Effects of Xenorhabdus and Photorhabdus bacterial metabolites on the ovipositional activity of Aedes albopictus

Viral diseases like yellow fever, dengue, zika have an alarming impact on public health affecting millions of people around the globe. These diseases can be transmitted by Aedes albopictus – a species originally endemic to South Asia but now widely distributed in several countries outside Asia. Xenorhabdus and Photorhabdus spp., are enteric Gram-negative bacterial symbionts of insect-preying nematodes and are known to produce an array of natural products (NP) with various activities including larvicidal activity. This study demonstrated that supernatants of different Xenorhabdus and Photorhabdus bacteria effectively deterred Ae. albopictus mosquito oviposition in a concentration dependent manner. Xenorhabdus cabanillasii displayed deterrent effects at 50-5% concentrations with oviposition activity index values ranging between -0.87 and -0.35, whereas the index values for X. nematophila were between -0.82 and -0.52 at concentrations of 50-10%. The other bacteria (X. szentirmaii, X. doucetiae, and P. kayaii) were effective at concentrations ≥ 20%. Using promoter exchange mutants of selected biosynthetic gene clusters in a Δ hfq background generated by the easyPACId approach, fabclavine from X. szentirmaii was identi�ed as the bioactive compound with evident deterrent effects. Such compounds with deterrence effects could be useful in mosquito control programs as preventing breeding site in speci�c habitats can greatly in�uence mosquito species establishment, population densities, and dispersion in conducive areas.


Introduction
Aedes albopictus (Diptera: Culicidae), with characteristic white bands on its legs and body, is a synanthropic Aedes species originally endemic to tropical and sub-tropical regions of South Asia.Due to its invasive and highly adaptive nature, it is now widely distributed in at least 30 countries throughout the tropics, subtropics, and temperate regions of the world outside Asia (Benedict et al. 2007;Fonseca et al. 2013; Touray et al. 2023).This spread has been greatly facilitated by rising temperatures due to climate change and transport of its immature stages (diapausing eggs and larvae capable of withstanding cold conditions in temperate regions) in bamboo plants, used tires, and arti cial containers during global shipping activities.Drastic deviations in global climate have also made its establishment in new habitats easier (Kraemer et al. 2015; Faraji and Unlu 2016; Beaty et al. 2016;Messina et al. 2019).Importantly, Ae. albopictus is a daytime-biting species that can take blood meals from a variety of host including humans, domestic and wild animals and a prominent vector of Zika, Chikungunya, Dengue, and Yellow Feverviral diseases whose epidemic potential and incidence has increased globally since the 1950s (Godfray 2013; Messina et al. 2019;Romanello et al. 2021;Bursali and Simsek 2023).Foremost among these arthropod infections, dengue is a predominantly urban disease of tropical and sub-tropical climates that has an alarming impact on public health with more than 3 billion people living in endemic areas and around 100 million cases and at least 40.000 deaths occur annually in 195 countries.Symptoms include a characteristic skin rash, high fever, headache, muscle and joint pains.Severe cases may be possibly life-threatening by causing serious bleeding and even death (Gubler 2002;Vos et al. 2015; Zeng et al.

2021).
Aside from yellow fever, these Aedes-transmitted diseases have no vaccine or effective therapeutics (Smith et  Xenorhabdus and Photorhabdus (Fam: Morganellaceae) are enteric Gram-negative bacterial symbionts of soil-dwelling and insect-preying nematodes in genera Steinernema and Heterorhabditis, respectively (Hazir et al. 2022).These nematode-bacteria complexes are distributed nearly worldwide, and as biological control agents are applied to soil to control pestiferous arthropods that are a menace to agriculture and public health (Shapiro-Ilan et al. 2019).Xenorhabdus and Photorhabdus bacteria are known to produce a starling array of enzymes and natural products (NP) that can suppress insect immunity, quickly kill host, and disintegrate tissue, as well as perform defense functions against competitors and natural enemies using polyketide synthases (PKSs) or non-ribosomal peptide synthetases (NRPS) (Bode 2009;Tobias et al. 2017;Cimen et al. 2022).So far, an important body of literature exists detailing the antimicrobial activities of these products from these bacteria against bacterial (Webster et al. 2002;Furgani et al. 2008)  This study investigated the effects of CFS of Xenorhabdus and Photorhabdus on the ovipositional behavior of Ae. albopictus and identi ed bioactive deterrence compounds.

