Regulation of Amylase Synthesis in Bacillus circulans F-2

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.     

Bacterial communities in predatory mites are associated with species and diet types

The symbiotic bacterial communities of phytophagous arthropods are affected by host species and feeding habits, but such effects have been poorly studied in natural enemies. Here, we investigated the entire bacterial microbiome of two species of predatory mites, Neoseiulus californicus and Neoseiulus barkeri, feeding on three types of diets (artificial diet, pollen and their natural prey, the spider mite Tetranychus urticae) by high-throughput sequencing of the 16S rRNA gene. We found that the bacterial diversity of predatory mites feeding on artificial diet was significantly different from pollen and spider mite feeding groups in both N. californicus and N. barkeri, while bacterial diversity also differed strikingly between the two species even when feeding on the same artificial diet. This finding suggests that the bacterial community of predatory mites is determined by both species and diet. Alphaproteobacteria and Gammaproteobacteria were the two dominant bacterial classes in both predatory mite species, except for N. californicus feeding on artificial diet. The bacterium Bosea sp. was detected in all samples as the core microbial species in predatory mites. Additionally, we discuss whether Bradyrhizobiaceae and Rhodobacteraceae bacteria could be used as probiotics in the artificial diet of N. californicus for better mass rearing.

     

DC-EPG assisted comparison of European spittlebugs and sharpshooters feeding behaviour on grapevine

Xylem-feeding is apparently the only requirement making an insect a competent vector of the bacterium Xylella fastidiosa, an organism responsible for the devastation of the Southern Italian olive forest and nowadays considered one of the most feared threats to agriculture and landscape in Europe, including vineyards. Here, we used the direct current-electrical penetration graph (DC-EPG) technique to compare and describe the feeding behaviour on grapevine of four xylem-feeding species considered candidate vectors of X. fastidiosa widespread in Europe, namely two spittlebugs (the meadow spittlebug Philaenus spumarius and the spittlebug Neophilaenus campestris) and two sharpshooter leafhoppers (the rhododendron leafhopper Graphocephala fennahi and the green leafhopper Cicadella viridis). We created a standard for the analysis of EPG waveforms recorded with a DC-EPG device, describing feeding activities performed by these insects from stylet insertion into the plant to withdrawal. This standard, along with freely available software, has been developed to harmonize the calculation of feeding behavioural parameters in xylem-feeders. The most relevant differences between the two vector taxa were the probing frequency and the dynamics of xylem ingestion. Sharpshooters tended to perform significantly more probes than spittlebugs. In contrast, the latter spent longer times in low-frequency xylem ingestion, characterized by scattered contractions of the cibarial dilator muscle interspersed with periods of pump inactivity. Cicadella viridis was the species displaying the highest frequency of the electrical pattern found to be associated with X. fastidiosa inoculation in spittlebugs (Xe). Feeding behavioural data presented here represent an important step forward for deepening our knowledge of xylem-sap feeding insects' interaction with both the host plants and the bacterium they transmit.     

Transmission of <Emphasis Type="Italic">Methylobacterium mesophilicum</Emphasis> by <Emphasis Type="Italic">Bucephalogonia xanthophis</Emphasis> for paratransgenic control strategy of Citrus variegated chlorosis

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.     

An Experimental Study on the Food Chain Among Bacteria,Paramecium and Daphnia

A quantitative study is made of the feeding of Paramecium caudatum on bacteria and of the feeding of Daphnia pulex on Paramecium and bacteria. The growth yield of P. caudatum on a diet of an unidentified yellow-colored bacterium was about 55% on a dry weight basis. Paramecium would not grow if the concentration of bacteria is less than 1 . 10₇ cells ml. Several other bacterial species were tested. Paramecium could not grow successfully on all of them, but could on Rhodopseudomonas spheroides. Adults of Daphnia pulex could grow and reproduce on a diet of P. caudatum alone, but young grew successfully only when bacteria were present.     

Beeinflussung des Stickstoffstoffwechsels wachsender Hähne durch die Jahreszeit

Although Enterococcus faecium is used as a probiotic feed supplement in animal production, feeding of the bacterium to piglets resulted in a more severe infection with Salmonella Typhimurium DT104 during a challenge experiment. To enlighten the mode of action by which E. faecium affected the piglets’ health, we investigated the influence of the probiotic bacterium on the development of intestinal and circulating immune cells during a challenge experiment with S.Typhimurium DT104. To minimise varying impacts of the maternal immunity on the course of infection, only piglets were implemented that descended from Salmonella-free sows. In addition, the potency of purified blood and intraepithelial immune cells to control the growth of Salmonella was tested in vitro. In animals treated with E. faecium, a reduction of intraepithelial CD8αβ T cells, reduced circulating CD8αβ T cells and a less efficient control of intracellular Salmonella growth, mediated by peripheral blood mononuclear cells, were observed.     

