Association Between Wolbachia Infection and Susceptibility to Deltamethrin Insecticide in Phlebotomus papatasi (Diptera: Psychodidae),the Main Vector of Zoonotic Cutaneous Leishmaniasis

Abstract

Background:

Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of zoonotic cutaneous leishmaniasis. Wolbachia is a symbiotic alphaproteobacteria of arthropods that can be involved in susceptibility or resistance. This study aimed to investigate the relationship between Wolbachia and Deltamethrin susceptibility/resistance in Ph. papatasi. Deltamethrin filter papers (0.00002%) were used to test sand fly field collected from southern Iran. After the test, PCR amplification of the Wolbachia surface protein gene (wsp) was used to measure Wolbachia infection rate in the killed, surviving, and control groups.

Result:

The rates of infection by Wolbachia strain (wPap, super group A) differed between killed (susceptible) and surviving (resistant) Ph. papatasi specimens. The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) in resistant individuals with the same genetic background. This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. In addition, the results showed that Deltamethrin can act as a PCR inhibitor during detection of Wolbachia in Ph. papatasi.

Conclusion:

Results of this study show that Wolbachia is associated with Deltamethrin susceptibility level in Ph. papatasi. Also, as Deltamethrin has been identified as a PCR inhibitor, great care must be taken in interpreting Wolbachia infection status in infected populations. The results of this study may provide information for a better understanding of the host-symbiont relationship, as well as application of host symbiosis in pest management.

Introduction

Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major, is a common zoonotic vector-borne disease in Iran. Close contact with infected reservoir hosts increases the probability of transmission of Leishmania parasite infections to susceptible humans (Abdolahnejad et al., 2021; Bakhshi et al., 2013; Ghassemi et al., 2023; Hajjaran et al., 2013; Maleki-Ravasan et al., 2015; Nezamzadeh-Ezhiyeh et al., 2021; Oshaghi et al., 2011).

Phlebotomus papatasi (Diptera: Psychodidae) is the main vector of ZCL; it is semidomesticated in most of its distribution areas and is the dominant species in human habitats (Abedi-Astaneh et al., 2015; Khamesipour et al., 2020; Rassi et al., 2011; Shirani-Bidabadi et al., 2020). Ph. papatasi has been exposed to four main groups of organic pesticides in Iran (carbamates, organophosphates, organochlorines, and pyrethroids) and although most test results show no resistance to these (Shirani-Bidabadi et al., 2022), some resistance has been reported in a few cases (Ali et al., 2022; Saeidi et al., 2021; Salim Abadi et al., 2022; Shirani-Bidabadi et al., 2022). Several factors are directly and indirectly involved in the development of resistance in insects, including internal symbiotic bacteria (Panini et al., 2016; Soltani et al., 2017).

Wolbachia is one of the most important symbiotic bacteria in insects (Karami et al., 2016; Karimian et al., 2022; Ross et al., 2019; Soh et al., 2022). Wolbachia is a symbiotic alphaproteobacterium, infecting arthropods and nematodes; it is transmitted through the mother and was first reported in Culex pipiens reproductive tissues by Hertig and Wolbach (Bowman, 2011; Hertig and Wolbach, 1924).

Wolbachia affects the host's biological processes, such as growth, reproduction, and immunity (Yen and Barr, 1971), and it expands in the host population by taking over the host reproductive system (Zug and Hammerstein, 2015). Wolbachia can make the host more susceptible and reduce its competence against viral infections (Glaser and Meola, 2010; Kriesner et al., 2016; Moreira et al., 2009), and it can affect the host's sensitivity to pesticides (Berticat et al., 2002; Duron et al., 2006; Echaubard et al., 2010). Cytoplasmic incompatibility (CI) phenomenon is another characteristic of Wolbachia that when Wolbachia-infected males mate with uninfected females, the resulting eggs will die. Also, Wolbachia-infected mosquitoes have a lower fecundity rate and shorter lifespan than uninfected strains (Zhang and Lui, 2020).

Currently, people involved in vector-borne disease control have successfully released these Wolbachia-infected mosquitoes into the field in numerous countries and have attained a high level of vector suppression or inhibited the transmission of a variety of RNA viruses like dengue virus, Zika virus, and other human diseases caused by RNA viruses in mosquitoes (Ferguson et al., 2015; Zhang and Lui, 2020). Wolbachia-infected insects may have both advantages and disadvantages. For instance, deployments of Wolbachia wMel-infected Aedes aegypti mosquitoes, the main vector of dengue, resulted in significant reduction in the application of insecticide (Indriani et al., 2023).

