on the fly. These investigations offer molecular insights into the physiological roles of the DH system and may assist in the fundamental aspects of developing D. suzukii management in the field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Host-gut microbiota interactions are more complex than good or bad. Both gut symbiotic bacteria and pathobionts can provide essential functions to their host in one scenario and yet be detrimental to host health in another. So, these gut-dwelling bacteria must be tightly controlled to avoid harmful effects on the host. However, how pathobionts and other symbiotic bacteria coordinate to establish a host immune defense system remains unclear. Here, using a Tephritidae fruit fly Bactrocera dorsalis, we report that both pathobionts and other gut symbiotic bacteria release tyramine, which is recognized by the host insects. These tyramines induce the formation of insect-conserved Malpighian tubules-gut countercurrent flow upon bacterial infection, which requires tyramine receptors and aquaporins. At the same time, pathobionts but not gut symbiotic bacteria induce the generation of reactive oxygen species, which are preserved by the countercurrent flow, promoting bacteria elimination through increasing gut peristalsis. More importantly, our results show that the Malpighian tubules-gut countercurrent flow maintains proper microbiota composition. Our work suggests a model where pathobiont-induced reactive oxygen species are preserved by Malpighian tubules-gut countercurrent flow involving both pathobionts and symbiotic bacteria. Furthermore, our work provides a Malpighian tubules-gut interaction that ensures efficient maintenance of the gut microbiota., (© The Author(s) [2024]. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Eurydema spectabilis (Heteroptera: Pentatomidae) has a piercing-sucking mouth type and feeds on plant sap. In this study, the morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis was examined using light and scanning electron microscopy. Salivary glands consist of the principal and accessory salivary glands. In E. spectabilis, digestion begins in the mouth and ends in the anus. Alimentary canal is divided into three parts: foregut, midgut, and hindgut. The foregut consists of pharynx, esophagus, and proventriculus. The esophagus connects to the proventriculus and resembles a narrow tube. The wall of the proventriculus has a recessed structure and is surrounded by a single cylindrical layer of epithelium and muscle. The midgut is divided into three regions: the first, second, and third ventricles (V1-V3). V1 and V2 consist of single-layered cylindrical epithelium. V3 contains a single layer of cuboidal epithelium. Gastric caeca were found in the midgut. The hindgut consisted of a pylorus followed by a well-developed rectum. The wall of the rectum consists of a single-layer cuboidal epithelium and muscle. Its lumen contains numerous bacteria and uric acid crystals. The pylorus consists of a single-layered cylindrical epithelium. It is also the origin of Malpighian tubules. Malpighian tubules consist of two regions: proximal and distal. The morphological structure of the salivary glands, alimentary canal, and Malpighian tubules of E. spectabilis, which has not been studied before, was examined and discussed in comparison with other orders. It is also aimed to contribute to future studies. RESEARCH HIGHLIGHTS: In E. spectabilis, the salivary glands are divided into principal and accessory salivary glands. Microvilli and numerous secretory granules were found in Malpighian tubules. Numerous uric acid crystals and bacteria were found in the rectum lumen., (© 2024 The Author(s). Microscopy Research and Technique published by Wiley Periodicals LLC.)

Triatoma infestans (Klug) is an insect recognized as not only an important vector of South American trypanosomiasis (Chagas disease) but also a model of specific cellular morphofunctional organization and epigenetic characteristics. The purpose of the present review is to highlight certain cellular processes that are particularly unveiled in T. infestans, such as the following: (1) somatic polyploidy involving nuclear and cell fusions that generate giant nuclei; (2) diversification of nuclear phenotypes in the Malpighian tubules during insect development; (3) heterochromatin compartmentalization into large bodies with specific spatial distribution and presumed mobility in the cell nuclei; (4) chromatin remodeling and co-occurrence of necrosis and apoptosis in the Malpighian tubules under stress conditions; (5) epigenetic markers; and (6) response of heterochromatin to valproic acid, an epidrug that inhibits histone deacetylases and induces DNA demethylation in other cell systems. These cellular processes and epigenetic characteristics emphasize the role of T. infestans as an attractive model for cellular research. A limitation of these studies is the availability of insect supply by accredited insectaries. For studies that require the injection of drugs, the operator’s dexterity to perform insect manipulation is necessary, especially if young nymphs are used. For studies involving in vitro cultivation of insect organs, the culture medium should be carefully selected to avoid inconsistent results.

Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites. [ABSTRACT FROM AUTHOR]

Parasites of the genus Porcisia, together with the genus Endotrypanum, form a sister clade to the species-rich and medically important genus Leishmania. Both Porcisia species, P. hertigi and P. deanei, are dixenous parasites of Neotropical porcupines. Almost 50 years after their first discovery, knowledge of their life cycle remains poor and their insect vectors are unknown. Because competent vectors of their closest phylogenetic relatives, genera Endotrypanum and Leishmania, are phlebotomine sand flies (Diptera: Psychodidae) and/or biting midges (Diptera: Ceratopogonidae), we examined here the potential of both sand flies and biting midges to transmit Porcisia parasites. The insects (Lutzomyia longipalpis, L. migonei and Culicoides sonorensis) were exposed to parasites through the chicken skin membrane and dissected at various time intervals post bloodmeal. Potentially infected females were also allowed to feed on the ears of anaesthetized BALB/c mice and the presence of parasite DNA was subsequently confirmed in the mice by PCR. Porcisia hertigi did not survive defecation in L. longipalpis or L. migonei, suggesting that these sand fly species are unlikely to serve as natural vectors of this parasite. Similarly, P. hertigi infections were lost in Culicoides midges. In contrast, mature P. deanei infections developed in 51-61% of L. longipalpis females, 7.3% of L. migonei females and 7.7% of Culicoides sonorensis females. In all three vector species, P. deanei colonized predominantly Malpighian tubules and produced metacyclic infective forms. Transmission of P. daenei to BALB/c mice was demonstrated via the prediuresis of L. longipalpis females. This mode of transmission, as well the colonization of Malpighian tubules as the dominant tissue of the vector, is unique among trypanosomatids. In conclusion, we demonstrated the vector competence of L. longipalpis for P. deanei but not for P. hertigi, and further studies are needed to evaluate competence of other Neotropical vectors for these neglected parasites.

The role of the mosquito excretory organs (Malpighian tubules, MT and hindgut, HG) in ammonia transport as well as expression and function of the Rhesus (Rh protein) ammonia transporters within these organs was examined in Aedes aegypti larvae and adult females. Immunohistological examination revealed that the Rh proteins are co-localized with V-type H+-ATPase (VA) to the apical membranes of MT and HG epithelia of both larvae and adult females. Of the two Rh transporter genes present in A. aegypti , AeRh50–1 and AeRh50–2 , we show using quantitative real-time PCR (qPCR) and an RNA in-situ hybridization (ISH) assay that AeRh50–1 is the predominant Rh protein expressed in the excretory organs of larvae and adult females. Further assessment of AeRh50–1 function in larvae and adults using RNAi (i.e. dsRNA-mediated knockdown) revealed significantly decreased [NH 4 +] (mmol l−1) levels in the secreted fluid of larval MT which does not affect overall NH 4 + transport rates, as well as significantly decreased NH 4 + flux rates across the HG (haemolymph to lumen) of adult females. We also used RNA sequencing to identify the expression of ion transporters and enzymes within the rectum of larvae, of which limited information currently exists for this important osmoregulatory organ. Of the ammonia transporters in A. aegypti , AeRh50–1 transcript is most abundant in the rectum thus validating our immunohistochemical and RNA ISH findings. In addition to enriched VA transcript (subunits A and d1) in the rectum, we also identified high Na+-K+-ATPase transcript (α subunit) expression which becomes significantly elevated in response to HEA, and we also found enriched carbonic anhydrase 9, inwardly rectifying K+ channel Kir2a , and Na+-coupled cation-chloride (Cl−) co-transporter CCC2 transcripts. Finally, the modulation in excretory organ function and/or Rh protein expression was examined in relation to high ammonia challenge, specifically high environmental ammonia (HEA) rearing of larvae. NH 4 + flux measurements using the scanning-ion selective electrode (SIET) technique revealed no significant differences in NH 4 + transport across organs comprising the alimentary canal of larvae reared in HEA vs freshwater. Further, significantly increased VA activity, but not NKA, was observed in the MT of HEA-reared larvae. Relatively high Rh protein immunostaining persists within the hindgut epithelium, as well as the ovary, of females at 24–48 h post blood meal corresponding with previously demonstrated peak levels of ammonia formation. These data provide new insight into the role of the excretory organs in ammonia transport physiology and the contribution of Rh proteins in mediating ammonia movement across the epithelia of the MT and HG, and the first comprehensive examination of ion transporter and channel expression in the mosquito rectum. [Display omitted] • Malpighian tubules & rectum secrete ammonia implicating these organs in ammonia excretion. • The A. aegypti AeRh50-1 is the predominant ammonia transporter expressed in the Malpighian tubules & rectum. • Many ammonia transporter genes are expressed in the rectum, including K+ transporters that may transport NH4+. [ABSTRACT FROM AUTHOR]

Kidney stone disease is a global problem affecting about 12% of the world population. Novel treatments to control this disease have a huge demand. Here we argue that the fruit fly, as an emerging kidney stone model, can provide a platform for the discovery of new drugs. The renal system of fruit fly (Malpighian tubules) is similar to the mammalian renal tubules in both function and structure. Different fruit fly models for different types of kidney stones including calcium oxalate (CaOx) stones, xanthine stones, uric acid stone, and calcium phosphate (CaP) stones have been successfully established through dietary or genetic approaches in the last ten years, notably improved our understanding of the formation mechanisms of kidney stone diseases. The fruit fly CaOx stones model, which is mediated by treatment with dietary lithogenic agents, is also one of the most potential models for drug development. Various potential antilithogenic agents have been identified using this model, including new chemical compounds and medicinal plants. The fruit fly kidney stone models also afford opportunities to study the therapeutic mechanism of these drugs in deeper.

