Isothiazolinones As Novel Candidate Insecticides for the Control of Hemipteran Insects

antibiotics

Article Isothiazolinones as Novel Candidate Insecticides for the Control of Hemipteran Insects

Wenze He, Lilong Pan , Wenhao Han and Xiaowei Wang *

Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China; [email protected] (W.H.); [email protected] (L.P.); [email protected] (W.H.) * Correspondence: [email protected]

Abstract: Hemipteran insects, such as whiteflies, aphids and planthoppers, resemble one of the most important pest groups threating food security. While many insecticides have been used to control these pests, many issues such as insecticide resistance have been found, highlighting the urgent need to develop novel insecticides. Here, we first observed that a commercial tetramycin solution was highly effective in killing whitefly. The major bioactive constituents were identified to be isothiazolinones, a group of biocides. We then tested the toxicity of several isothiazolinones to five hemipteran insects. The results show that Kathon, a widely used biocide against microorganisms, and its two constituents, chloromethylisothiazolinone (CMIT) and methylisothiazolinone (MIT), can cause considerable levels of mortality to whiteflies and aphids when applied at concentrations close to, or lower than, the upper limit of these chemicals permitted in cosmetic products. The results also indicate that two other isothiazolinones, benzisothiazolinone (BIT) and octylisothiazolinone

(OIT) can cause considerable levels of mortality to whiteﬂy and aphids but are less toxic than Kathon. Further, we show that Kathon marginally affects whiteﬂy endosymbionts, suggesting its insecticidal

Citation: He, W.; Pan, L.; Han, W.; activity is independent of its biocidal activity. These results suggest that some isothiazolinones Wang, X. Isothiazolinones as Novel are promising candidates for the development of a new class of insecticides for the control of Candidate Insecticides for the Control hemipteran pests. of Hemipteran Insects. Antibiotics 2021, 10, 436. https://doi.org/ Keywords: benzisothiazolinone; hemiptera; insecticide; isothiazolinone; Kathon; octylisothiazolinone 10.3390/antibiotics10040436

Academic Editors: Roger C Levesque and Marc Maresca 1. Introduction As the global population will continue to grow to roughly nine billion by the middle of Received: 20 March 2021 this century, sustaining crop production is vital, which entails, among others, efficient crop Accepted: 10 April 2021 Published: 14 April 2021 protection against insect pests [1,2]. Among insect pests, hemipterans such as whiteflies, aphids and planthoppers, are one of the most significant groups threating food security as

Publisher’s Note: MDPI stays neutral they damage crops directly by feeding and indirectly by transmitting plant diseases [3,4]. with regard to jurisdictional claims in Some species of the Bemisia tabaci whitefly complex are pests of many crops worldwide [5,6]. published maps and institutional affil- Whitefly feeding on crops may result in reduced plant vigor and physiological perturbation; iations. more importantly, whiteflies can transmit many plant viruses belonging to the genera Begomovirus (Geminiviridae), Crinivirus (Closteroviridae), Torradovirus (Secoviridae), among others [7–9]. Aphids represent a serious challenge for the sustainable production of many important cereal and vegetable crops; they can remove plant sap, secret phytotoxic salivary components and transmit viruses such as potyviruses [10]. Likewise, planthoppers are Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. economically important pests of many cereals such as rice; they feed directly or serve This article is an open access article as vectors of pathogenic microorganisms including viruses, resulting in significant crop distributed under the terms and damage and yield losses [11]. conditions of the Creative Commons While the importance of integrated pest management is being increasingly realized, Attribution (CC BY) license (https:// the application of chemical pesticides remains necessary, and in many cases, the only creativecommons.org/licenses/by/ crop protection measure in agricultural practices [1]. Although many compounds, such as 4.0/). organophosphates, carbamates, pyrethroids, and more recently, neonicotinoids are effective

Antibiotics 2021, 10, 436. https://doi.org/10.3390/antibiotics10040436 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 12

