Antibiotic and insecticidal bioactivities of the extracellular secondary metabolites produced by entomopathogenic bacteria belonging to genus Xenorhabdus have been identified; however, their novel applications such as mosquito feeding-deterrence have not been reported. Here, we show that a mixture of compounds isolated from Xenorhabdus budapestensis in vitro cultures exhibits potent feeding-deterrent activity against three deadly mosquito vectors: Aedes aegypti, Anopheles gambiae, and Culex pipiens. We demonstrate that the deterrent active fraction isolated from replicate bacterial cultures is highly enriched in two compounds consistent with the previously described fabclavines, strongly suggesting that these are the molecular species responsible for feeding-deterrence. The mosquito feeding-deterrent activity in the putative fabclavine-rich fraction is comparable to or better than that of N,N-diethyl-3-methylbenzamide (also known as DEET) or picaridin in side-by-side assays. These findings lay the groundwork for research into biologically derived, peptide-based, low–molecular weight compounds isolated from bacteria for exploitation as mosquito repellents and feeding-deterrents.

In a screen for repellent activities produced by Xenorhabdus, we observed that Xbu extracts deterred adult female Aedes, Anopheles, and Culex mosquitoes from feeding on an artificial diet in in vitro feeding experiments. The compounds exhibiting feeding-deterrent activity from bacterial cultures were concentrated via a three-step procedure, including reversed-phase chromatography, and analyzed by mass spectrometry (MS). In addition to other molecules, the feeding-deterrent active fraction consistently contained two highly abundant molecular species that we provisionally identify as members of the previously described class of molecules called fabclavines ( 11 ). Here, we present data on the enrichment and characterization of mosquito feeding-deterrent activity produced by Xenorhabdus and present evidence that fabclavines may be potent mosquito feeding-deterrents. Our discovery adds bacteria as a notable, potential source of novel mosquito feeding-deterrents and lays the groundwork for future exploration of bacterially derived secondary metabolites as feeding-deterrents for other pest insects.

Repellents (topical and area) can provide protection from mosquito (and other blood-feeding insects) bites and thus from the disease agents that they transmit while feeding ( 15 , 16 ). Mosquitoes transmit pathogens that cause devastating human diseases, including dengue, Chikungunya, West Nile, and Zika viral infections, that continue to affect millions of people worldwide. Limiting the impact of mosquito-borne diseases is an important goal for global public health agencies, and the use of mosquito repellents is one important tactic. Among U.S. Environmental Protection Agency–registered topical insect repellents, a majority of more than 500 products contain the active ingredient N,N-diethyl-3-methylbenzamide (also known as DEET; www.epa.gov/insect-repellents/skin-applied-repellent-ingredients ), which is the most widely used and effective repellent against mosquitoes and other disease vectors. Other commercially successful ( 17 ) insect repellents include IR3535 [insect repellent 3535, ethyl butylacetylaminoproprionate (EBAAP), a derivative of β-alanine ( 18 )], (p)icaridin (and other piperidine derivatives) ( 19 ), and para-menthane-3,8-diol (distilled from Eucalyptus citriodora). Historically, research into biologically derived DEET alternatives has primarily focused on plant metabolites. Despite the exploitation of many genera for pharmaceutical exploration, bacteria have thus far remained unexplored in the search for insect repellent chemistries.

Genome studies ( 6 , 10 ) suggest that there is an enormous range of chemical diversity and bioactivity that still remains to be explored and may lead to the discovery of novel bioactive compounds. In Xenorhabdus, genome mining has uncovered gene clusters predicted to participate in the synthesis of several compounds of unknown biological functions ( 11 ). The products encoded by these gene clusters exhibit diverse and structurally complex chemistries. One example produced by Xenorhabdus budapestensis (Xbu) is a unique class of hybrid compounds called fabclavines. These compounds were shown to exhibit a broad range of bioactivities, including antibiotic and insecticidal activities ( 3 , 4 , 11 , 12 ). Uniquely, culture supernatants of two insect-killing bacteria, Xenorhabdus nematophila and Photorhabdus luminescens, also deterred feeding of ants, crickets, and wasps ( 13 , 14 ). However, the bioactive compounds responsible for feeding-deterrence in those studies were not identified. Together, these studies suggested that secondary metabolites produced by Xenorhabdus might act as mosquito feeding- deterrents.

