A Malpighian Tubule-Specific P450 Gene SlCYP9A75a Contributes to Xenobiotic Tolerance in Spodoptera litura.

Phytophagous insects are more predisposed to evolve insecticide resistance than other insect species due to the "preadaptation hypothesis". Cytochrome P450 monooxygenases have been strongly implicated in insecticide and phytochemical detoxification in insects. In this study, RNA-seq results reveal that P450s of Spodoptera litura, especially the CYP3 clan, are dominant in cyantraniliprole, nicotine, and gossypol detoxification. The expression of a Malpighian tubule-specific P450 gene, SlCYP9A75a, is significantly upregulated in xenobiotic treatments except α-cypermethrin. The gain-of-function and loss-of-function analyses indicate that SlCYP9A75a contributes to cyantraniliprole, nicotine, and α-cypermethrin tolerance, and SlCYP9A75a is capable of binding to these xenobiotics. This study indicates the roles of inducible SlCYP9A75a in detoxifying man-made insecticides and phytochemicals and may provide an insight into the development of cross-tolerance in omnivorous insects.

Cytotoxic and genotoxic effects of a pendimethalin-based herbicide in Apis mellifera.

Public concern about the effects of pesticides on non-target organisms has increased in the recent years. Nevertheless, there is a limited number of studies that address the actual toxic effects of herbicides on insects. This study investigated the side effects of herbicides on non-target organisms inhabiting agroecosystems and performing essential ecological and economic functions such as crop pollination. We analysed morphological alterations in the gut, Malpighian tubules and circulating haemocytes of Apis mellifera workers as markers of exposure effects. A commercial formulation of a pendimethalin-based herbicide (PND) was administered orally under laboratory conditions at a realistic concentration admitted in the field (330gL-1 of active ingredient., 4 L ha-1 for cereal and vegetable crops). The worker bees were exposed to a single application of PND for a period of one week, to simulate the exposure that can occur when foraging bees accidentally drink drops of contaminated water upon treatments. Histopathological analyses of the midgut, ileum and Malpighian tubules showed alterations over time (from 24 to 72 h after the beginning of exposure) such as loss of epithelial organisation, cellular vacuolisation and altered pyknotic nuclei as well as disruption of the peritrophic membrane over time. Semiquantitative analyses of the midgut showed a significant increase in the organ injury index 24 and 72 h after the initial exposure in PND-exposed bees compared to control bees. In addition, a change in positivity to Gram staining was observed in the midgut histological sections. A recovery of cytotoxic effects was observed one week after the initial exposure, which was favoured by the periodic renewal of the intestinal epithelium and the herbicide dissipation time. Cytochemical staining with Giemsa of haemocytes from PND-treated workers over 24 and 72 h showed significant nuclear alterations such as lobed or polymorphic nuclei and micronuclei compared to bees in the control group. These results show that the dose of PND used to protect crops from weeds can lead to significant cytotoxic and genotoxic effects in non-target organisms such as honey bees. In croplands, the sublethal effects on cell morphology can impair vital physiological processes such as nutrition, osmoregulation, and resistance to pathogens, contributing to the decline in biodiversity and abundance of species that play a prominent ecological role, such as pollinators.

Ammonia transport in the excretory system of mosquito larvae (Aedes aegypti): Rh protein expression and the transcriptome of the rectum.

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 [NH4+] (mmol l-1) levels in the secreted fluid of larval MT which does not affect overall NH4+ transport rates, as well as significantly decreased NH4+ 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. NH4+ flux measurements using the scanning-ion selective electrode (SIET) technique revealed no significant differences in NH4+ 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.

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow.

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that the ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

Effects of dietary calcium (Ca2+) and blood feeding on the immunochemical expression of the plasma membrane Ca2+-ATPase (PMCA) in Malpighian tubules of adult female mosquitoes (Aedes aegypti).