Antibiotics 2021, 10, 436 2 of 12 protection measure in agricultural practices [1]. Although many compounds, such as organophosphates, carbamates, pyrethroids, and more recently, neonicotinoids are effective in manymany circumstances,circumstances, to controlcontrol ofof hemipteranhemipteran pests,pests, extensiveextensive applicationsapplications ofof thesethese chemicals have led to the emergence of resistance in these insects in addition to off-target effects on on non-target non-target organisms organisms and and the the enviro environmentnment [11–14]. [11–14 Based]. Based on this on thisscenario, scenario, it is itimperative is imperative to develop to develop new classes new classes of insecticides, of insecticides, preferably preferably with novel with modes novel modesof action. of action.However, However, nowadays nowadays the identification the identification and development and development of new ofinsecticides new insecticides is accom- is accomplishedplished by only by a onlylimited a limited number number of companies, of companies, probably probably due to duethe fact to the that fact the that devel- the developmentopment of a new of a insecticide new insecticide is often is oftenvery costly, very costly, in terms in terms of both of bothfinancial financial input input and time and time[15]. Hence, [15]. Hence, identifying identifying new ch newemicals chemicals with insecticidal with insecticidal properties properties from fromexisting existing com- compounds,pounds, presently presently not notused used as asinsecticides, insecticides, may may contribute contribute to to the the development development of novel insecticides. Tetramycin was obtained from the fermentation of Streptomyces ahygroscopicus subsp. Wuzhouensis. ItIt exhibitsexhibits significant significant antibiotic antibiotic activity activity against against a widea wide range range of microorgan-of microor- ismsganisms such such as bacteria as bacteria and and fungi fungi [16]. [16]. In China, In China, tetramycin tetramycin has beenhas been widely widely used used to treat to fungaltreat fungal diseases diseases in plants in plants [17]. [17]. In an In experiment an experiment to eliminate to eliminate endosymbionts endosymbionts in whiteflies, in white- weflies, accidentally we accidentally noticed noticed thattreatment that treatment of whiteflies of whiteflies with awith commercial a commercial tetramycin tetramycin solutionsolution resulted resulted in high in high mortality mortality within within 48 h.48 Sinceh. Since tetramycin tetramycin has has no no insecticidal insecticidal activity, activ- weity, postulatedwe postulated that that some some other other components components in in the the formulated formulated tetramycin tetramycin solutionsolution maymay kill whiteflies.whiteflies. We analyzedanalyzed thethe tetramycintetramycin solutionsolution usingusing LiquidLiquid chromatography-masschromatography-mass spectrometry (LC-MS) and surprisingly found that isothiazolinones were present inin thethe solution and have insecticidal activity. Isothiazolinones are a class of broadbroad spectrumspectrum microbicides that have been widelywidely used to control the growth of microorganisms suchsuch asas bacteria, fungifungi andand yeast in cooling water systems and and cosmetics, cosmetics, among among others others [18]. [18]. However, However, whether whether they they can can be beused used as asinsecticides insecticides for forthe thecontrol control of agricultural of agricultural pests, pests, remains remains unexplored. unexplored. We tested We tested the most the mostwidely widely used isothiazolinone used isothiazolinone Kathon, Kathon, which which is a combination is a combination of 5-chloro-2-methyl-4-iso- of 5-chloro-2-methyl- 4-isothiazolin-3-onethiazolin-3-one (Chloromethylisothiazolinone (Chloromethylisothiazolinone,, CMIT) CMIT) (Figure (Figure 1A) and1A) 2-methyl-4-isothi- and 2-methyl-4- isothiazolin-3-oneazolin-3-one (Methylisothiazolinone, (Methylisothiazolinone, MIT) (Figure MIT) (Figure 1B) in 1anB) approximate in an approximate 3:1 ratio. 3:1 We ratio. as- Wesessed assessed the toxicity the toxicity of Kathon of Kathon to five tospecies five species of hemipteran of hemipteran insects insectsincluding including three species three speciesfrom the from B. tabaci the B. whitefly tabaci whitefly complex, complex, the green the greenpeach peach aphid aphid MyzusMyzus persicae persicae and andthe rice the ricebrown brown planthopper planthopper NilaparvataNilaparvata lugens lugens. Further,. Further, we assessed we assessed the toxicity the toxicity of another of another two twowidely widely used used isothiazolinones isothiazolinones 1,2-Benzisothiazolin-3-One 1,2-Benzisothiazolin-3-One (Benzisothiazolinone, (Benzisothiazolinone, BIT) (Fig- BIT) (Figure1C) and 2-N-octyl-4-isothiazolin-3-one (Octylisothiazolinone, OIT) (Figure1D) to ure 1C) and 2-N-octyl-4-isothiazolin-3-one (Octylisothiazolinone, OIT) (Figure 1D) to whitefly and the green peach aphid. Finally, we explored the effects of Kathon treatment whitefly and the green peach aphid. Finally, we explored the effects of Kathon treatment on the density of endosymbionts in whitefly. Our results provide novel insights into the on the density of endosymbionts in whitefly. Our results provide novel insights into the insecticidal properties of isothiazolinones, and highlight the potential of these compounds insecticidal properties of isothiazolinones, and highlight the potential of these compounds in the development of novel insecticides for the control of hemipteran pests. in the development of novel insecticides for the control of hemipteran pests.

Figure 1. Chemical structure of CMIT (A), MIT ((B),), BITBIT ((C)) andand OITOIT ((D).). Antibiotics 2021, 10, 436 3 of 12

2. Materials and Methods 2.1. Insects Three species of whiteflies of the B. tabaci complex, namely Middle East-Asia Minor 1 (MEAM1), Mediterranean (MED) and Asia II 1, were tested. The mtCOI GenBank accession codes were KM821540 for MEAM1, GQ371165 for MED and DQ309077 for Asia II 1. A laboratory culture of each of the three species of whiteflies was originally established from field samples, collected in Zhejiang, China in 2012 (MEAM1), 2009 (MED) and 2010 (Asia II 1). Whiteflies were maintained on cotton plants (Gossypium hirsutum cv. Zhemian1793) in climate-controlled chambers at 26 ± 2 ◦C, 60–80% relative humidity and a 14/10 h light/dark cycle without any contact with insecticides. Unless specified otherwise, adult whiteflies 1–7 days post emergence were used in the experiments. A laboratory culture of the green peach aphid M. persicae was originally established from a field sample collected from field-grown tobacco plants (Nicotiana tabacum) on the Zijingang Campus of Zhejiang University, Hangzhou, China in 2018. The aphid culture was maintained on tobacco plants (N. tabacum cv. NC89) in a climate-controlled chamber at 23 ± 2 ◦C, 50–60% relative humidity and a 16/8 h light/dark cycle without any contact with insecticides. Test insects of the rice brown planthopper N. lugens were provided by Professor Hai-Jun Xu (Institute of Insect Sciences, Zhejiang University) [19]. The culture of brown planthopper was collected in 2008 and maintained thereafter on rice seedlings (Oryza sativa cv. Xiushui 134) in a climate-controlled chamber at 26 ± 0.5 ◦C, 50–60% relative humidity and a 16/8 h light/dark cycle without any contact with insecticides.