Atrazine degradation in saline wastewater by Pseudomonas sp strain ADP

Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products, which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance ¹H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance. Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater. Received 26 August 1997/ Accepted in revised form 06 December 1997     

Independent Origins of Vectored Plant Pathogenic Bacteria from Arthropod-Associated <Emphasis Type="Italic">Arsenophonus</Emphasis> Endosymbionts

The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sap-sucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood and Bayesian criteria, revealed that this bacterium is not a sister taxon to “Candidatus Phlomobacter fragariae,” a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropod-associated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, “Candidatus Arsenophonus phytopathogenicus” novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.     

Bacterial feeders, the nematode Caenorhabditis elegans and the flagellate Cercomonas longicauda, have different effects on outcome of competition among the Pseudomonas biocontrol strains CHA0 and DSS73

How bacterial feeding fauna affects colonization and survival of bacteria in soil is not well understood, which constrains the applicability of bacterial inoculants in agriculture. This study aimed to unravel how food quality of bacteria and bacterial feeders with different feeding habits (the selective feeding flagellate Cercomonas longicauda versus the non-selective feeding nematode Caenorhabditis elegans) influence the abundance of two bacteria that compete for resources in simple model communities. Microcosms consisted of either one gfp-tagged bacterial strain (Pseudomonas fluorescens DSM50090 or one of two biocontrol strains P. fluorescens CHA0 or Pseudomonas sp. DSS73) or combinations of two bacterial strains. DSM50090 is a suitable food bacterium, DSS73 is of intermediate food quality, and CHA0 is inedible to the bacterial feeders. Bacterial and protozoan cell numbers were measured by flow cytometry. In the presence of flagellates, CHA0 increased its abundance as compared to the other biocontrol strain DSS73 or to DSM50090, which were both eaten by the flagellates. In contrast, the number of CHA0 declined as compared to DSS73 when the model community was subjected to nematode predation pressure. Hence, the results suggested that the outcome of competition among bacteria depended on their ability to cope with the prevailing bacterial predator.     

The feeding behavior of Spumella sp. as a function of particle size: Implications for bacterial size in pelagic systems

Direct observation was used to measure feeding rates of the flagellate Spumella on three sizes of bacteria plus 0.3 µm latex beads using video microscopy. Feeding rate was maximum on the intermediate-sized bacteria. Maximum ingestion rates (I_m) for the large- (0.53 µm³), intermediate- (0.08 µm³) and small-sized (0.02 µm³) bacteria and 0.014 µm³ latex beads were 11, 38 and 14 bacteria and 9 beads flagellate^–1 h^–1, respectively. The growth rates of Spumella sp. feeding on monoxenic cultures of the large- vs. the intermediate-sized bacteria were indistinguishable but Spumella sp. could not sustain its population density when feeding on the small bacterium as the sole food source. Our data are consistent with the hypothesis that Spumella sp., and possibly other flagellate protozoa, tend to feed selectively on larger prey. One consequence of this hypothesis is that differential grazing by bactivores may select for small bacteria in natural waters.     

Acquisition and transmission of bacteria through artificial membranes by leafhopper vectors of Pierce's disease

The leafhopper Graphocephala atropunctata transmitted the bacterium Erwinia amylovora to plant sap enclosed between Parafilm^® membranes after feeding on a suspension of the same bacterium. They did not do so, however, following intrahaemocoelic injection or surface inoculation. The bacterium Lactobacillus hordniae was similarly acquired and transmitted. Various bacteria were recovered from the gut but not from the haemocoel of the leafhoppers. Bacteria transmitted through artificial membranes had identical morphology and colony characteristics as bacteria isolated from the guts of the transmitting leafhoppers. The results of these experiments suggest that bacterial transmission is via egestion during feeding.