Secondary or specialized metabolites ( 1 ) are a chemically diverse group of organic compounds produced by some microbes, plants, and animals ( 2 ). Generally considered nonessential for growth and development, secondary metabolites play other roles such as conferring protection against varied environmental risks. Many secondary metabolites of microbial or plant origin have been exploited for myriad applications in the pharmaceutical industry, including antibiotics, chemotherapeutic drugs, immunosuppressants, and other medicines ( 2 ). Secondary metabolites produced by Xenorhabdus, a group of bacteria that symbiotically associate with entomopathogenic nematodes, exhibit a range of antibiotic, antifungal, and insecticidal activities ( 3 – 9 ).

RESULTS AND DISCUSSION

In vitro membrane feeding system as a screening assay for determining mosquito feeding-deterrent activity in Xbu extracts In the context of insect repellents, a deterrent is defined as “something that inhibits feeding or oviposition when present in a place where the insect would, in its absence, feed, rest or oviposit” (20). Here, we define compounds produced by Xbu as mosquito feeding-deterrents because, in the presence of Xbu compounds, female mosquitoes failed to feed on the food source described in this section. Among methodologies to evaluate mosquito deterrents/repellents, in vitro assays offer important advantages. Because of the risk associated with accidental exposure to infectious agents during standard arm-in-cage assays (21, 22), and because of inconsistency in results among test subjects due to varying mosquito attraction to human subjects (23), researchers have developed artificial feeding systems. Blood is contained in a warming chamber and accessed by adult female mosquitoes through a natural or artificial membrane. Membrane feeders provide flexible systems that can be modified in terms of feeding solutions (24–26), membrane types (24, 25, 27), and solution temperature (24, 25, 28). These systems eliminate the need to expose animals (29) or human volunteers, an especially important consideration when the compounds in the initial screening phases are of unknown toxicological or dermatological properties. Thus, we chose to modify our existing Hemotek membrane feeding system (Discovery Workshops, Accrington, UK) to screen feeding-deterrent compounds produced by Xbu bacteria. The main features of this system (Fig. 1) included (i) easy setup, (ii) minimal laboratory space requirements, (iii) a thermostat-regulated temperature source that eliminates the need for a water heater and circulation pump, and (iv) capacity for up to five screening assays at a time. The modifications used for the final bioassays included introduction of a loosely woven cotton cloth for application of the feeding-deterrent compounds, a collagen membrane, and use of a cocktail diet containing a red food dye (24) instead of blood (27). Fig. 1 Description of the membrane feeding and feeding-deterrent screening system. (A) Components of the feeding system, including (top/bottom panel, left to right) Hemotek temperature controller, feeder-housing assembly, metal feeder assembled with cocktail diet (red color) secured with collagen casing and O-ring, two layers of cotton cloth, metal feeder with cotton cloth secured via rubber bands (not visible), and metal feeder assembly secured to feeder housing. ID, inside diameter. Photo credit: Mayur Kumar Kajla, University of Wisconsin–Madison. (B and C) Results of the feeding assays. (B) Plot showing the number of A. aegypti mosquitoes that fed when water was applied to the cotton cloth as control, or DEET or picaridin (0.95 mg/cm2) [equivalent to 1.0% (v/v)] was applied in three replicate experiments. Each replicate consisted of 20 mosquitoes for each of the control as well as tests. (C) Representative image depicting appearance of fed (red abdomens) versus unfed Aedes mosquitoes resulting from the bioassay. Images show engorged abdomens and red dye in fed mosquitoes. Absence of both color and engorgement of abdomens indicates that no feeding occurred with 1% DEET (or picaridin) as a positive control. We first tested this system with positive [DEET or picaridin at 0.95 mg/cm2 in water; equivalent to 1.0% (v/v)] and negative (water) controls applied to cloth covering the membrane feeder. Mosquitoes were allowed to feed for 30 min and then frozen at −20°C before scoring and counting. Mosquito feeding success was then scored by counting fed and unfed mosquitoes following bioassays in the presence or absence of the repellent compounds. The outcomes of the screening assay are shown in Fig. 1 (B and C). Using this system, we obtained reproducible and consistently high mosquito feeding success when the cotton cloth was treated with water and 0% feeding when DEET or picaridin was tested as positive repellent controls. Results of three replicate experiments (20 mosquitoes per replicate; total number of mosquitoes tested in three replicate experiments = 60) are presented in Fig. 1B and show an average feeding success of 96.67%, with water controls with Aedes aegypti, Anopheles gambiae, and Culex pipiens also fed well (75 to 80%; Fig. 2), indicating that the bioassay can be used with multiple mosquito species. On the basis of these results, we determined that the bioassay provided a robust and reproducible test arena to screen mosquito feeding-deterrent activities in the Xbu extracts at various stages of purification. Fig. 2 Mosquito feeding-deterrent activity of Xbu Peak#3 with C. pipiens, A. gambiae, and A. aegypti. (A) Plots show feeding-deterrent activity of Xbu compounds tested at 0.057 mg/cm2. Data from three replicate experiments are shown. Each replicate consisted of 20 mosquitoes for each of the control (water only) as well as tests (Xbu Peak#3), respectively. A. aegypti mosquitoes were included for comparison. (B) Appearance of fed (red abdomens) and unfed Anopheles and Culex mosquitoes. In this experiment, one-layer muslin cloth was used. Fisher’s exact test was used to assess differences between groups using Stata statistical software. In addition, this bioassay was optimal to screen minimal amounts of compounds, a major concern when working with naturally produced microbial compounds not easily obtainable in large quantities. The method also provided rapid results as well as consistent measures of feeding-deterrence that were reproducible across assay dates.