2.2. Chemicals Tetramycin (0.3% in water) was purchased from Liaoning Wkioc Bioengineering Co., Ltd. (Liaoning, China). Kathon (3% in glycol) and MIT (Figure1B) (9.5% in water) were purchased from Sigma-Aldrich. CMIT (Figure1A) (1.5% in water), BIT (Figure1C) (85% in water) and OIT (Figure1D) (100%) were purchased from Hubei Jusheng Technology Co., Ltd. (Hubei, China). Acetamiprid (20% in water) was purchased from Shandong Pharmaceutical Co., Ltd. (Shandong, China).

2.3. Analysis of Insecticidal Activity of Tetramycin and Kathon against Whiteflies via Oral Route To assess the insecticidal activity of tetramycin solution against whitefly, 15% sucrose solutions containing 80, 40, 20, 10, 5 and 0 mg/L tetramycin were prepared. Similarly, 15% sucrose solutions containing 600.00, 300.00, 150.00 and 0 mg/L Kathon were prepared. Membrane feeding was conducted as mentioned before [20]. Five replicates were conducted for each concentration and 50 whiteflies were tested in each replicate.

2.4. Liquid Chromatography-Mass Spectrometry (LC-MS) LC-MS was employed to identify the bioactive chemicals in tetramycin solution as reported before [21]. Waters Ultra Performance Liquid Chromatography (UPLC) and AC- QUITY UPLC HSS SB-C18 column (1.7 µm, 2.1 × 100 mm) (Waters, MA, USA) was used in the chromatographic experiments. The mobile phases were 0.1% formic acid-water and 0.1% formic acid-acetonitrile. Sample injection volume, 3 µL; column oven temperature, 30 ◦C; ﬂow rate, 0.4 mL/min; and UV detector was set at 254 nm. AB TripleTOF 5600plus System (AB SCIEX, Framingham, MA, USA) was employed for mass spectrometry. The op- timal MS conditions: negative ion mode: Source voltage was −4.5 kV, and the source temperature was 550 ◦C. Positive ion mode: Source voltage was +5.5 kV, and the source temperature was 600 ◦C. Maximum allowed error was set to ±5 ppm. Decluttering potential (DP), 100 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the parameters were the same except that the collision energy (CE) was set at 40 ± 20 V, ion release delay (IRD) at 67, ion release width (IRW) at 25. The IDA-based auto-MS2 was performed on the eight most intense metabolite ions in a cycle of full scan (1s). The scan range of m/z of precursor ion and product ion were set as 100–2000 Da and 50–2000 Da. The exact mass Antibiotics 2021, 10, 436 4 of 12

calibration was performed automatically before each analysis employing the Automated Calibration Delivery System. The data were analyzed using Peakview1.2 (AB SCIEX, Framingham, MA, USA).

2.5. Analysis of Insecticidal Activity of Kathon against Whiteflies via Spraying To assess the insecticidal activity of Kathon against the adults of MEAM1 whitefly via spraying, the chemical was firstly diluted with water containing 1% surfactant (Tween-80) to six concentrations, namely 150.00, 75.00, 37.50, 18.75 and 9.38 mg/L. Water containing 1% surfactant (Tween-80) was used as negative control, and Acetamiprid (30.00 mg/L in water) was included as positive control. For MEAM1 nymphs, Asia II 1 and MED adults, 18.75 and 9.38 mg/L Kathon were used. For tests with whitefly adults, the test insects were confined on cotton leaves and then Kathon solutions were sprayed onto leaves. Specifically, in each replicate, approximately 100 whitefly adults were collected and released into a clip cage, which was covered with gauze and placed on the undersurface of a cotton leaf. Two hours later, live whiteflies were counted as the starting number of insects to be tested. The clip cage was then sprayed with a test solution using a hand-held sprayer as previously reported [22,23]. The gauze used allowed for efficient penetration of chemical solutions. For each of the four treatments of a whitefly species/stage, three replicates were conducted. Forty-eight hours later, the number of live and dead adult whiteflies were counted, and individuals were counted as dead when they were observed with a lack of discernible movement or obvious desiccation [24]. For the tests with MEAM1 whitefly nymphs, approximately 100 adult whiteflies were firstly collected and released into a clip cage placed on the undersurface of a cotton leaf. Fourteen days later, whitefly adults were removed, and the nymphs were then counted as the test insects (60–180 nymphs of all instars). Spray of Kathon solutions was then conducted as described above. The number of live nymphs was counted 48 h later, and dehydrated nymphs were considered dead.