Material and Method
Maintenance of Aedes albopictus colony Adult Ae. albopictus were reared in insect cages (40 × 40 × 40 cm) in a controlled insectary with a temperature of 30°C, 70 ± 10% relative humidity and a 12 h light: 12 h dark photoperiod.Mosquitoes were fed every 2-3 days on de brinated sheep blood using an arti cial blood feeder (Shah et al. 2021).Sugary water (10%) was available at all times.Mosquitoes laid eggs on lter papers in water lled cups.Eggs were hatched in tap water in small plastic containers (10 × 15 × 15 cm).Emerged larvae fed on crushed sh food and maintained at 24 ± 1°C.Newly emerged adults (~ 1 week old) were used in the experiments.

Preparation of Xenorhabdus and Photorhabdus supernatants
Xenorhabdus cabanillasii JM26-1, X. doucetiae DSMZ 17909, X. nematophila ATCC 19061, X. szentirmaii DSMZ16338 and Photorhabus kayaii DSMZ 15194 were used in this study.Cell free supernatants of these bacteria were obtained as described in Hazir et al., (Hazir et al. 2017).Brie y, bacteria were cultured on Luria-Bertani (LB) agar for 24 h at 28°C and then a single colony was inoculated into a fresh LB culture to prepare an overnight culture.One ml of this overnight culture was then inoculated into a fresh 100 ml LB medium which was incubated at 28°C for 72 h.These cultures were then centrifuged at 10.000 rpm at 4°C for 10 min and the supernatants were separated into new Falcon tubes.

Generation of Xenorhabdus spp. Δhfq pCEP-KM promoter exchange supernatants
To identify bioactive ovipositional deterrent compound(s) in the bacterial supernatants, Δhfq promoter exchange mutants of Xenorhabdus bacteria were used (Table 1).These mutants were generated using the easyPACId approach (easy Promoter Activated Compound Identi cation) ( was adjusted to 0.1.These newly inoculated cultures were incubated for 1 h at 30°C and afterwards induced with 0.2% L-arabinose (Wenski et al. 2020).These induced cultures were incubated for 72 h at 150 rpm and 30°C and then CFS was obtained and used in the oviposition experiments.

Effects of wild-type and mutant bacterial supernatants on the ovipositional activity of Aedes albopictus
Experimental design was based on (Kramer and Mulla 1979)) using a binary choice design with 10 newly blood-fed female mosquitoes in insect cages.After blood feeding, females were transferred to new cages from stock colonies using an aspirator and allowed 4 days to digest blood.Sugary water (10%) was available at all times.Two plastic cups (100 ml) were introduced into the cages (two-choice test): one had CFS of wild-type X.szentirmaii, X. cabanillasii, X. nematophila, X. doucetiae or P. kayaii diluted (50, 20, 10, 5 and 1% concentrations) in 40 ml distilled water and the other had LB diluted in distilled water (control).Besides, commercial larvicidal compounds were compared with distilled water to determine if they have any ovipositional deterrent activity.The active ingredient in the larvicides used were Spinosad, Lysinibacillus sphaericus, or Bacillus thuringiensis subsp.israelensis (Table 2).These products were used at doses recommended by the manufacturer.Field-collected water was also assessed against distilled water.Edges of the cups were lined with Whatman No. 2 lter papers as a substrate for collection of deposited eggs.These plastic cups were placed equidistant from each other at the corner of the cages and their positions were alternated between replicates to nullify any position biases.The cages were placed in the insectarium at 27 ± 1 ℃ temperature, 70% relative humidity and 12 h photoperiod.After 72 h, the lter papers in each plastic cup were collected, and the eggs deposited on lter papers were counted under a stereomicroscope.Each treatment had four replicates and the experiments were conducted three times.The oviposition attractant/deterrence feature of the bacterial supernatant was evaluated by calculating the oviposition activity index (OAI) using the number of eggs laid in the cups (Kramer and Mulla 1979; Hwang et al. 1982).The OAI was determined using the formula and scores were used for analysis of variance The same method was used with cell-free supernatants obtained from promoter exchange mutants shown in Table 1 to identify the compound responsible for the attractant/deterrence activity.Different derivatives of the bioactive compound obtained from different bacteria (i.e., X. hominckii, X. cabanillasii, X. budapestensis, X. szentirmaii, X. bovienii, and X. stockiae) were also assessed.