Malpighamoeba mellificae is a protozoan that infects the Malpighian tubules of honey bees. The amoebae, ingested as cysts, develop into trophozoites that feed upon tubule epithelia. The resulting damage of the Malpighian tubules can induce an imbalance of waste excretion and hemolymph exchange. This causes the so-called amoebiasis disease in adult bees, which may co-occur with Nosema infections. Most reports of this amoeba are from the 1960s and earlier, and knowledge of the disease and its spreading is very poor. The lack of any genetic marker for the species hampers its sensitive identification using molecular tools and gaining knowledge on its epidemiology. Here, we present a diagnostic RT-qPCR assay, consisting of two primers and one probe that were developed based on 18S rRNA sequences of the amoeba, generated with metagenomic sequencing of Malpighian tubules with and without M. mellificae cysts. The assay was initially tested and adjusted with samples microscopically tested for the presence of M. mellificae cysts. Later, it was validated and material with unknown infection status was tested. The sensitive diagnostic Malpighamoeba disease 18S assay is now ready to be applied for honey bee health monitoring purposes and to investigate the prevalence of M. mellificae in more detail. [ABSTRACT FROM AUTHOR]

Amilenus aurantiacus overwinter in diapause, a natural starvation period, in hypogean habitats. The structure of spherites in the midgut diverticula (MD) and Malpighian tubules (MT) has been studied comparatively by light microscopy and TEM to detect eventual differences in mineral consumption in the beginning and at the end of the starvation period in these organs (MD and MT) associated with digestive processes. The chemical composition of spherites was examined by combining energy-dispersive X-ray spectroscopy (EDXS), electron energy-loss spectroscopy (EELS) and energy-filtered TEM (EFTEM). The structure of the spherites changed during overwintering in both organs. At the beginning of overwintering, the spherites were composed of densely packed concentric layers of electron-dense and electron-lucent material. In the middle and at the end of overwintering, the electron-lucent layers between the layers of material indicated the loss of some material. The chemical composition of the spherites changed only in the MD; at the beginning of overwintering, these contained Si, O, C and Fe, while later there was no more Fe. In contrast, spherites in the MT were composed of Si, O, C and Ca throughout overwintering. A less intensive exploitation of the MD spherites was probably due to complete cessation of digestive and other cell activity in this organ during the winter diapause; activity of the MT slowed, but continued removing the cell metabolites. [ABSTRACT FROM AUTHOR]

Tabanids, commonly known as horseflies and belonging to the family Tabanidae, are blood-feeding arthropods (BFA) found worldwide. They are known for their ability to mechanically and biologically transmit various animal pathogens. Tabanids are potential vectors for diseases such as Francisella tularensis, Anaplasma marginale, Theileria spp., and contributors to lumpy skin diseases. Despite their involvement in common BFA studies, tabanids have not been extensively explored in microbiome research. In this study, the microbiota structure and composition in various organs of four distinct genera of tabanids: Atylotus , Haematopota , Tabanus , and Hybomitra were examined. High-throughput sequencing of the bacterial 16S rRNA gene was performed to gain insights into the microbial communities associated with the different tabanid species. Result display that microbiota composition and diversity, including Firmicutes, Proteobacteria, and Bacteroidetes, varied significantly among the different organs, with the ovaries exhibiting significantly higher diversity. Apart from the Haematopota genus, Tenericutes were enriched in the midgut of other tabanid species, whereas the Malpighian tubules exhibited a higher abundance of Bacteroides. Notably, the ovarian microbiota structure was conserved among the four tabanid species, indicating its potential association with reproductive development. Evaluation of the potential pathogen risk revealed putative pathogens in over 100 genera associated with these tabanid commensal organisms. Twenty genera were annotated as zoonotic agents with a high abundance of Citrobacter and Brucella , highlighting the presence of this important group of zoonotic pathogens. Functional predictions of vector-microbiota interactions indicate that microbiota significantly affects vector biological traits and can influence pathogen transmission via direct interactions or by regulating host immunity and nutrition. For the first time, the distribution characteristics and functions of four genera of horsefly microbiota were analyzed, revealing the presence of multiple potential pathogenic microorganisms. These findings provide valuable insights for future research and the development of symbiotic-based strategies to control insect-borne diseases among tabanids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qiu, Lv, Wang, Ju, Luo, Liu, Na, Chang and Jiang.)