2.6. Insecticidal Activity of CMIT and MIT against MEAM1 Whitefly To assess the insecticidal activity of CMIT and MIT against MEAM1 whitefly adults, each of the two chemicals was diluted with water containing 1% surfactant (Tween-80) to two concentrations, namely 9.38 and 18.75 mg/L. Negative and positive controls were prepared as described above. Protocols of application of the test solutions and observa- tion of mortality were conducted via spraying as above for Kathon. For each of the four treatments of CMIT or MIT, five replicates were conducted.

2.7. Insecticidal Activity of Kathon against the Green Peach Aphid Kathon solutions of various concentrations (18.75 and 9.38 mg/L), negative (water containing 1% Tween-80) and positive controls (30.00 mg/L Acetamiprid in water) were prepared, as described above. Leaves (30–50 cm2) of tobacco plants grown in greenhouses without any insecticide treatment were collected, soaked in Kathon solutions for 10 s and air-dried. Wingless aphid adults or nymphs were collected using a ﬁne brush and placed onto the treated leaves, 15 individuals per leaf. The leaves were placed on agarose medium in 9 cm petri dishes to retain freshness. Live aphids were counted 48 h post transfer, and aphids that were completely immobile when touched upon or dehydrated were considered dead. For each of the four treatments for CMIT or MIT, three replicates were conducted.

2.8. Insecticidal Activity of Kathon against the Rice Brown Planthopper A preliminary trial indicated that the rice brown planthopper was far less susceptible to Kathon than whiteﬂies or the green peach aphid. For the formal test, here, Kathon was diluted with water, containing the 1% surfactant (Tween-80) to two concentrations, namely 150.00 and 300.00 mg/L. Negative and positive controls were prepared, as described above. Rice plants that were grown in greenhouses without any insecticide treatment and at the booting stage were collected and washed thoroughly. Rice stems (about 10 cm in length) Antibiotics 2021, 10, 436 5 of 12

with roots were cut, air-dried, dipped in Kathon solutions in groups of three for 30 s and then air-dried again for 2 h. The rice stems were then covered with moistened cotton wool around the roots and placed into 500 mL plastic cups, one group per cup. Next, 20 third-instar nymphs or adults of the rice brown planthopper were transferred into each plastic cup using a ﬁne brush. Live planthoppers were counted at 48 h post transfer, and planthoppers that were completely immobile when touched upon or dehydrated were considered dead. Three replicates were conducted for each of the four treatments for adults or nymphs.

2.9. Insecticidal Activity of BIT and OIT against the MEAM1 Whitefly and the Green Peach Aphid By consulting the results of a preliminary trial, BIT and OIT were diluted with water containing 1% surfactant (Tween-80) to a final concentration of 150.00 mg/L. Negative and positive controls were prepared as described above. The mortality of MEAM1 whitefly adults or aphid adults was assessed as described above. Three replicates were conducted for each of the four treatments for the MEAM1 whitefly or the green peach aphid.

2.10. Analysis of Relative Endosymbiont Density Following Kathon Treatment Kathon solutions of 4.69 and 9.38 mg/L were prepared and water containing 1% surfactant (Tween-80) was used as negative control. Whiteflies were collected and released into leaf-clip cages, and then Kathon solutions were applied by spraying as mentioned above. Forty-eight hours later, whiteflies were collected in groups of 15 and then subjected to DNA extraction and analysis of endosymbionts as reported before [25]. Briefly, lysis buffer (5 mmol/L of Tris-HCl (pH 8.0), 0.5 mmol/L EDTA, 0.5% Nonidet P-40 and 1 mg/mL of proteinase K) was used for whitefly DNA extraction. Endosymbionts were quantified by quantitative real-time PCR (qPCR) with SYBR Premix Ex TaqTM (Takara, Japan) and Bio-Rad CFX96 Real- Time System (Bio-Rad, Hercules, CA, USA) with primers listed in Shan et al. (2019)

2.11. Statistical Analysis For the analysis of whitefly mortality, all percentage data were arcsine square root transformed prior to statistical analysis, and back-transformed for presentation. Relative endosymbiont density was calculated as normalized to whitefly actin. For the comparisons of whitefly mortality and endosymbiont density among different treatments, one-way analysis of variance, followed by Fisher’s least significance divergence (LSD) test was used. The differences between treatments were considered significant when p < 0.05. All data are presented as mean ± standard error of mean. All statistical analyses were performed using SPSS 20.0.