Insect Malpighian tubules contribute to Ca2+ homeostasis via Ca2+ storage in intracellular compartments, Ca2+ secretion into the tubule lumen, and Ca2+ reabsorption into the hemolymph. A plasma membrane Ca2+-ATPase (PMCA) is hypothesized to be a Ca2+-transporter involved in renal Ca2+ transport of insects, however few studies have investigated its immunochemical expression in Malpighian tubules. Here we characterized the abundance and localization of PMCA-like immunoreactivity in Malpighian tubules of adult female mosquitoes Aedes aegypti using an antibody against Drosophila melanogaster PMCA. Western blotting revealed expression of a relatively abundant 109 kDa isoform and a relatively sparse 115 kDa isoform. Feeding mosquitoes 10% sucrose with 50 mM CaCl2 for 7 days did not affect PMCA immunoreactivity. However, at 24, 48, and 96 h post-blood feeding (PBF), the relative abundance of the 109 kDa isoform decreased while that of the 115 kDa isoform increased. Immunolabeling of Malpighian tubules revealed PMCA-like immunoreactivity in both principal and stellate cells; principal cell labeling was intracellular, whereas stellate cell labeling was along the basal membrane. Blood feeding enhanced immunolabeling of PMCA in stellate cells but weakened that in principal cells. Moreover, a unique apicolateral pattern of PMCA-like immunolabeling occurred in principal cells of the proximal segment at 24 h PBF, suggesting potential trafficking to septate junctions. Our results suggest PMCA isoforms are differentially expressed and localized in mosquito Malpighian tubules where they contribute to redistributing tubule Ca2+ during blood meal processing.

Voltage-gated ion channels are expressed in the Malpighian tubules and anal papillae of the yellow fever mosquito (Aedes aegypti), and may regulate ion transport during salt and water imbalance.

Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs - the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

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.

Identification of inducible CYP3 and CYP4 genes associated with abamectin tolerance in the fat body and Malpighian tubules of Spodoptera litura.

Abamectin, as a broad-spectrum bioinsecticide, has been widely used for the control of Lepidoptera insects, resulting in different levels of resistance to abamectin in Spodoptera litura. Cytochrome P450 monooxygenases (P450s) are known for their important roles in insecticide detoxification. In this study, the expression of SlCYP6B40, SlCYP4L12 and SlCYP9A32 in the fat body, and SlCYP4S9, SlCYP6AB12, SlCYP6AB58, SlCYP9A75a and SlCYP9A75b in Malpighian tubules was found to be significantly upregulated after abamectin exposure. SlCYP6AE44 and SlCYP6AN4 were simultaneously upregulated in these two tissues after abamectin exposure. Ectopically overexpressed SlCYP6AE44, SlCYP9A32 and SlCYP4S9 in transgenic Drosophila conferred tolerance to abamectin. In addition, homology modeling and molecular docking results suggested that SlCYP6AE44, SlCYP9A32 and SlCYP4S9 may be capable of binding with abamectin. These results demonstrate that upregulation of CYP3 and CYP4 genes may contribute to abamectin detoxification in S. litura and provide information for evidence-based insecticide resistance management strategies.

Expression of the non-neuronal cholinergic system components in Malpighian tubules of Mythimna separata and evidence for non-neuronal acetylcholine synthesis.

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.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.

A novel antidiuretic hormone governs tumour-induced renal dysfunction.

Maintenance of renal function and fluid transport are essential for vertebrates and invertebrates to adapt to physiological and pathological challenges. Human patients with malignant tumours frequently develop detrimental renal dysfunction and oliguria, and previous studies suggest the involvement of chemotherapeutic toxicity and tumour-associated inflammation1,2. However, how tumours might directly modulate renal functions remains largely unclear. Here, using conserved tumour models in Drosophila melanogaster3, we characterized isoform F of ion transport peptide (ITPF) as a fly antidiuretic hormone that is secreted by a subset of yki3SA gut tumour cells, impairs renal function and causes severe abdomen bloating and fluid accumulation. Mechanistically, tumour-derived ITPF targets the G-protein-coupled receptor TkR99D in stellate cells of Malpighian tubules-an excretory organ that is equivalent to renal tubules4-to activate nitric oxide synthase-cGMP signalling and inhibit fluid excretion. We further uncovered antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homologue of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumours. Together, our results demonstrate a novel antidiuretic pathway mediating tumour-renal crosstalk across species and offer therapeutic opportunities for the treatment of cancer-associated renal dysfunction.

Inducible Cytochrome P450s in the Fat Body and Malpighian Tubules of the Polyphagous Pests of Spodoptera litura Confer Xenobiotic Tolerance.