Environmental gradients cause evolutionary and developmental changes in the cellular composition of organisms, but the physiological consequences of these effects are not well understood. Here, we studied experimental populations of Drosophila melanogaster that had evolved in one of three selective regimes: constant 16 °C, constant 25 °C, or intergenerational shifts between 16 °C and 25 °C. Genotypes from each population were reared at three developmental temperatures (16 °C, 20.5 °C, and 25 °C). As adults, we measured thorax length and cell sizes in the Malpighian tubules and wing epithelia of flies from each combination of evolutionary and developmental temperatures. We also exposed flies from these treatments to a short period of nearly complete oxygen deprivation to measure hypoxia tolerance. For genotypes from any selective regime, development at a higher temperature resulted in smaller flies with smaller cells, regardless of the tissue. At every developmental temperature, genotypes from the warm selective regime had smaller bodies and smaller wing cells but had larger tubule cells than did genotypes from the cold selective regime. Genotypes from the fluctuating selective regime were similar in size to those from the cold selective regime, but their cells of either tissue were the smallest among the three regimes. Evolutionary and developmental treatments interactively affected a fly's sensitivity to short-term paralyzing hypoxia. Genotypes from the cold selective regime were less sensitive to hypoxia after developing at a higher temperature. Genotypes from the other selective regimes were more sensitive to hypoxia after developing at a higher temperature. Our results show that thermal conditions can trigger evolutionary and developmental shifts in cell size, coupled with changes in body size and hypoxia tolerance. These patterns suggest links between the cellular composition of the body, levels of hypoxia within cells, and the energetic cost of tissue maintenance. However, the patterns can be only partially explained by existing theories about the role of cell size in tissue oxygenation and metabolic performance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The dimeric cytokine ligand Spätzle (Spz) is responsible for Toll pathway activation and antimicrobial peptide (AMP) production upon pathogen challenge in Tenebrio molitor. Here, we indicated that Tm Spz5 has a functional role in response to bacterial infections. We showed that the highest expression of TmSpz5 is induced by Candida albicans. However, TmSpz5 knockdown reduced larval survival against Escherichia coli and Staphylococcus aureus. To evaluate the molecular mechanism underlying the observed survival differences, the role of TmSpz5 in AMP production was examined by RNA interference and microbial injection. T. molitor AMPs that are active against Gram-negative and -positive bacteria, including Tm tenecins, Tm attacins, Tm coleoptericins, Tm taumatin-like-proteins, and Tm cecropin-2, were significantly downregulated by TmSpz-5 RNAi in the Malpighian tubules (MTs) following a challenge with E. coli and S. aureus. However, upon infection with C. albicans the mRNA levels of most AMPs in the ds TmSpz5 -injected group were similar to those in the control groups. Likewise, the expression of the transcription factors NF-κB, TmDorX2 , and TmRelish were noticeably suppressed in the MTs of TmSpz5 -silenced larvae. Moreover, E. coli -infected TmSpz5 knockdown larvae showed decreased antimicrobial activity in the MTs and hindgut compared with the control group. These results demonstrate that Tm Spz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli. [ABSTRACT FROM AUTHOR]

Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

The stick insects or phasmids (Phamsatodea) have a series of pyriform ampulles with long, thin filaments on the posterior end of their midgut referred to as the "appendices of the midgut." Found only in phasmids, their function had never been determined until now. Their similarity to the Malpighian tubules, which are ubiquitous insect organs of excretion, suggested a similar function. To differentiate between the appendices and the Malpighian tubules and compare functional differences between the two tissue types, vital staining (the injection of histological stains into living organisms) was done in conjunction with light and scanning electron microscopy in multiple phasmid species. The results showed that the appendices originated in the basal phasmids (Timematidae) and grew more numerous in derived species. The appendices stain selectively, notably failing to pick up the indicators of the two known systems of invertebrate excretory function, indigo carmine and ammonium carmine. Appendices sequester stains in the ampule portion before eliminating the compounds into the midgut. We conclude by confirming that the appendices do have an excretory function, but one unlike any other known in invertebrates. Their function is likely cation excretion, playing a role in calcium regulation and/or organic alkaloid sequestration. The appendices must thus be considered distinct organs from the Malpighian tubules.

Triglycerides (TGs) are the main energy storage form that accommodates changing organismal energy demands. In Drosophila melanogaster, the TG lipase Brummer is centrally important for body fat mobilization. Its gene brummer (bmm) encodes the ortholog of mammalian adipose TG lipase, which becomes activated by α/β-hydrolase domain-containing 5 (ABHD5/CGI-58), one member of the paralogous gene pair, α/β-hydrolase domain-containing 4 (ABHD4) and ABHD5. In Drosophila, the pummelig (puml) gene encodes the single sequence-related protein to mammalian ABHD4/ABHD5 with unknown function. We generated puml deletion mutant flies, that were short-lived as a result of lipid metabolism changes, stored excess body fat at the expense of glycogen, and exhibited ectopic fat storage with altered TG FA profile in the fly kidneys, called Malpighian tubules. TG accumulation in puml mutants was not associated with increased food intake but with elevated lipogenesis; starvation-induced lipid mobilization remained functional. Despite its structural similarity to mammalian ABHD5, Puml did not stimulate TG lipase activity of Bmm in vitro. Rather, Puml acted as a phospholipase that localized on lipid droplets, mitochondria, and peroxisomes. Together, these results show that the ABHD4/5 family member Puml is a versatile phospholipase that regulates Drosophila body fat storage and energy metabolism.