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Résumé La cicadelle Graphocephala atropunctata transmet les bactéries Erwinia amylovora, à de la sève maintenue entre des membranes de Parafilm^(r), après alimentation sur une suspension des mêmes bactéries. Il n'y a pas transmission, par contre, après injection intrahaemocélique ou inoculation superficielle. Les bactéries Lactobacillus hordniae sont acquises et transmises de la même façon. Différentes bactéries sont retrouvées dans le tube digestif, mais non dans l'haemocoele des cicadelles. Les bactéries transmises à travers des membranes artificielles sont identiques morphologiquement et ont les caractéristiques des colonies isolées des tubes digestifs des cicadelles vectrices. Les résultats de ces expériences suggèrent que la transmission bactérienne s'effectue par égestion pendant l'alimentation.

     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).     

Interaction of acid exudates in chickpea with biological activity of Bacillus thuringiensis towards Helicoverpa armigera

The gram pod borer, Helicoverpa armigera, is one of the most important constraints to chickpea production. High acidity of chickpea exudates is associated with resistance to pod borer, H. armigera; however, acidic exudates in chickpea might influence the biological activity of the bacterium, Bacillus thuringiensis (Bt), applied as a foliar spray or deployed in transgenic plants for controlling H. armigera. Therefore, studies were undertaken to evaluate the biological activity of Bt towards H. armigera on chickpea genotypes with different amounts of organic acids. Significantly lower leaf feeding, larval survival and larval weights were observed on ICC 506EB, followed by C 235, and ICCV 10 across Bt concentrations. Leaf feeding by the larvae and larval survival and weights decreased with an increase in Bt concentration. However, rate of decrease in leaf feeding and larval survival and weights with an increase in Bt concentration was greater on L 550 and ICCV 10 than on the resistant check, ICC 506EB, suggesting that factors in the resistant genotypes, particularly the acid exudates, resulted in lower levels of biological activity of Bt possibly because of antifeedant effects of the acid exudates. Antifeedant effects of acid exudates reduced food consumption and hence might reduce the efficacy of Bt sprays on insect‐resistant chickpea genotypes or Bt‐transgenic chickpeas, although the combined effect of plant resistance based on organic acids, and Bt had a greater effect on survival and development of H. armigera than Bt alone.     

Effects of carrot psyllid (Trioza apicalis) feeding on carrot yield and content of sugars and phenolic compounds

Carrot psyllid, Trioza apicalis, is a serious pest of carrot in Northern Europe, as it can significantly damage young carrot seedlings in a period as short as 3 days. This study was conducted to investigate effects of carrot psyllid feeding at different plant growth stages on carrot yield and to assess changes in content of sugars, phenolics and related compounds in carrot roots resulting from the psyllid feeding. In addition, reflectance of carrot leaves was measured to assess the intensity of discolouration in damaged leaves. Results showed that carrot yield was significantly reduced by a 3‐day carrot psyllid feeding period when the seedlings were exposed to psyllids at 1‐ or 2‐leaf stage. However, at 4‐leaf stage feeding by one carrot psyllid did not reduce yield. Sucrose concentration in the damaged roots was significantly decreased, whereas concentrations of some phenolic compounds were significantly increased. The reflectance of leaves of damaged carrots differed significantly from those of undamaged control leaves. These observations indicate that carrot psyllid damage has potential to lower not only the carrot yield, but also the carrot crop quality. No phytoplasma was detected in the carrots exposed to psyllids, but recently, T. apicalis has been associated with ‘Candidatus Liberibacter solanacearum’. The role of carrot psyllid feeding and the psyllid‐associated bacterium in the damage formation are discussed.     

The effect of suspended clay on ciliate population growth rates

• 1 Renewed batch cultures of three algivorous and one bacterivorous ciliate showed that ciliate reproductive rates increase with increasing food concentration up to an asymptote. Maximum r_m values for Strobilidium gyrans, Bursaridium difficile and Euplotes eurystomus feeding on the cryptomonad alga Rhodomonas minuta were 0.87, 0.86 and 0.45 day-¹, respectively, while Paramecium aurelia feeding on the bacterium Enterobactor aerogenes had an r_m of 0.40 day-¹. B. difficile had a food threshold (r_m= 0) of 0.38 μg Cml-¹ and P. aurelia had a threshold of 1.6μg Cml-¹.
• 2 The presence of suspended fine (particle size <1 μm) and coarse (particle size 1–2μm) clay suppressed the half maximum reproductive rate of the ciliate Strobilidium gyrans by up to 69% but had no effect on B. difficile, E. eurystomus or P. aurelia. Populations of ciliates unaffected by clay may be released in situations where suspended particles negatively affect their metazooplankton predators and competitors.