Performance of bioactive oviposition deterrent compounds
After identifying the bioactive compound/s, multiple choice experiments were also conducted.In this case, four plastic cups (100 ml) with the components below were added to cages with 10 female mosquitoes: Choice experiment-1 Field collected water, clean water, water with 10 larvae (3rd -4th stage), and LB-water (control).
The total liquid volume in each cup was 40 ml.The plastic cups were placed in the corners of the insect cages.Eggs were counted after 3 days and the OAI was calculated as described previously.These experiments had 5 replicates and the experiments were conducted twice.

Statistical analyses
Analysis of variance with Tukey's test (p = 0.05) was used to compared OAI as well as a number of eggs laid in multiple choice experiments in the statistical analysis using SPSS program (version 23).

Effects of wild-type and mutant strain bacterial supernatants on the ovipositional activity of Aedes albopictus
The oviposition deterrence activity of CFS from different wild-type Xenorhabdus and Photorhabdus bacteria against Ae.albopictus is presented in Fig. 1.All bacteria supernatants effectively deter oviposition of female mosquitoes in a concentration-dependent manner with signi cant difference between all treatments (F = 9.328; df = 29,303; P < 0.001).Supernatant from X. cabanillasii displayed deterrent effects between 50 and 5% concentrations with oviposition activity index values ranging between − 0.87 and − 0.35.Index values for X. nematophila were − 0.82, -0.85, and − 0.52 at concentrations of 50, 20 and 10%, respectively.Xenorhabdus szentirmaii, X. doucetiae, and P. kayaii were effective at concentrations ≥ 20%.Among the controls, the mosquitoes only abstained from treatments with L. sphaericus with an OAI of -0.47.Aedes albopictus females were neutral to the effects of B. thuringensis israelensis and spinosad.Field collected water was signi cantly more attractive than distilled water (Fig. 1).
Various mutants generated from X. szentirmaii were used to identify the respective deterrent compounds present in supernatants.Fabclavine (OAI= -0.72) as the only compound with evident deterrent effects in X. szentirmaii (Figs. 2 and 3).

Performance of bioactive ovipositional deterrent compounds
There was a statistically signi cant difference in the number of eggs laid by Ae. albopictus females in the multiple-choice experiments with 4 different containers that had either eld collected water, supernatant (treated), water with larvae (10), LB-water (control) or clean water (Fig. 5).In the rst combination (choice experiment-1), which served as a control, eld water was found to be the most attractive breeding site for the female with at least 140 eggs laid here.In choice experiments 2 and 4, females were observed to abstain from containers where fabclavine had been added (Fig. 5), even reducing the attractiveness of eld collected water.The number of eggs laid in the containers with eld collected water and fabclavine in the choice experiment-3 was nearly halved (73 eggs) compared to the other combinations.