Cytochrome P450 plays vital roles in detoxifying xenobiotics. In this study, SlCYP340A and SlCYP340L expression in the Spodoptera litura fat body and SlCYP332A1, SlCYP6AB12, SlCYP6AB58, SlCYP6AB59, and SlCYP6AN4 expression in the Malpighian tubules were significantly upregulated after cyantraniliprole exposure, and SlCYP6AB58 and SlCYP6AB59 expression levels were simultaneously increased in the Malpighian tubules after gossypol treatment. Drosophila ectopically expressing candidate P450 genes showed that SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A conferred cyantraniliprole tolerance. The overexpression of SlCYP6AB58 and SlCYP6AB59 in Drosophila increased the number of eggs laid under the gossypol treatment. Moreover, the knockdown of SlCYP332A1, SlCYP6AB12, SlCYP6AB59, SlCYP6AN4, and SlCYP340A increased S. litura mortality under the cyantraniliprole treatment. Homology modeling and molecular docking results suggested that candidate P450 has the potential to bind with cyantraniliprole. These results indicate that the CYP3 and CYP4 genes participate in cyantraniliprole detoxification and that SlCYP6AB59 may be simultaneously involved in the gossypol tolerance of S. litura.

Requirement of Snakeskin for normal functions of midgut and Malpighian tubules in Henosepilachna vigintioctopunctata.

Septate junctions (SJs) are located between epithelial cells and play crucial roles in epithelial barrier formation and epithelia cell homeostasis. Nevertheless, the molecular constituents, especially those related to smooth SJs (sSJs), have not been well explored in non-Drosophilid insects. A putative integral membrane protein Snakeskin (Ssk) was identified in a Coleoptera foliar pest Henosepilachna vigintioctopunctata. RNA interference-aided knockdown of Hvssk at the third-instar larval stage arrested larval development. Most resultant larvae failed to shed larval exuviae until their death. Silence of Hvssk at the fourth-instar larvae inhibited the growth and reduced foliage consumption. Dissection and microscopic observation revealed that compromised expression of Hvssk caused obvious phenotypic defects in the midgut. A great number of morphologically abnormal columnar epithelial cells accumulated throughout the midgut lumen. Moreover, numerous vesicles were observed in the malformed cells of the Malpighian tubules (Mt). All the Hvssk depleted larvae remained as prepupae; they gradually darkened and eventually died. Furthermore, depletion of Hvssk at the pupal stage suppressed adult feeding and shortened adult lifespan. These findings demonstrated that Ssk plays a vital role in the integrity and function of both midguts and Mt, and established the conservative roles of Ssk in the formation of epithelial barrier and the homeostasis of epithelial cells in H. vigintioctopunctata.

Calcium storage in Malpighian tubules and the putative use for pupal chamber formation in a wood-feeding insect.

Cerambycid beetles form a chamber to spend their pupal stages in various forms according to the species. The red-necked longhorn beetle Aromia bungii (Coleoptera: Cerambycidae), which is an invasive pest that severely damages Rosaceae trees, makes a pupal chamber at the end of a tunnel deep in the xylem. Beetle larvae and the closely related species form a calcareous lid at the entrance of a pupal chamber. Previous studies on the closely related species conducted more than a century ago suggested that Malpighian tubules (MTs) play a vital role in calcium carbonate accumulation. However, the association between this Ca2+ accumulation and pupal chamber lid formation utilizing the possible calcium compounds stored in MTs have not yet been demonstrated. First, we artificially reared A. bungii larvae from eggs in host branches for 100 days and identified the larval developmental status and pupal chamber formation, using X-ray computed tomography. Second, we collected larvae from the branches and observed the internal organs by direct dissection under a microscope. Finally, we analyzed the elemental distribution, particularly calcium, in the larval gut with MTs, using energy dispersive X-ray fluorescence. The results suggest that immature larvae of A. bungii can accumulate Ca2+ in the MTs through wood tunneling and feeding activities. Ca2+ was stored at the proximal regions in two of the six MTs located posteriorly in the body. Additionally, larvae that formed a calcareous lid at the entrance of pupal chambers in the branches did not store Ca2+ in the MTs, suggesting that the larvae of A. bungii used the stored Ca2+ in their MTs for lid formation.

Adaptive Changes in Detoxification Metabolism and Transmembrane Transport of Bombyx mori Malpighian Tubules to Artificial Diet.

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.

Transport of tetraethylammonium by the Malpighian tubules of Trichoplusia ni: Regional specialization and the influence of diet.

Insect Malpighian tubules (MTs) play a major role in elimination of many potentially toxic compounds, including the organic cation tetraethylammonium (TEA). This paper examines transport of TEA by different segments of the MTs of the cabbage looper, Trichoplusia ni. The results show that the proximal ileac plexus (PIP) region of the MTs plays a dominant role in secretion of the organic cation TEA and that the rate of secretion is altered by feeding; principal cells of the proximal ileac plexus in tubules from larvae with full guts secreted TEA at higher rates than did the same cells in tubules of larvae in which the gut was empty. Michaelis-Menten analysis revealed that TEA secretion by the PIP was saturable and was blocked in a concentration-dependent manner by the organic cation cimetidine. For larvae reared from eggs on TEA-rich diet, higher concentrations of TEA in fluid secreted by the ileac plexus of tubules, and lower concentrations of TEA in the hemolymph, relative to larvae reared on control diet, is consistent with an upregulation of TEA transport in response to higher levels of dietary intake of an exogenous organic cation. The distal and proximal regions of the ileac plexus were also differentiated on the basis of transepithelial and basolateral membrane potentials and the influence of these electrical potentials on organic cation transport are discussed.