In this study, morphological and histological studies of the alimentary canal and associated salivary gland and Malpighian tubules were conducted for the firebug Pyrrhocoris apterus (Hemiptera: Pyrrhocoridae), which is their diets are mainly linden trees and mallows. Understanding its digestive process will help elucidate the ecological and economical significance of this species. Therefore, the insects were dissected to examine all the sections under stereomicroscope, light and scanning electron microscopies. Morphological and histological results show that the salivary glands of P. apterus find in the thorax of the insect with a pair of principal glands, a pair of accessory glands, a pair of principal canals and a pair of accessory canals. The alimentary canal is divided into three regions: foregut, midgut, and hindgut. The foregut consists of oral cavity, pharynx, esophagus, and proventriculus. The esophagus consists of a long tube and is surrounded by a layer of longitudinal muscle. The proventriculus is formed by a single layer of columnar cells with oval nuclei surrounded by a layer of muscles. The midgut is divided into four regions: the first, second, third, and fourth ventriculi. The midgut is composed of monolayer epithelial cells surrounded by muscles. P. apterus midgut lacks gastric caeca. The hindgut has a pylorus followed by a well-developed rectum. The wall of the rectum is surrounded by a single layer cubic epithelium which has deep infoldings. There is a pair of Malpighian tubules linked at each side of mediolateral of pylorus. Malpighian tubules consist of two regions as proximal and distal. Malpighian tubules consist of a monolayer cuboidal epithelium with cubic nuclei in middle. [ABSTRACT FROM AUTHOR]

PURPOSE: The limitations imposed by human clinical studies and mammalian models of nephrolithiasis have hampered the development of effective medical treatments and preventive measures for decades. The simple but elegant Drosophila melanogaster is emerging as a powerful translational model of human disease, including nephrolithiasis. It may provide important information essential to our understanding of stone formation. We present the current state of research using D. melanogaster as a model of human nephrolithiasis. MATERIALS AND METHODS: We comprehensively reviewed the English language literature using PubMed®. When necessary, authoritative texts on relevant subtopics were consulted. RESULTS: The genetic composition, anatomical structure and physiological function of Drosophila malpighian tubules are remarkably similar to those of the human nephron. The direct effects of dietary manipulation, environmental alteration and genetic variation on stone formation can be observed and quantified in a matter of days. Several Drosophila models of human nephrolithiasis have been developed, including genetically linked and environmentally induced stones. A model of calcium oxalate stone formation is among the most recent fly models of human nephrolithiasis. CONCLUSIONS: The ability to readily manipulate and quantify stone formation in D. melanogaster models of human nephrolithiasis presents the urological community with a unique opportunity to increase our understanding of this enigmatic disease.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro‐computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio‐opaque concretions. Moreover, the anatomy of O. ovis third‐instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length. [ABSTRACT FROM AUTHOR]

Malpighamoeba mellificae is a protozoan that infects the Malpighian tubules of honey bees. The amoebae, ingested as cysts, develop into trophozoites that feed upon tubule epithelia. The resulting damage of the Malpighian tubules can induce an imbalance of waste excretion and hemolymph exchange. This causes the so-called amoebiasis disease in adult bees, which may co-occur with Nosema infections. Most reports of this amoeba are from the 1960s and earlier, and knowledge of the disease and its spreading is very poor. The lack of any genetic marker for the species hampers its sensitive identification using molecular tools and gaining knowledge on its epidemiology. Here, we present a diagnostic RT-qPCR assay, consisting of two primers and one probe that were developed based on 18S rRNA sequences of the amoeba, generated with metagenomic sequencing of Malpighian tubules with and without M. mellificae cysts. The assay was initially tested and adjusted with samples microscopically tested for the presence of M. mellificae cysts. Later, it was validated and material with unknown infection status was tested. The sensitive diagnostic Malpighamoeba disease 18S assay is now ready to be applied for honey bee health monitoring purposes and to investigate the prevalence of M. mellificae in more detail.

BackgroundThe mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti.Methodology/Principal FindingsUsing a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis.Conclusions/SignificanceOur results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.

Rab11, a member of Rab-GTPase family, and a marker of recycling endosomes has been reported to be involved in the differentiation of various tissues in Drosophila. Here we report a novel role of Rab11 in the differentiation of stellate cells via the non-canonical Notch pathway in Malpighian tubules. During Malpighian tubule development caudal visceral mesodermal cells intercalate into the epithelial tubule of ectodermal origin consisting of principal cells, undergo mesenchymal to epithelial transition and differentiate into star shaped stellate cells in adult Malpighian tubule. Two transcription factors, Teashirt and Cut (antagonistic to each other) are known to be expressed in stellate cells and principal cells, respectively, from early stages of development and serve as markers for these cells. Inhibition of Rab11 function or over-expression of activated Notch in stellate cells resulted in the expression of Cut that leads to down-regulation of Teashirt or vice-versa that leads to hampered differentiation of stellate cells. The stellate cells do not transform to star/bar shaped and remain in mesenchymal state in adult Malpighian tubule. Over-expression of Deltex, which plays important role in non-canonical Notch signaling pathway, shows similar phenotype of stellate cells as seen in individuals with down-regulated Rab11, while down-regulation of Deltex in genetic background of Rab11 RNAi rescues Teashirt expression and shape of stellate cells. Our experiments suggest that an inhibition or reduction of Rab11 function in stellate cells results in the faulty recycling of Notch receptors to plasma membrane as they accumulate in early and late endosomes, leading to Deltex mediated non-canonical Notch activation. • Rab11 is indispensable for maintenance of shape and required for MET of SCs. • Rab11 genetically interact with Deltex during SCs morphogenesis. • Rab11 is responsible for proper localization of Notch on early and late endosomes in SCs. [ABSTRACT FROM AUTHOR]