However, Wolbachia may increase or decrease their hosts against xenobiotics and insecticides. Studies on sand flies have shown relatively high, with different percentages of Wolbachia infection (Bell-Sakyi et al., 2021; Karimian et al., 2018; Vivero et al., 2017; Vivero-Gomez et al., 2021), but no study has been done to survey the association between Wolbachia and the level of susceptibility to pesticides. This study aims to investigate the role of Wolbachia in the resistance/susceptibility of Ph. papatasi to Deltamethrin.

Methods

Study area

In this study, we used natural sand fly populations because laboratory colony populations are very weak and highly susceptible to insecticides. Moreover, after few generations, the rate of Wolbachia infection would be increased as Wolbachia transmission happens across generations due to CI, and the rate of infected sand flies rises to become the dominant sand fly strain in the laboratory population. The ages and diets of natural populations may differ; however, the condition was the same for both the control and treatment groups.

Due to the abundance of Ph. papatasi and the use of insecticides against it (Saeidi et al., 2021), the Matin Abad village in southern Iran was selected as an endemic area for sample collection. Live sand fly specimens were collected in June 2022. During the night, sand flies were caught alive using an aspirator device from outside and inside parked vehicles (car) and were transferred to cages that had previously been placed in nylon to maintain humidity and temperature. The cages were transferred to the Research Station laboratory, and after a 2-h rest, the sand flies were prepared for the susceptibility test.

Insecticide susceptibility tests

Sand flies were exposed to filter paper impregnated with technical-grade Deltamethrin insecticide (BATCH No: DE 527), which were obtained by Centers for Disease Control and Prevention (CDC), Ministry of Health and Medical Education, Iran, by the World Health Organization (WHO) collaborating center in University Sains Malaysia, Penang, Malaysia.

Control papers were equipped using “silicone oil and acetone”-impregnated paper (0.66 mL oil +1.34 mL acetone) according to the standard method of World Health Organization Pesticides Evaluation Scheme (WHOPES) Institute of Research for Development (IRD), Montpellier, France. The diagnostic dose (0.05%) for sand flies was used as the starting reference point (WHO, 2016) for initial insecticide exposure; however, due to high susceptibility rate observed in the sand flies tested, the concentrations of the insecticide to which Ph. papatasi were exposed were adjusted as 0.00000625 0.0000125, 0.000025, 0.00005, 0.0001, 0.001, and 0.01% (Saeidi, 2021). These concentrations were used to derive the dose–response survival curves for the Ph. papatasi population.

Following exposure, the sand flies were let to recover for 24 h, after which mortality was documented. From dose–response survival curves for Ph. papatasi created with the Probit software, the lethal concentration causing 50% mortality (LC50) was determined as 0.00002% for Deltamethrin, which can separate susceptible individuals from nonsusceptible ones after exposure. Following diagnosis LC50, in the final tests, for each contact and control tube, 25 sand fly specimens (unfed blood) were placed for 60 min. Based on WHO guidelines (WHO, 2016), the tests were performed in 12 replicates for the treatment group (red point tubes) and three replicates for the control group (green point tubes). After the test, the sand flies were transferred to holding tubes under insectarium conditions (temperature 28 ± 5°C and relative humidity 70 ± 5).

After 24 h, the dead sand flies were removed, and then the holding tubes were transferred to the freezer to kill the live sand flies. The two groups—live and dead—of sand fly specimens were identified by morphological characteristics (Lewis, 1982; Theodor and Mesghali, 1964) and Ph. papatasi specimens were kept in two separate groups in 1.5 mL Eppendorf tubes with 96% Ethanol for further analysis to determine their Wolbachia infection. The number of dead adults was recorded after 24 h of testing, the rate of adults' death was calculated for each group, and the results were analyzed statistically.

Wolbachia detection

To identify Wolbachia in the two groups, 100 susceptible (killed in the bioassay) and 100 resistant (live specimens in bioassay) insects were selected randomly, with each group containing 50 male and 50 female Ph. papatasi specimens. As an additional control for the effect of insecticide on the PCR, a group of field-collected sand flies with no exposure to Deltamethrin (n = 73) was tested for Wolbachia infection. Total genomic DNA of the specimens was extracted individually using the Collins method (Collins et al., 1987) and subjected to PCR amplification of the Wolbachia surface protein gene (wsp) to identify the bacteria (Zhou et al., 1998).