The morphology of the free-living females of Strepsiptera (Insecta).

The morphology of the adult free-living females of Mengenilla moldrzyki and Eoxenos laboulbenei (Strepsiptera, Mengenillidae) was documented with µCT-based 3D reconstructions and histological serial sections. External and internal features of both species are characterized by far-reaching specialization and structural simplification. The well-developed mandibles are moved by large muscles. Other mouthparts and their corresponding musculature are simplified or absent. The brain is partly shifted into the prothorax. It is followed by a single postcerebral ganglionic complex also containing the subesophageal ganglion and an unpaired abdominal nerve. Postcephalic sclerites are absent, except for the plate-like pronotum and small pleural sclerites. Wings and associated muscles are missing. The lumina of the large midgut and the anterior hindgut are disconnected. Seven bulb-shaped Malpighian tubules in M. moldrzyki is the highest number yet described for Strepsiptera. The 10-segmented abdomen lacks appendages. An unpaired birth organ opens ventrally on abdominal segment VII. The entire body cavity is filled with numerous freely floating eggs, 1386 in the specimen of M. moldrzyki and 721 in E. laboulbenei. Genital ducts, defined gonads, and genital glands are missing. The morphology of female Mengenillidae is discussed with respect to sexual dimorphism and structural features of the postembryonic stages. Phylogenetic implications are outlined.

Developmental and Nutritional Dynamics of Malpighian Tubule Autofluorescence in the Asian Tiger Mosquito Aedes albopictus.

Malpighian tubules (MTs) are arthropod excretory organs crucial for the osmoregulation, detoxification and excretion of xenobiotics and metabolic wastes, which include tryptophan degradation products along the kynurenine (KYN) pathway. Specifically, the toxic intermediate 3-hydroxy kynurenine (3-HK) is metabolized through transamination to xanthurenic acid or in the synthesis of ommochrome pigments. Early investigations in Drosophila larval fat bodies revealed an intracellular autofluorescence (AF) that depended on tryptophan administration. Subsequent observations documented AF changes in the MTs of Drosophila eye-color mutants genetically affecting the conversion of tryptophan to KYN or 3-HK and the intracellular availability of zinc ions. In the present study, the AF properties of the MTs in the Asian tiger mosquito, Aedes albopictus, were characterized in different stages of the insect's life cycle, tryptophan-administered larvae and blood-fed adult females. Confocal imaging and microspectroscopy showed AF changes in the distribution of intracellular, brilliant granules and in the emission spectral shape and amplitude between the proximal and distal segments of MTs across the different samples. The findings suggest AF can serve as a promising marker for investigating the functional status of MTs in response to metabolic alterations, contributing to the use of MTs as a potential research model in biomedicine.

Aedes aegypti Malpighian tubules are immunologically activated following systemic Toll activation.

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.

Chloroquine disrupts zinc storage granules in primary Malpighian tubule cells of Drosophila melanogaster.

Contrasting reports exist in the literature regarding the effect of chloroquine treatment on cellular zinc uptake or secretion. Here, we tested the effect of chloroquine administration in the Drosophila model organism. We show that larvae grown on a diet supplemented with 2.5 mg/ml chloroquine lose up to 50% of their stored zinc and around 10% of their total potassium content. This defect in chloroquine-treated animals correlates with the appearance of abnormal autophagolysosomes in the principal cells of the Malpighian tubules, where zinc storage granules reside. We further show that the reported increase of Fluozin-3 fluorescence following treatment of cells with 300 µM chloroquine for 1 h may not reflect increased zinc accumulation, since a similar treatment in Madin-Darby canine kidney cells results in a 36% decrease in their total zinc content. Thus, chloroquine should not be considered a zinc ionophore. Zinc supplementation plus chloroquine treatment restored zinc content both in vivo and in vitro, without correcting autophagic or other ionic alterations, notably in potassium, associated with the chloroquine treatment. We suggest that chloroquine or hydroxychloroquine administration to patients could reduce intracellular zinc storage pools and be part of the drug's mechanism of action.