Bacteriocyte-associated symbionts are essential for the health of many sap-sucking insects, such as cicadas, leafhoppers and treehoppers, etc., but little is known about the bacterial community in the gut and other related organs in these insects. We characterized the bacterial communities in the salivary glands, alimentary canal and the Malpighian tubules of two populations of the cicada Subpsaltria yangi occurring in different habitats and feeding on different hosts. A high degree of similarity of core microbiota was revealed between the two populations, both with the top three bacteria belonging to Meiothermus, Candidatus Sulcia and Halomonas. The bacterial communities in various organs clustered moderately by populations possibly reflect adaptive changes in the microbiota of related S. yangi populations, which provide a better understanding of the speciation and adaptive mechanism of this species to different diets and habitats. When compared with two phylogenetically distant cicada species, Hyalessa maculaticollis and Meimuna mongolica, the core microbiota in S. yangi was significantly different to that of these species. In addition, our results confirm that Ca. Sulcia distributes in the digestive and excretory organs besides the bacteriomes and gonads, which provide potential important information onto the trophic functions of this obligate endosymbiont to the host insects. [ABSTRACT FROM AUTHOR]

Modifications in the Malpighian tubules of Rhodnius prolixus during the insect's feeding cycle and under hyperosmotic conditions were analyzed. From the time of one hour after feeding, the proximal region presents a gradual reduction in epithelium volume, with intense vacuolization in the basal region and partial disorganization of the microvilli. Twenty-four hours after feeding many vacuoles without electron-dense content are observed. At 72 hours the volume is large and the mitochondria are found spread throughout the epithelium. The distal region presents concretions of amorphous minerals with an appearance of concentric lamellae. When the tubules are submitted to hyperosmotic shock they present partial disorganization of the microvilli in the proximal region, without alteration of the basal vacuolization. In the distal region, the concretions are expelled into the lumen, together with pieces of membrane, without breaking the integrity of the epithelium, thus maintaining the parallel pattern of the basal interdigitations and suggesting that the ions present are utilized in volume regulation. These data reinforce the importance of feeding in relation to morphological modifications and regulation of cell volume in the luminal and basolateral membrane of the cells of the Malpighian tubules of R. prolixus. In this way, these observations demonstrate that in the physiology of the insect the blood supply is not only related to the nutrient acquisition but to the regulation of the cellular volume and the intense ion exchange due to the osmotic shock that this feeding triggers. [ABSTRACT FROM AUTHOR]

Drosophila metamorphosis is associated with substantial metabolic activity involving cell death and cell proliferation leading to differentiation of adult tissues and structures. Unlike other larval tissues, Malpighian tubules (MTs) exhibit apoptotic immunity and do not undergo cell death but are carried over to the adult with some cell reorganisation. They persist despite the fact that they express apoptotic proteins and caspases. In the present study, we analysed the global transcription changes in MTs and compared with salivary glands, to decipher the biology of MTs. Gene set enrichment analysis indicated reduced expression of many ecdysone induced genes, including the critical regulator of cell death, E93 in MTs. We hypothesize that reduction of E93 could be because of over expression of ecdysone oxidase, which is high in MTs and is responsible for regulation of hormone titer by degradation of ecdysone. Ectopic expression of E93 in MTs results in cell death through autophagy. Fork head, which is crucial for survival, is enriched in the MT transcriptome, and its down regulation in MTs could be consequent to over expression of E93. Together our data suggests that the cascade of events initiated by ecdysone mediates survival of MTs through concerted action of multiple factors. [ABSTRACT FROM AUTHOR]

We investigated gene expression related to endoplasmic reticulum (ER) stress, apoptosis, and autophagy to understand how Gryllus bimaculatus (two-spotted field cricket) overcomes starvation stress. Male adults were used in starvation experiments. Expression levels of genes related to ER stress, apoptosis, and autophagy were investigated for each tissue after starvation stress (consisting of 1, 3, and 6 days of starvation, and 1 and 2 days of refeeding after 6 days of starvation) by reverse-transcription PCR. Furthermore, Malpighian tubules were stained with LysoTracker Red to observe autophagy. Starvation significantly upregulated gene expression of ER chaperones in the Malpighian tubules of G. bimaculatus. Gene expression levels of BiP, ERap2, and SERCA were upregulated up to 2.5-fold, ATF6 was upregulated up to 2-fold, and PDI and Derl2 were upregulated up to 1.5-fold in the starvation group compared to controls. Although autophagy was induced by starvation, apoptosis was not. Notably, gene expression of ATG9 was upregulated up to 15-fold in the Malpighian tubules of G. bimaculatus, which is the highest among the autophagy-related genes. These results suggest that ER stress-induced autophagy appears to be acutely involved in the mechanism by which insects overcome starvation stress. [ABSTRACT FROM AUTHOR]