For PCR, each 25 μL reaction solution contained 12.5 μL of master mix (AMPLIQON, Denmark), 2 μL (0.5 μM) each of the two general wsp primers (wsp81F: 5′-TGG TCC AAT AAG TGA TGA AGA AAC-3′ and wsp691R: 5′-AAA AAT TAA ACG CTA CTC CA-3′) (Zhou et al., 1998), 2 μL DNA as template, and 8.5 μL double distilled water. These primers amplify a DNA fragment ranging from 590 to 632 bp. The amplification conditions were 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 45s, annealing at 47°C for 50s, and extension at 72°C for 60s, with a final extension step at 72°C for 10 min. Individual specimens of Anopheles stephensi and Drosophila melanogaster were used as PCR-negative and PCR-positive controls, respectively.

All PCR products were run on 1.5% agarose gel electrophoresis with a safe stain solution and viewed under UV light before Sanger sequencing by a commercial laboratory (Codon Company, Iran). The sequences were checked and edited using Chromas software and the consensus of confident sequences was analyzed using NCBI (Nucleotide collection) database and categorized at supergroup and subgroup level. A representative wsp gene sequence of Wolbachia found in the Ph. papatasi population was submitted to GenBank. Statistical parameters were extracted from the toxicity rates and the rate of positive PCR in the two groups was analyzed with each other using a chi-square test in SPSS software.

Results

The lethal concentration causing 50% mortality (LC50) was determined as 0.00002% for Deltamethrin against the Ph. papatasi population. In this study, exposure of the wild sand fly's population with LC50 for Deltamethrin showed that out of 300 sand flies in the treatment group and 75 sand flies in the control group, 296 and 73 were Ph. papatasi, respectively. The results of the susceptibility tests showed that in the treatment group, 141 Ph. papatasi were killed (47.6%) and 155 remained alive (54.4%). Of the 141 killed Ph. papatasi, 74 were male and 67 were female. In addition, of 155 Ph. papatasi in the surviving group, 66 were female and 89 were male (Table 1).

Table 1.

The Results of the Susceptibility Test to Deltamethrin (0.00002%) of Phlebotomus papatasi Sand Flies from Southern Iran, 2022

Group	n	No of specimens in susceptibility tests		Susceptibility status of Ph. papatasi					Susceptibility status of Ph. papatasi males and females
		Ph. papatasi	Other species	n	Killed		Alive		Killed		Alive
		Ph. papatasi	Other species	n	n	%	n	%	Male	Female	Male	Female
Control	75	73	2	25	2	8.0	23	92.0	2	0	13	10
				23	2	8.7	21	91.3	1	1	9	12
				25	3	13.0	22	88.0	2	1	11	11
Subtotal				73	7	9.6	66	90.4	5	2	33	33
Treatment	300	296	4	25	14	56.0	11	44.0	6	8	7	4
				25	11	44.0	14	56.0	5	6	9	5
				25	13	52.0	12	48.0	8	5	6	6
				24	10	41.6	14	58.4	4	6	8	6
				24	13	54.1	11	45.9	6	7	6	5
				25	12	48.0	13	52.0	5	7	7	6
				25	12	48.0	13	52.0	6	6	8	5
				25	9	36.0	16	64.0	4	5	7	9
				23	12	52.1	11	47.9	9	3	8	3
				25	11	44.0	14	56.0	7	4	5	9
				25	10	40.0	15	60.0	5	5	11	4
				25	14	56.0	11	440	9	5	7	4
Subtotal				296	141	47.6	155	52.4	74	67	89	66
Total	375	369	6	369	148	—	221	—	79	69	122	99

The number of killed and alive specimens in the treatment group was not different significantly using a chi-square test (p > 0.05).

The results of molecular studies showed that the assay amplified a fragment of about 600 bp from the wsp gene of Wolbachia genome. A subset of PCR products was subjected to sequencing and the sequences obtained were BLAST checked and the consensus sequence was submitted to GenBank (GenBank ID: OP959792). The results confirmed that the amplicons were of Wolbachia. The BLAST search revealed that the sequences were 100% homologous to that of Wolbachia wPap (super group A) strain (a Wolbachia strain isolated from Ph. papatasi) previously recorded in GenBank (accession number: AF237882).