     

Comparative study of the entomopathogenic nematode, Steinernema carpocapsae, reared on mutant and wild-type Xenorhabdus nematophila

The symbiotic interaction between Steinernema carpocapsae and Xenorhabdus nematophila was investigated by comparing the reproduction, morphology, longevity, behavior, and efficacy of the infective juvenile (IJ) from nematodes reared on mutant or wild-type bacterium. Nematodes reared on the mutant X. nematophila HGB151, in which an insertion of the bacterial gene, rpoS, eliminates the retention of the bacterium in the intestinal vesicle of the nematode, produced IJs without their symbiotic bacterium. Nematodes reared on the wild-type bacterium (HGB007) produced IJs with their symbiotic bacterium. One or the other bacterial strain injected into Galleria mellonella larvae followed by exposing the larvae to IJs that were initially symbiotic bacterium free produced progeny IJs with or without their Xenorhabdus-symbiotic bacterium. The two bacterial strains were not significantly different in their effect on IJ production, sex ratio, or IJ morphology. IJ longevity in storage was not influenced by the presence or absence of the bacterial symbiont at 5 and 15 °C, but IJs without their bacterium had greater longevity than IJs with their bacterium at 25 and 30 °C, suggesting that there was a negative cost to the nematode for maintaining the bacterial symbiont at these temperatures. IJs with or without their symbiotic bacterium were equally infectious to Spodoptera exigua larvae in laboratory and greenhouse and across a range of soil moistures, but the absence of the bacterial symbiont inhibited nematodes from producing IJ progeny within the host cadavers. In some situations, such as where no establishment of an alien entomopathogenic nematode is desired in the environment, the use of S. carpocapsae IJs without their symbiotic bacterium may be used to control some soil insect pests.     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.     

Gut passage times in Gammarus pulex (Crustacea,Amphipoda) and aspects of summer feeding in a stony stream

Gut passage times in Gammarus pulex (Crustacea, Amphipoda) were calculated by counting and measuring voided faecal pellets. Following feeding on elm leaves conditioned in a stream, or summer fine detritus from the same source, calculated gut passage times were usually short, about 2 h or less at 15 °C. Using harder oak and beech leaves, not previously conditioned in the stream, apparent gut passage times were much longer, 8–48 h, but since intermittent feeding occurred these figures are not comparable to the others.In connection with summer feeding of the animal in the stream, the microbiological status of the fine detritus there was studied. It contained fungal hyphae, which were mostly empty, viable fungal spores of terrestrial derivation, and bacteria, all largely carried on fragments of vascular plant tissues. In experiments, such fungal spores (of Aureobasidium pullulans and of Mucor sp.), incorporated into fine detritus, were not digested by the animal. The bacterium Bacillus cereus was present in the fine detritus and when the latter was consumed it survived passage through the gut of the animal. Using B. cereus as a proportion marker it was concluded that other unicellular bacteria in the fine detritus were not digested either. In other experiments, not immediately related to the summer season, nutrient extraction by the animal from hyphae of the fungus Nectria lugdunensis was examined; it probably occurs through sub-microscopic pores connecting the cells. This mode of extraction may apply when the animal consumes vascular plant tissues.     

Non‐pathogenic aquatic bacteria activate the immune system and increase predation risk in damselfly larvae

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Who is eating fructose within the Aedes albopictus gut microbiota?

The Asian tiger mosquito Aedes albopictus is a major public health concern because of its invasive success and its ability to transmit pathogens. Given the low availability of treatments against mosquito-borne diseases, vector control remains the most suitable strategy. The methods used thus far are becoming less effective, but recent strategies have emerged from the study of mosquito-associated microorganisms. Although the role of the microbiota in insect biology does not require further proof, much remains to be deciphered in mosquitoes, especially the contribution of the microbiota to host nutrient metabolism. Mosquitoes feed on plant nectar, composed of mostly fructose. We used stable isotope probing to identify bacteria and fungi assimilating fructose within the gut of Ae. albopictus. Mosquitoes were fed a ¹³C-labelled fructose solution for 24 h. Differences in the active microbial community according to the sex of mosquitoes were highlighted. The bacterium Lelliottia and the fungi Cladosporium and Aspergillus dominated the active microbiota in males, whereas the bacterium Ampullimonas and the yeast Cyberlindnera were the most active in females. This study is the first to investigate trophic interactions between Ae. albopictus and its microbiota, thus underscoring the importance of the microbial component in nectar feeding in mosquitoes.