Discussion
Our data shows that Ae. albopictus abstained from laying eggs in containers with supernatants of Xenorhabdus and Photorhabdus.All bacteria supernatants effectively deterred oviposition of female mosquitoes in a concentration-dependent manner.Shah et al. 2021).Our study, as a rst, demonstrates that Xenorhabdus and Photorhabdus spp.produce highly effective ovipositional deterrent compounds against mosquito Ae albopictus.The easyPACId method showed clearly that the bioactive deterrent compound was fabclavine.These non-ribosomally synthesized peptide/polyketide with polyamine moieties has broad-spectrum bioactivities.An important body of literature exists detailing the possible application of fabclavines against various bacterial (Donmez Ozkan et al. 2019), fungal (Cimen et al., 2021), and protozoal organisms (Gulsen et al. 2022) of medical, veterinary, and agricultural importance.These compounds are produced by certain Xenorhabdus and Photorhabdus bacteria to kill adversaries of the EPN-bacteria complex and help maintain a monoxenic environment within the infected host (Fuchs et (Bentley and Day, 1989;Day, 2016;Baik and Carlson, 2020).The ies can even sense the presence of con-and heterospeci c immature stages and predators and chemical cues associated with these organisms, and they are attracted, repelled or deterred from depositing eggs in such sites (A fy and Galizia 2015; Russell et al. 2022).Our study demonstrates that the application of fabclavine and L. sphaericus in uence the ovipositional behavior of mosquitoes.The effects of B. thuringiensis israelensis and spinosad were neutral.Likewise, Nazni et al. (2009) showed that Ae. albopictus females were not repelled from Bti treated containers.However, we detected that the females did not lay eggs or laid a few in containers where fabclavine had been added.Fabclavine derivatives obtained from X. hominckii, X. cabanillasii, X. budapestensis, X. szentirmaii, X. bovienii, and X. stockiae had deterrent activities on Ae. albopictus with OAI that ranged between − 0.53 and − 0.89.Fabclavine from X. szentirmaii even reduced the attractiveness of eld collected water.We observed that the mosquitoes landed on the wet lter papers on the sides of treated containers at which point they sensed fabclavine, which are highly polar compounds that are miscible in water, through sensilla in tarsal segments of their legs before abstaining from egg laying and moving away.The mode of action of fabclavine on female mosquitoes needs to be elucidated.Xenorhabdus nematophila, X. doucetie and P. kayaii do not produce fabclavines hence a different compound/s is responsible for the deterrence effects.
Interestingly, the oviposition deterrence activity of CFS from X. nematophila, X. doucetiae and P. kayaii (Fig. 1) that all do not encode a biosynthesis gene cluster responsible for fabclavine production (Tobias et al. 2017), suggests another compound to display the deterrence activity that has not been identi ed yet.
As far as we are aware this is the rst report of the oviposition deterrent effects of bacterial secondary metabolite on mosquitoes.Poonam et al. (2002) investigated the effects of CFS of Azospirillum brasilense, B. cereus, B. megaterium, Pseudomonas uorescens, B. thuringiensis var.israelensis and B. sphaericus, on the oviposition activity of Culex quinquefasciatus females.They observed that depending on concentration CSF of these bacteria exhibited attractancy comparable to p-cresol, a known oviposition attractant.On the other hand, numerous compounds mainly from plants have been reported to have ovipositional deterrent activity against mosquitoes (Waliwitiya et  Development of compounds with deterrence effects could be useful in mosquito control programs as such compounds can prevent gravid mosquito females from breeding in certain habitats.Choice of breeding site can greatly in uence mosquito species establishment, population densities and dispersion in conducive areas.

Declarations Figures
al. 2016; Huang et al. 2023) hence mosquito control professionals around the world monitor Aedes populations and evaluate novel control strategies to mitigate pathogen transmission.Control strategies include elimination of mosquito breeding sites or use of chemical larvicides in these breeding sites or insecticides against adult mosquitoes(Beaty et al. 2016; Weeratunga et al. 2017).Nevertheless, Ae. albopictus mosquitoes are not well-controlled by the above strategies as location and complete elimination of breeding sites especially arti cial containers in urban and poorly developed areas is often a challenging task, and continuous use of chemical insecticides has resulted in development of resistance to insecticides in many countries and most insecticides have damaging impacts on the environment (Smith et al. 2016; Silva et al. 2020).These issues have encouraged the use of environmentally friendly biological alternatives like the fungus, Metarhizium anisopliae and the bacteria, Bacillus thuringiensis subsp.israelensis (Bti) and Lysinibacillus sphaericus as well as insect growth regulators (Lacey 2007; Scholte et al. 2007; Benelli et al. 2016) and much effort has been invested in the search for many more.
et al. 2020; Baik and Carlson 2020; Girard et al. 2021).The literature on mosquito oviposition behavior is vast and several studies have established that factors such as presence of con-and heterospeci c immature stages, microbes, predators and chemical cues associated with these organisms attract, repel or deter gravid mosquitoes from depositing eggs (Eitam and Blaustein 2004; A fy and Galizia 2015; Russell et al. 2022).Application of potential deterrence compounds to breeding sites might in uence the ovipositional behavior of mosquitoes.Numerous compounds obtained from plants have been reported to have oviposition deterrent activity against mosquitoes (Waliwitiya et al. 2009; Cheah et al. 2013; Dias and Moraes 2014).Development of compounds with deterrence effects could be useful in mosquito control programs.Such compounds can prevent breeding in certain habitats and as such help reduce mosquito population densities and dispersion in conducive areas.