The rate of Wolbachia infection in susceptible individuals was more than twice (2.3) (39% vs. 17%) that of resistant individuals with the same genetic background (Table 2). This difference was highly significant (p < 0.001), indicating a positive association between Wolbachia infection and susceptibility to Deltamethrin. The results of molecular studies showed that, of 50 live (Deltamethrin resistant) male specimens of Ph. papatasi after the susceptibility test, 42 (84.0%) were not infected with Wolbachia, and only 8 (16.0%) were infected. Also, of 50 female samples prepared from live (Deltamethrin resistant), 41 (82.0%) were not infected with Wolbachia and only 9 (18.0%) were infected.

Table 2.

Wolbachia Infection Rate in Susceptible and Resistant Strains of Ph. papatasi After Susceptibility Test by Impregnated Deltamethrin Paper (0.00002%) World Health Organization Standard Kit

Group		Sex	Total	Positive for Wolbachia		Negative for Wolbachia		Total negative for Wolbachia		Total positive for Wolbachia
Group		Sex	Total	n	%	n	%	n	%	n	%
Exposed to Deltamethrin	Alive	Male	50	8	16.00	42	84.00	17	17.00	83	83.00
	Alive	Female	50	9	18.00	41	82.00
	Killed	Male	50	18	36.00	32	64.00	39	39.00	61	61.00
	Killed	Female	50	21	42.00	29	58.00
No exposure	Control	Male	41	25	60.97	16	39.02	42	57.53	31	42.46
No exposure	Control	Female	32	17	53.12	15	46.87

The rate of Wolbachia infection between alive and killed groups was significantly different using a chi-square test (p < 0.001).

The results also showed that of 50 Deltamethrin-susceptible male Ph. papatasi, 32 (64.0%) did not harbor Wolbachia and 18 (36%) were infected, and of 50 female Deltamethrin-susceptible Ph. papatasi, 29 (58.0%) were not infected and 21 (42.0%) were infected. The overall results showed that 83% of Deltamethrin-resistant Ph. papatasi were not infected with Wolbachia and only 17% were infected. In addition, 61% of Deltamethrin-susceptible Ph. papatasi were Wolbachia negative, while 39% were Wolbachia positive (Table 2). The Wolbachia infection rate in the control group (not exposed to Deltamethrin) was 57%, which is almost 1.5 times that of the exposed specimens, indicating that Deltamethrin can act as a PCR inhibitor for Wolbachia detection.

Discussions

In this study, we investigated the relationship between Wolbachia infection and insecticide susceptibility in Ph. papatasi. Our results show that Wolbachia infection is more than twice as common in the Deltamethrin-susceptible specimens as in the resistant specimens, indicating that Wolbachia is associated with Deltamethrin susceptibility levels in Ph. papatasi, and the bacterium increases the insect's susceptibility to Deltamethrin. This study provides a basis for a symbiont-mediated mechanism in influencing insecticide susceptibility, which was previously unknown to Ph. papatasi.

The association between Wolbachia infection and susceptibility to Deltamethrin insecticides in this sand fly species is congruent with some previous studies in other insect species. In a study by Shemshadian et al. (2021), a positive association was found between Wolbachia infection and susceptibility to Deltamethrin in Culex quinquefasciatus, but they showed neutral effect on dichlorodiphenyltrichloroethane susceptibility. On the other hand, Soh and Veera Singham showed that Wolbachia increased the susceptibility of insects to fenitrothion and imidacloprid, but not to Deltamethrin (Soh and Veera Singham, 2022). In another study by Dângelo et al. (2021), it was shown that Wolbachia was absent in whitefly Bemisia tabaci resistance to lambda-cyhalothrin and spiromesifen insecticides.

However, other studies found no association between Wolbachia and susceptibility to insecticides: for example, in a study testing the relationship between Wolbachia and buprofezin susceptibility in Laodelphax striatellus, which suggested not only that Wolbachia effects on chemical resistance are complex and unpredictable but also that they can be substantial (Li et al., 2018). This suggestion is supported by Endersby and Hoffmann (2013), who studied the differences in response to four classes of insecticides (bifenthrin as a pyrethroid, Bacillus thuringiensis as a bioagent, temephos as an organophosphate, and methoprene as an insect growth regulator) between Wolbachia-infected and uninfected lines of Ae. aegypti. They reported that, while differences in response between lines were noted for some insecticides, no obvious or constant effect related to Wolbachia infection was detected.