3. Results 3.1. Effects of Tetramycin Treatment on the Survival of MEAM1 Whitefly via Oral Route and Identification of Bioactive Constituents We first explored the effects of tetramycin treatment on the survival of MEAM1 whitefly (Figure2A). The mortality of whitefly increased with increasing concentrations of tetramycin, reaching 100.0% when the concentration was 80 mg/L. Since no insecticidal activity of tetramycin has been reported, but has been used in the field for many years, we speculated some other components in the formulated tetramycin solution may be responsible for killing whiteflies. We used LC-MS and identified two chemicals in the formulated tetramycin solution we used (Figure2B), and followed MS analysis identified these chemicals as 2- methyl-4-isothiazolin-3-one (Figure S1) and 2-methyl-4-isothiazolin-3- one hydrochloride (Figure S2). Both chemicals are members of isothiazolinones, a group of broad-spectrum biocides. Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 12

tetramycin solution we used (Figure 2B), and followed MS analysis identified these chemicals as 2- methyl-4-isothiazolin-3-one (Figure S1) and 2-methyl-4-isothiazolin-3-one hy- Antibiotics 2021, 10, 436 drochloride (Figure S2). Both chemicals are members of isothiazolinones,6 of 12 a group of broad-spectrum biocides.

Figure 2. 2.Effects Effects of tetramycinof tetramycin treatment treatment on whitefly on survivalwhitefly and survival LC-MS analysis and LC-MS of the formulated analysis of the formu- tetramycinlated tetramycin solution. solution. Solutions Soluti of 15%ons sucrose, of 15% containing sucrose, various containing concentration various of concentration formulated of formu- tetramycinlated tetramycin solution weresolution prepared were and prepared presented and to whitefly presented orally to by whitef membranely orally feeding by ( Amembrane). LC- feeding MS(A). analysis LC-MS profile analysis of formulated profile of tetramycin formulated solution tetramycin (B). Different solution letters above(B). Different bars in A indicateletters above bars in A significantindicate significant difference ( pdifference< 0.05, one-way (p < 0.05, analysis one-way of variance analysis and Fisher’s of varian LSDce test). and PeaksFisher’s in BLSD test). Peaks representin B represent different different chemicals chemicals detected in liquiddetected chromatography. in liquid chromatography. 3.2. Effects of Kathon on the Survival of Three Species of Whiteflies 3.2. EffectsWe then of tested Kathon the on efficacy the Surv of Kathon,ival of Three the most Species widely of Whiteflies used isothiazoline with a combinationWe then of CMITtested and the MIT efficacy in an approximate of Kathon, 3:1 the ratio, most against widely the used adults isothiazoline of MEAM1 with a com- whitefly via oral route, and found that Kathon killed 100% of whiteflies when concen- trationsbination were of 600.00,CMIT 300.00 and andMIT 150.00 in an mg/L approximate (Figure3A). 3:1 Next, ratio, a spraying against method the wasadults of MEAM1 employedwhitefly forvia whitefly oral route, bioassay and and found lower that concentrations Kathon killed were 100% used. Acetamipridof whiteflies were when concentra- includedtions were as a600.00, positive 300.00 control. and When 150.00 MEAM1 mg/L adults (Figure were 3A). treated Next, with a Kathonspraying at the method was em- concentrationsployed for whitefly of 75.00, 37.50,bioassay 18.75 and and 9.38lower mg/L, concentrations considerable levels were of used. mortality Acetamiprid were were in- observed,cluded as though a positive not as highcontrol. as that When observed MEAM1 with application adults were of acetamiprid treated (Figurewith Kathon3B) . at the con- Likewise, treatment of MEAM1 whitefly nymphs with 18.75 mg/L Kathon, resulted in a considerablecentrations levelof 75.00, of mortality, 37.50, and18.75 no and significant 9.38 mg/L, mortality considerable was observed levels with the of treat-mortality were ob- mentserved, of 9.38 though mg/L Kathonnot as (Figurehigh as3C). that Considerable observed levels with of mortalityapplication were of also acetamiprid observed (Figure 3B). inLikewise, adults of bothtreatment MED (Figure of MEAM13D) and Asiawhitefly II 1 (Figure nymphs3E) when with they 18.75 were mg/L treated Kathon, with resulted in a 9.38considerable and 18.75 mg/L level Kathon. of mortality, and no significant mortality was observed with the treatment of 9.38 mg/L Kathon (Figure 3C). Considerable levels of mortality were also observed in adults of both MED (Figure 3D) and Asia II 1 (Figure 3E) when they were treated with 9.38 and 18.75 mg/L Kathon. Antibiotics 2021, 10, 436 7 of 12 Antibiotics 2021, 10, x FOR PEER REVIEW 7 of 12