Figure 1 The
Figure 1

Figure 2 Activity
Figure 2

Figure 4 The
Figure 4 (Gulcu et al. 2012006;Gulsen et al. 2022) as well as insect and mite pests(Sergeant et al. 2006; Hinchliffe et al. 2010; Incedayi et al. 2021) of medical, veterinary and agricultural importance.For instance, several recent works (da Silva et al. 2013; Wagutu et al. 2017; Luiz Rosa da Silva et al. 2017; Vitta et al. 2018; Shah et al. 2021) have demonstrated the larvicidal e cacy of cell-free bacterial supernatants and/or bacterial cell suspensions of Xenorhabdus and Photorhabdus on different mosquito species.Attempts to identify the bioactive natural product as potential larvicidal agents from these bacteria are ongoing.Besides larvicidal activity of compounds from these bacteria against mosquito larvae, it was observed that the calliphorid y, Chrysomya albiceps did not lay eggs on treating lamb meat with P. luminescens supernatant(Gulcu et al. 2012) and Lobesia botrana laid 55-95% fewer eggs on grapes treated with X. nematophila and P. laumondii supernatants when compared with untreated grapes (Vicente-Díez et al. 2023).
(Incedayi et al. 2021;Cimen et al. 2021b;Gulsen et al. 2022cedayi et al. 2021;Cimen et al. 2021b;Gulsen et al. 2022).Brie y, X. szentirmaii Δhfq mutants were generated which results in no NP production and subsequently, the native promoter regions of selected NP BGCs listed in Table1were replaced with a chemically inducible promoter P BAD via the integration of the plasmid pCEP-KM; the biosynthesis of the selected BGC and subsequent selective (over) production of the associated single NP compound class can be activated by the addition of L-arabinose(Bode etal.2015; Tobias et al. 2017; Bode et al. 2019; Bode et al. 2023).
Cimen et al. (2021)ere rst cultured in LB agar + kanamycin (50 µg/ml nal concentration) and incubated at 30°C for 48 h according toCimen et al. (2021).Then a single colony was transferred into 10 ml LB medium + kanamycin (50 µg/ml nal concentration) and incubated at 150 rpm and 30°C to obtain an overnight culture which was transferred into 100 ml fresh LBs and the nal optical density (OD 600 )

Table 2
Commercial larvicidal products used in the study.SC; Suspension Concentrate, WDG; Water-dispersible granule.
Several reports have highlighted the importance of the bioactive natural product of Xenorhabdus and Photorhabdus as potential larvicidal agents on different mosquito species (da Silva et al. 2013; Wagutu et al. 2017; Luiz Rosa da Silva et al. 2017; Vitta et al. 2018; al. 2014; Wenski et al. 2020; Wenski et al. 2021).Female mosquitoes are highly sensitive to environmental factors.They can sense the myriad of olfactory, visual, gustatory, and tactile signals emanating from breeding sites as well as various environmental factors such as uctuations in temperature, rainfall, relative humidity that can in uence ovipositional behavior using the sensilla in antennae, maxillary palps, proboscis, and taste organs including tarsal segments of the legs, and cibarium (Bentley and Day 1989; Day 2016; Baik and Carlson 2020; Parker et al. 2020; Girard et al. 2021).They generally seek out suitable aquatic habitats rich with nutrients for her offspring and devoid of potential predators