Also, studies of Tantowijoyo et al. (2022) showed no difference in the insecticide resistance phenotypes of Ae. aegypti in the first and second years after wMel deployments in Indonesia. In another study, it was shown the Wolbachia presence in Ae. albopictus and Cx. pipiens did not provide a protective advantage against entomopathogenic fungal infection (Ramirez et al., 2021). These studies suggest that endosymbionts generally rise host susceptibility to chemical insecticides, but cases of increased resistance also exist (Liu and Guo, 2019). For example, it was shown that Wolbachia increases the expression of NlCYP4CE1 to promote the detoxification metabolic response to imidacloprid stress in the brown planthopper (BPH), Nilaparvata lugens (Cai et al., 2021).

Other studies also showed that Wolbachia has much greater densities in insecticide-resistant Cx. pipiens mosquitoes than in insecticide-susceptible individuals (Echaubard et al., 2010; Shan-Chao et al., 2014; Wang et al., 2021). A noteworthy positive association was observed between Wolbachia titer and the resistance level of whiteflies to neonicotinoids, such as acetamiprid, imidacloprid, and thiamethoxam (Barman et al., 2022; Ghanim and Kontsedalov, 2009). Also, some symbionts, such as Burkholderia in the bean bug Riptortus pedestris (Hemiptera: Coreidae) and Citrobacter sp. in Bactrocera dorsalis (Diptera: Tephritidae), can help the insects to degrade Fenitrothion and Trichlorfon, respectively, and confer resistance (Cheng et al., 2017; Kikuchi et al., 2012).

In contrast, Pang et al. (2018) suggested that one Arsenophonus strain (S-type) adversely affected the insecticide resistance in the BPH, Nilaparvata lugens, a major pest insect for rice crop in Asian countries, and significantly decreased the host insecticide resistance to imidacloprid. It is suggested that the symbiont-mediated insecticide susceptibility/resistance differs with host as well as symbiont species, density of endosymbionts, upregulation or downregulation of xenobiotic metabolism, and type of insecticide. Impacts of endosymbionts on host metabolism, fitness, gene expression, and immune system may regulate how endosymbionts affect insecticide susceptibility/resistance (Liu and Guo, 2019). However, in this study, based on the mortality rates of Ph. papatasi, it can be concluded that the presence of Wolbachia increases its susceptibility to Deltamethrin.

In this study, the Wolbachia infection rate of screened individuals exposed to Deltamethrin was 28%, which is inconsistent with that of the control group (57%) in the same population, and with a previous study from the same area that showed an 83% infection rate (Karimian et al., 2018). We suggest that the incongruence between the exposed and unexposed specimens is probably the result of Deltamethrin exposure, and the disagreement between our report and previous one is due to sample size and seasonal factors. It has been shown that insecticide residues can act as inhibitors of PCR and may influence its results, depending on the pesticide groups (Espeland et al., 2010; Nabil et al., 2011).

These results emphasize the importance of assessing the existence of inhibitors in PCR assays by using a known positive standard to allow the accurate determination of Wolbachia infection status in infected populations (Beckmann and Fallon, 2014). It is worth mentioning that the reported rates of Wolbachia infection in sand flies is quite variable: 0.91% in Mexico (Lozano-Sardaneta et al., 2022), 17% in different species and locations (Ono et al., 2001), 20% in Colombia (Vivero et al., 2017), 21.4% in South of Iran (Alipour et al., 2021), 66.7% in Brazil (Cruz et al., 2021), 80% in Iran (Karimian et al., 2018), and 60.3% and 81.7% in Ph. perniciosus and Ph. papatasi, respectively, from Europe, Tunisia, and Iran (Benlarbi and Ready, 2003). This variation is probably due to the broad range of species and the geographical regions tested.

Conclusion

Wolbachia is associated with Deltamethrin resistance in Ph. papatasi and the bacterium increases the vector's susceptibility to the insecticide.

Footnotes

Acknowledgment

The article was edited by the ICGEB Editing service.

Ethical Approval

The protocols conducted in this study followed the guidelines of the institutional ethical committee (Tehran University of Medical Sciences, TUMS). The protocols were approved by TUMS ethical committee under registry IR.TUMS.SPH.REC. 1400.267.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This research was supported by Elite Researcher Grant Committee under award no. 4000968 from the National Institutes for Medical Research Development (NIMAD) and by Iran National Science Foundation (INSF) under award no. 4002768, Tehran, Iran. Also, this study was supported by the Tehran University of Medical Sciences, Iran (grant no. 54570).

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