The non-neuronal cholinergic system, widely distributed in nature, is an ancient system that has not been well studied in insects. This study aims to investigate the key components of the cholinergic system and to identify the non-neuronal acetylcholine (ACh)-producing cells and the acting sites of ACh in the Malpighian tubules (MTs) of Mythimna separata. We found that non-neuronal ACh in MTs is synthesized by carnitine acetyltransferase (CarAT), rather than choline acetyltransferase (ChAT), as confirmed by using enzyme inhibitors and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Fluorescence in situ hybridization revealed the presence of CarAT mRNA within MTs, specifically localized in the principal cells. Immunohistochemistry showed strong staining for A-mAChR, a muscarinic acetylcholine receptor, in the principal cells. Pharmacological analysis further demonstrated that ACh acts through A-mAChR in the principal cells to increase the intracellular Ca2+ concentration. These findings provide compelling evidence for the existence of a non-neuronal cholinergic system in the MTs of M. separata , and the principal cells play a crucial role in ACh synthesis via CarAT. [Display omitted] • Discovery of a non-neuronal cholinergic system in MTs. • The ACh synthetic enzyme in the MTs differs from the one in the head. • The principal cells of the MTs produce ACh. • The action site of ACh is on the basolateral membranes of principal cells. • ACh induced an increase of [Ca2+] i via A-mAChR in MTs. [ABSTRACT FROM AUTHOR]

Abstract Background Cold tolerance is a key determinant of temperate insect distribution and performance. Chill-susceptible insects lose ion and water homeostasis during cold exposure, but prior cold acclimation improves both cold tolerance and defense of homeostasis. The mechanisms underlying these processes are mostly unknown; cold acclimation is thought to enhance ion transport in the cold and/or prevent leak of water and ions. To identify candidate mechanisms of cold tolerance plasticity we generated transcriptomes of ionoregulatory tissues (hindgut and Malpighian tubules) from Gryllus pennsylvanicus crickets and compared gene expression in warm- and cold-acclimated individuals. Results We assembled a G. pennsylvanicus transcriptome de novo from 286 million 50-bp reads, yielding 70,037 contigs (~44% of which had putative BLAST identities). We compared the transcriptomes of warm- and cold-acclimated hindguts and Malpighian tubules. Cold acclimation led to a ≥ 2-fold change in the expression of 1493 hindgut genes (733 downregulated, 760 upregulated) and 2008 Malpighian tubule genes (1009 downregulated, 999 upregulated). Cold-acclimated crickets had altered expression of genes putatively associated with ion and water balance, including: a downregulation of V-ATPase and carbonic anhydrase in the Malpighian tubules and an upregulation of Na+-K+ ATPase in the hindgut. We also observed acclimation-related shifts in the expression of cytoskeletal genes in the hindgut, including actin and actin-anchoring/stabilizing proteins, tubulin, α-actinin, and genes involved in adherens junctions organization. In both tissues, cold acclimation led to differential expression of genes encoding cytochrome P450s, glutathione-S-transferases, apoptosis factors, DNA repair, and heat shock proteins. Conclusions This is the first G. pennsylvanicus transcriptome, and our tissue-specific approach yielded new candidate mechanisms of cold tolerance plasticity. Cold acclimation may reduce loss of hemolymph volume in the cold by 1) decreasing primary urine production via reduced expression of carbonic anhydrase and V-ATPase in the Malpighian tubules and 2) by increasing Na+ (and therefore water) reabsorption across the hindgut via increase in Na+-K+ ATPase expression. Cold acclimation may reduce chilling injury by remodeling and stabilizing the hindgut epithelial cytoskeleton and cell-to-cell junctions, and by increasing the expression of genes involved in DNA repair, detoxification, and protein chaperones.

In most laboratory studies with oral exposure of terrestrial invertebrates to metals an artificial food, which is easy to handle, is used. The bioavailability of metals from this artificial food may, however, be much higher than from more field relevant food sources. Such differences may affect toxicokinetic processes in different tissues. To test the effect of bioavailability of Cd in food on Cd toxicokinetics and internal distribution in terrestrial invertebrates, we performed the experiment using the ground beetle Pterostichus oblongopunctatus exposed to Cd via food differing in their soluble Cd pool. We showed that in carabids Cd accumulation and elimination pattern in different tissues is not governed by the metal availability in food. [ABSTRACT FROM AUTHOR]