Antibiotics 2021, 10, x FOR PEER REVIEW 7 of 12

FigureFigure 3. 3.Effects Effects of of Kathon Kathon on on the the survival survival of of three three species species of of whiteflies whiteflies of of the theBemisia Bemisia tabaci tabacicomplex complex via via oral oral route route and and spraying.spraying.Figure Membrane 3. Membrane Effects of feeding Kathonfeeding was on was the conducted conducted survival toof to determinethree determine species the the of effects whiteflieseffects of of Kathon Kathonof the onBemisia on the the survival tabaci survival complex of of MEAM1 MEAM1 via oral adults adults route via viaand oral oral routeroutespraying. (A ().A). Spraying Spraying Membrane application application feeding was was conducted performed performed to for determinefor MEAM1 MEAM1 the adults adults effects ((B Bof),), Kathon MEAM1MEAM1 on nymphs nymphsthe survival ((CC),), of MEDMED MEAM1 adults adults (D) andvia oral Asia II II 1 adults route ( A (E).). Spraying ForFor spraying,spraying, application KathonKathon was waswas performed diluteddiluted for withwith MEAM1 waterwater containingadults containi (B),ng MEAM1 1% 1% surfactant surfactant nymphs (Tween-80) (Tween-80)(C), MED adults to to two two (D concentrations. )concentrations. and Asia II AcetamipridAcetamiprid1 adults ( (30E ).(30 mg/L)For mg/L) spraying, was was used usedKathon as as positive positivewas diluted control. control. with The The water solutions solutions containi were wereng sprayed1% sprayed surfactant onto onto plants,(Tween-80) plants, and and theto the two mortality mortality concentrations. of of whitefly whitefly Acetamiprid (30 mg/L) was used as positive control. The solutions were sprayed onto plants, and the mortality of whitefly adultsadults and and nymphs nymphs was was assessed assessed at at 48 48 h posth post treatment. treatment. For For each each of of the the four four treatments treatments of of a givena given whitefly whitefly species/stage, species/stage, threeadults replicates and nymphs were wasconducted assessed with at 48 each h post containing treatment. approximately For each of the 100 four adults treatments or 50– 200of a nymphs.given whitefly Different species/stage, letters above three replicates were conducted with each containing approximately 100 adults or 50–200 nymphs. Different letters above barsthree indicate replicates significant were conducted difference with (p

FigureFigure 4. Effects 4. Effects of CMITof CMIT (A )(A and) and MIT MIT (B ()B on) on the the survival survival of of MEAM1 MEAM1 adults.adults. CMITCMIT and and MIT MIT were were diluted diluted with with water water Figure 4. Effects of CMIT (A) and MIT (B) on the survival of MEAM1 adults. CMIT and MIT were diluted with water containingcontaining 1% surfactant1% surfactant (Tween-80) (Tween-80) to two to concentrations, two concentrations, respectively. respectively. Acetamiprid: Acetamiprid: 30 mg/L. 30 mg/L. The solutions The solutions were sprayed were containing 1% surfactant (Tween-80) to two concentrations, respectively. Acetamiprid: 30 mg/L. The solutions were ontosprayed the plants, onto and the theplants, mortality and the of mortality whitefly of adults whitefly and adults nymphs and was nymphs assessed was assesse at 48 hd post at 48 treatment. h post treatment. For each For of each the fourof sprayed onto the plants, and the mortality of whitefly adults and nymphs was assessed at 48 h post treatment. For each of treatmentsthe four for treatments CMIT or for MIT, CMIT five or replicates MIT, five werereplicates conducted were conducted on groups on of groups approximately of approximately 100 adults 100 or adults 50–200 or nymphs.50–200 the four treatments for CMIT or MIT, five replicates were conducted on groups of approximately 100 adults or 50–200 Differentnymphs. letters Different above letters bars indicate above bars significant indicate difference significant (p difference< 0.05, one-way (p < 0.05, analysis one-way of varianceanalysis of and variance Fisher’s and LSD Fisher’s test). nymphs.LSD test). Different letters above bars indicate significant difference (p < 0.05, one-way analysis of variance and Fisher’s LSD test). Antibiotics 2021, 10, x FORAntibiotics PEER 2021 REVIEW, 10, x FOR PEER REVIEW 8 of 12 8 of 12 Antibiotics 2021, 10, 436 8 of 12

3.4. Effects of Kathon3.4. Effectson the ofSu Kathonrvival ofon the the GreenSurvival Peach of the Aphid Green Peach Aphid 3.4. Effects of Kathon on the Survival of the Green Peach Aphid When aphid adultsWhen were aphid treated adults withwere Kathontreated atwith concentrations Kathon at concentrations of 9.38 and of18.75 9.38 and 18.75 When aphid adults were treated with Kathon at concentrations of 9.38 and 18.75 mg/L, mg/L, the mortalitiesmg/L, thewere mortalities 46.7% and were 62.2 46.7%% (Figure and 62.2 5A).% (FigureWhen aphid5A). When nymphs aphid were nymphs were the mortalities weretreated 46.7% with and Kathon 62.2% at (Figure9.38 and5A). 18.75 When mg/L, aphid the mortalities nymphs were were treated 46.7% and with 66.7% (Figure treated with Kathon at 9.38 and 18.75 mg/L, the mortalities were 46.7% and 66.7% (Figure Kathon at 9.38 and5B). 18.75 mg/L, the mortalities were 46.7% and 66.7% (Figure5B). 5B).