Enrichment and characterization of mosquito feeding-deterrent active compounds from Xbu Next, we developed a procedure to isolate mosquito feeding-deterrent active compounds from Xbu cultures. Xenorhabdus bacteria are known to produce antibiotics and secondary metabolites in the late stationary phase of their growth cycle (11, 30, 31). Accordingly, we used 72-hour bacterial cultures to harvest mosquito feeding-deterrent compounds from the cell-free culture supernatants. Mosquito feeding-deterrent active compounds in the culture supernatants were concentrated via acetone precipitation. In the next step, water-soluble acetone precipitates yielded a broad peak detected at 280 nm on a reversed-phase C18 flash chromatography column, indicating that the mosquito feeding-deterrent active compounds coeluted with other compounds detectible at 280 nm. The feeding-deterrent activity concentrated and eluted as a single broad peak fraction. Representative images of C18 flash purification results are shown in fig. S1. The peak fraction was subjected to MS analysis for determination of molecular masses and structural characterization. Matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) MS revealed the presence of several abundant molecular species including compounds at m/z (mass/charge ratio) 1302.94, 1312.96, and 1430.07 in this fraction (Fig. 3A), with additional masses at m/z 44.03 units higher, leading to the idea that these were likely related. The difference of 44 atomic mass units is consistent with addition of one C 2 H 4 O moiety (11). Fig. 3 MALDI-TOF spectrum of flash and HPLC C18 reversed-phase chromatography separated mosquito feeding-deterrent active peak fraction. (A) MALDI-TOF analysis of the C18 flash fractionated feeding-deterrent active fraction yielded several major masses (and related molecular species) at m/z 1302.94 (1346.97), 1312.96 (1356.98), and 1430.07 (1474.10), as well as m/z 1238.953. The mass difference of m/z 44 in these pairs may be due to addition of C 2 H 4 O moieties (11). (B) MALDI-TOF analysis of HPLC-separated feeding-deterrent active Xbu Peak#3 shows enrichment of the same two abundant (and related) molecular species at m/z 1302.92 and 1346.95. The mosquito feeding-deterrent active fraction from the C18 flash chromatography was subjected to a second fractionation step using analytical reversed-phase high-performance liquid chromatography (HPLC) after an ultrafiltration step (see Materials and Methods for details). Four major peaks were observed, with most of the repellent activity concentrated in peak number 3 (hereafter referred as Xbu Peak#3), which eluted at ~24% acetonitrile (ACN). Representative images of HPLC C18 purification are shown in fig. S2. None of the other peaks showed feeding-deterrent activity (fig. S3, A and B) except peak 4. The activity in peak 4 is likely due to cross-contamination from peak 3 as a result of incomplete resolution of these peaks. MS analysis of Xbu Peak#3 showed two highly abundant masses at m/z 1302.9 and 1346.9 (Fig. 3B), indicating that these compounds, previously observed in the active fraction from C18 flash chromatography, had been enriched in the mosquito feeding-deterrent fraction, as well as many other lower-abundance molecular species. MALDI-TOF MS on mosquito feeding-deterrent active Peak#3 was performed on three different