• Lepidopteran kinin Helicokinin (HK) decreased fluid secretion rate in the Malpighian tubules. • HK receptor (HK-R) identified and localized to principal cells (PCs) and secondary cells (SCs) • HK-R abundance altered in response to changes in dietary ion content. • HK reduced K+ secretion by the principal cells (PCs) and Na+ reabsorption by the SCs. • HK reduced mRNA abundance of hk-r, NKA a , nkcc, nhe-7 and -8 , and aqp-1. Excretion in insects is accomplished by the combined actions of the Malpighian tubules (MTs) and hindgut, which together form the functional kidney. MTs of many insect groups consist of principal cells (PC) and secondary cells (SC). In most insect groups SCs are reported to secrete ions from haemolymph into the tubule lumen. Paradoxically, SCs in the MTs of the lepidopteran cabbage looper T. ni are used to reabsorb Na+ and K+ back into haemolymph. The current study was designed to investigate the effects and mode of action of the lepidopteran kinin, Helicokinin (HK), on ion transport in the SC-containing region of MT of T. ni. We identified a HK receptor (HK-R) homologue in T. ni and detected its expression in the SC-containing region of the MTs. The mRNA abundance of hk-r altered in response to changes in dietary K+ and Na+ content. HK-R immunolocalized to both PCs and SCs. Ramsay assays of preparations of the isolated distal ileac plexus (DIP) indicated that [HK] = 10−8 M: (i) decreased fluid secretion rate in unstimulated and serotonin-stimulated preparations, and (ii) increased [Na+]/[K+] ratio in the secreted fluid. Scanning ion-selective electrode technique measurements revealed that HK reduced: (i) K+ secretion by the PCs, and (ii) Na+ reabsorption by the SCs in intact tubules. In vitro incubation of the DIP with HK resulted in reduced mRNA abundance of hk-r as well as Na+/K+-ATPase subunit α (NKAα) , Na+/K+/Cl− co-transporter (nkcc) , Na+/H+ exchangers (nhe) 7 and 8 , and aquaporin (aqp) 1. Taken together, results of the current study suggest that HK is capable of altering fluid secretion rate and [Na+]/[K+] ratio of the fluid, and that HK targets both PCs and SCs in the DIP of T. ni. [ABSTRACT FROM AUTHOR]

The Malpighian tubules (MTs) and hindgut together act as the functional kidney in insects. MTs of caterpillars are notably complex and consist of several regions that display prominent differences in ion transport. The distal ileac plexus (DIP) is a region of MT that is of particular interest because it switches from ion secretion to ion reabsorption in larvae fed on ion-rich diets. The pathways of solute transport in the DIP are not well understood, but one potential route is the paracellular pathway between epithelial cells. This pathway is regulated by the septate junctions (SJs) in invertebrates, and in this study, we found regional and cellular heterogeneity in the expression of several integral SJ proteins. DIP of larvae fed ion-rich diets demonstrated a reduction in paracellular permeability, coupled with alterations in both SJ morphology and the abundance of its molecular components. Similarly, treatment in vitro with helicokinin (HK), an antidiuretic hormone identified by previous studies, altered mRNA abundance of many SJ proteins and reduced paracellular permeability. HK was also shown to target a secondary cell-specific SJ protein, Tsp2A. Taken together, our data suggest that dietary ion loading, known to cause ion transport reversal in the DIP of larval Trichoplusia ni, leads to alterations in paracellular permeability, SJ morphology and the abundance of its molecular components. The results suggest that HK is an important endocrine factor that coregulates ion transport, water transport and paracellular permeability in MTs of larval lepidopterans. We propose that co-regulation of all three components of the MT function in larval lepidopterans allows for safe toggling between ion secretion and reabsorption in the DIP in response to variations in dietary ion availability. [ABSTRACT FROM AUTHOR]

Ticks are commonly infected by Coxiella -like endosymbionts (Coxiella -LE) which are thought to supply missing B vitamin nutrients required for blood digestion.While this nutritional symbiosis is essential for the survival and reproduction of infected tick species, our knowledge of where Coxiella -LE is localized in tick tissues is partial at best since previous studies have focused on a limited number of Asian or American tick species. To fill this gap, we investigated the tissue localization of Coxiella -LE in three European tick species, Ornithodoros maritimus , Dermacentor marginatus and Ixodes hexagonus , using a diagnostic fluorescence in situ hybridization (FISH) assay, combined with PCR-based detection. Specific fluorescent foci were observed in several tick tissues. We visualized a pronounced tissue tropism of Coxiella -LE for tick ovaries and Malpighian tubules, a pattern suggestive of a high degree of lifestyle specialization toward mutualism: infection of the ovaries is indicative of transovarial transmission, whereas infection of the Malpighian tubules suggests a nutritional function. We postulate that Malpighian tubules are key organs for the nutritional symbiosis, notably the synthesis of B vitamins by Coxiella -LE, whereas the infection of the ovaries ensures vertical transmission of the symbionts to future generations. We also detected occasional infections in other organs, such as salivary glands and the midgut. Finally, we discuss the potential significance of the different tissue tropism for tick biology. [ABSTRACT FROM AUTHOR]

Given that hematophagous insects ingest large quantities of blood in a single meal, they must undergo a rapid post-prandial diuresis in order to maintain homeostasis. In the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae), the coordinated activity of the Malpighian tubules and anterior midgut maintains water and ion balance during the postprandial diuresis. Three to four hours after the meal the diuretic process finishes, and the animal enters an antidiuretic state to ensure water conservation until the next blood intake. The diuretic and antidiuretic processes are tightly regulated by serotonin and neuropeptides in this insect. In the present work, we report that the neuropeptide precursor CCHamide 2 is involved in the regulation of the post-prandial diuresis in the kissing bug R. prolixus. Our results suggest a dual effect of RhoprCCHamide2 peptide, enhancing the serotonin-induced secretion by Malpighian tubules, and inhibiting serotonin-induced absorption across the anterior midgut. To our knowledge, this is the first report of a hormone presenting opposite effects in the two osmoregulatory organs (i.e. midgut and Malpighian tubules) in insects, probably reflecting the importance of a well-tuned diuretic process in hematophagous insects during different moments after the blood meal. [ABSTRACT FROM AUTHOR]