Figure 5.Figure Effects 5.of EffectsKathon ofon Kathonthe survival on the of the survival green ofpeach the aphid green Myzus peach persicae aphid :Myzus adult ( persicaeA) and nymph: adult ( (BA).) Acetamiprid and (30 mg/L)nymph was used (B). as Acetamiprid a positive control. (30 mg/L) The solutions was used were as a applied positive using control. dipping, The solutions and the mortality were applied of aphid using adults and Figure 5. Effects of Kathon on the survival of the green peach aphid Myzus persicae: adult (A) and nymph (B). Acetamiprid nymphs was assessed at 48 h post treatment. For each of the four treatments of adult or nymph, three replicates were (30 mg/L) was used as a positivedipping, control. and theThe mortality solutions of were aphid applied adults using and nymphs dipping, was and assessed the mortality at 48 h of post aphid treatment. adults and For each conducted on groups of 15 adults or nymphs. Different letters above bars indicate significant difference (p < 0.05, one-way nymphs was assessed at 48of h thepost four treatment. treatments For of each adult of or the nymph, four treatments three replicates of adult were or conducted nymph, three on groups replicates of 15 were adults or analysis of variance and Fisher’s LSD test). conducted on groups of 15 adultsnymphs. or nymphs. Different Different letters above letters bars above indicate bars indicate significant significant difference difference (p < 0.05, (p < one-way 0.05, one-way analysis of analysis of variance and Fisher’svariance LSD and test). Fisher’s LSD test). 3.5. Effects of Kathon on the Survival of the Rice Brown Planthopper 3.5. Effects of Kathon onAs theadults SurvivalSurv ofival the of rice the planthopper Rice BrownBrown PlanthopperPlanthopper were treated with Kathon at a concentration of 300 mg/L, a moderate level of mortality was observed, though significantly lower than that As adults of the rice planthopper were treated with Kathon at a concentration of As adults ofobserved the rice followingplanthopper application were treated of acetamiprid. with Kathon Whereas, at a concentration no significant ofmortality 300 was ob- 300 mg/L, a moderate level of mortality was observed, though significantly lower than mg/L, a moderateserved level when of mortality treated with was Kathonobserved, at the though concentration significantly of 150.00 lower mg/L than (Figure that 6A). Simi- that observed following application of acetamiprid. Whereas, no significant mortality observed followinglarly, application when nymphs of acetamiprid. of the rice brown Whereas, planthopper no significant were treated mortality with was Kathon ob- at the con- was observed when treated with Kathon at the concentration of 150.00 mg/L (Figure6A). served when treatedcentrations with Kathonof 150.00 at and the 300.00concentration mg/L, moderate of 150.00 levels mg/L of (Figure mortality 6A). were Simi- observed and Similarly, when nymphs of the rice brown planthopper were treated with Kathon at the larly, when nymphsthe level of the of mortality rice brown increased planthopper at the higher were treatedconcentration, with Kathon though atstill the significantly con- lower concentrations of 150.00 and 300.00 mg/L, moderate levels of mortality were observed and centrations of 150.00than that and observed 300.00 mg/L, following moderate application levels of of acetamiprid mortality were(Figure observed 6B). and thethe level of mortality increased at the higher concentration, though still significantlysignificantly lowerlower thanthan thatthat observedobserved followingfollowing applicationapplication ofof acetamipridacetamiprid (Figure(Figure6 6B).B).

Figure 6. Effects of Kathon on the survival of rice brown planthopper Nilaparvata lugens: adult (A) and nymph (B). Acetamiprid (30 mg/L) was used as a positive control. The solutions were applied to rice-stems by dipping, and mortality of brown planthopper adults and nymphs was assessed at Figure 6. Effects of48 Kathon h post treatment. on the survival For each of of riceof rice the brown brown four treatmen planthopper planthopperts of adults NilaparvataNilaparvata or nymphs, lugens lugens three: :adult adult replicates (A (A) ) were conducted on groups of 20 individuals. Different letters above bars indicate significant difference and nymph (B).). AcetamipridAcetamiprid (30(30 mg/L)mg/L) was was used used as as a a positive positive control. control. The The solutions solutions were were applied applied to rice-stems by dipping,(p < 0.05, and one-way mortality analysis of brown of vari planthopperance and Fisher’s adults LSD and test). nymphs was assessed at to rice-stems by dipping, and mortality of brown planthopper adults and nymphs was assessed 48 h post treatment. For each of the four treatments of adults or nymphs, three replicates were at 48 h post treatment. For each of the four treatments of adults or nymphs, three replicates were conducted on groups of 20 individuals. Different letters above bars indicate significant difference conducted(p < 0.05, one-way on groups analysis of 20 of individuals. variance and Different Fisher’s letters LSD test). above bars indicate signiﬁcant difference (p < 0.05, one-way analysis of variance and Fisher’s LSD test). Antibiotics 2021, 10,Antibiotics 436 2021, 10, x FOR PEER REVIEW 9 of 12 9 of 12