batches of the fractionated compounds. In each replicate, these two masses were the most abundant ions detected. Spectra for these experiments are presented in fig. S4. A number of other lower-abundance molecular species are observed in each replicate fractionation of Xbu cultures (see fig. S4). Several of these compounds do not reproduce across all fractionations, eliminating them from consideration as the active component. Others, most notably at m/z 1430 and 1474, are observed in all of the active fractions. However, their relative enrichment in the active HPLC fraction is decreased compared to m/z 1302 and 1346. This can be seen when comparing the signal intensities for m/z 1302, 1346, 1430, and 1474 in the C18 flash fraction before and after HPLC (Fig. 3, A and B, and fig. S4). The decrease in m/z 1430 and 1474 relative to m/z 1302 and 1346 indicates only a partial coelution with the activity. Because we were interested in identifying the compounds, we subjected the mosquito feeding-deterrent active fraction to MS/MS analysis by unassisted nanospray on an Orbitrap mass spectrometer. Nanospray MS displayed the same major molecular species observed by MALDI-TOF MS, with each compound in a dominant charge state of 2, as well as other lower-abundance species. Charge states from 1 to 4 were detected (fig. S5). MS/MS was performed on the doubly charged species at m/z 651.9 and 673.9 using both collision-induced dissociation (CID) and high-energy collisional dissociation (HCD) fragmentations. The MS/MS spectra revealed that these compounds were structurally very similar, with several ions common to both compounds, and many ions showing a difference of 44 Da between them, as previously observed by MALDI-TOF MS. The MS/MS spectra and the inferred fragment ion assignments show excellent agreement with the recently described “fabclavines” isolated from a similar strain of Xbu (11). Using the naming convention described in that work and the structures for fabclavines Ib and IIb, the observed fragment ions were consistent with assignment of m/z 1302 as fabclavine IIb and m/z 1346 as fabclavine Ib (fig. S6, A and B), suggesting that the two major constituents of the active fraction are fabclavines. Results of N-terminal Edman degradation analyses on mosquito feeding-deterrent active Peak#3 that contains two related peptides (provided in table S2) were inconclusive in determining the sequence of amino acids, possibly explained by an inaccessible N terminus of the molecule due to macrocyclization (11). Total amino acids were measured from mosquito feeding-deterrent active Peak#3 by two different strong cation ion exchange chromatography methods, sodium- and lithium-based elution systems (Molecular Structure Facility, University of California, Davis, CA, USA). Both systems resulted in significant signals for amino acids Asx and His (fig. S7, A and B). In addition, two unidentified signals were observed, which could be 2,3-diaminobutyric acid (elution profiles were consistent with this compound). Together, these data indicate that two of the amino acids Asx and His and possibly 2,3-diaminobutyric acid are likely to be present in the feeding-deterrent active fraction—amino acids that are also described in fabclavines (11), which, in addition to these, also contain phenylalanine or histidine, and a modified proline residue.