Antibiotics 2021, 10, x FOR PEER REVIEW 9 of 12

3.6. Efficacy of BIT3.6. and Efficacy OIT againstof BIT and Adults OIT of against the MEAM1 Adults Whiteflyof the MEAM1 and the Whitefly Green Peach and Aphidthe Green Peach Aphid 3.6. Efficacy of BIT and OIT against Adults of the MEAM1 Whitefly and the Green Peach Aphid When adults ofWhen the MEAM1 adults of whitefly the MEAM1 were treated whitefly with were BIT treated or OIT with at a concentrationBIT or OIT at a concentration When adults of the MEAM1 whitefly were treated with BIT or OIT at a concentration of 150 mg/L, considerableof 150 mg/L, considerable levels of mortality levels of were mortalit observed,y were the observed, mortality the caused mortality by caused by OIT of 150 mg/L, considerable levels of mortality were observed, the mortality caused by OIT OIT was significantlywas significantly higher than higher that causedthan that by caused BIT, but by significantly BIT, but significantly lower than lower that than that ob- was significantly higher than that caused by BIT, but significantly lower than that ob- observed followingserved application following application of acetamiprid of acetamiprid (Figure7A). (Figure When 7A). adults When of theadults green of the green peach served followingaphis application were treated of acetamiprid with BIT (Figureor OIT, 7A). high When levels adults of mortality of the green were peach observed, and the mor- peachaphis aphiswere treated were treated with BIT with or OIT, BIT orhigh OIT, levels high of levels mortality of mortality were observed, were observed, and the mor- and the tality caused by by BIT approached 100%, which was observed following application of mortalitytality caused caused by by by BIT by BITapproached approached 100%, 100%, which which was observed was observed following following application application of acetamiprid (Figure 7B). ofacetamiprid acetamiprid (Figure (Figure 7B).7B).

FigureFigure 7.7. Effects ofFigure of BIT BIT and 7. and Effects OIT OIT on onof the BIT the surv and survivalival OIT of MEAM1on of MEAM1the surv whiteflyival whitefly of adults MEAM1 adults (A) andwhitefly (A )adults and adults adults of the ( ofA) theand adults of the greengreen peachpeach aphidgreen ( BB).). Acetamipridpeach Acetamiprid aphid (30(B (30). mg/L) Acetamiprid mg/L) was was used used (30 as mg/L)a as positive a positive was control. used control. as Whitefly a positive Whitefly and control. aphid and aphid Whitefly and aphid mortality was recorded at 48 h post treatment. For each of the four treatments of the whitefly or mortality was recordedmortality at 48 was h post recorded treatment. at 48 Forh post each treatment. of the four For treatments each of the of the four whitefly treatmen or aphid,ts of the whitefly or aphid, three replicates were conducted. Each replicate of whitefly comprised approximately 100 three replicates wereaphid, conducted. three replicates Each replicate were conducted. of whitefly Each comprised replicate approximately of whitefly comprised 100 adults approximately and 100 adults and each replicate of aphid comprised 15 aphid adults. Different letters above bars indicate adults and each replicate of aphid comprised 15 aphid adults. Different letters above bars indicate eachsignificant replicate difference of aphid (p comprised< 0.05, one-way 15 aphid analysis adults. of vari Differentance and letters Fisher’s above LSD barstest). indicate significant difference (p < 0.05,significant one-way difference analysis of(p variance< 0.05, one-way and Fisher’s analysis LSD of test). variance and Fisher’s LSD test). 3.7. Effects of Kathon Treatment on the Density of Endosymbionts in Whitefly 3.7. Effects of Kathon3.7. Effects Treatment of Kathon on the Treatment Density of on Endosymbionts the Density of in Endosymbionts Whitefly in Whitefly To explore whether Kathon treatment would affect the density of endosymbionts in To explore whether Kathon treatment would affect the density of endosymbionts in whitefly, three endosymbiontsTo explore Portiera whether, Hamiltonella Kathon treatment and Rickettsia would were affect analyzed. the density For the of endosymbionts in Portiera Hamiltonella Rickettsia whitefly,primary endosymbiont three endosymbiontswhitefly, Portiera three endosymbionts, Kathon, treatment Portiera did andnot, Hamiltonella significantlywere and change analyzed.Rickettsia its density were For the analyzed. For the primaryin whitefly endosymbiont (Figureprimary 8A). PortieraendosymbiontLikewise,, Kathon the density Portiera treatment of, Kathon secondary did not treatment significantlyendosymbionts did not change significantlyHamiltonella its density change its density indid whitefly not significantly (Figurein whitefly8A). change Likewise, (Figure upon the Kathon 8A). density Likewise, treatment of secondary the (Figure density endosymbionts 8B). of However, secondary Hamiltonellaa significantendosymbionts did Hamiltonella notdecrease significantly of Rickettsiadid change not density significantly upon was Kathon observed; change treatment specifically, upon (Figure Kathon8 9.38B). However, treatmentand 4.69 mg/L a(Figure significant Kathon 8B). decreasetreat- However, a significant ofmentRickettsia decreaseddensitydecrease Rickettsia was of density observed; Rickettsia by 25.7%, density specifically, and was 26.0%, observed; 9.38 respectively and 4.69specifically, mg/L (Figure Kathon 9.38 8C). and treatment 4.69 mg/L Kathon treat- decreased Rickettsiamentdensity decreased by 25.7%,Rickettsia and density 26.0%, by respectively 25.7%, and (Figure 26.0%,8 C).respectively (Figure 8C).

Figure 8. Effects of Kathon treatment on the density of endosymbionts in whitefly. Whiteflies were treatment with Kathon

via spraying, and 48 h later the density of Portiera (A), Hamiltonella (B) and Rickettsia (C) were analyzed. Different letters above bars indicateFigure significant 8. EffectsFigure difference of 8. KathonEffects (p treatment< of 0.05, Kathon one-way on treatment the analysis density on of theof vari endosymbionts densityance and of endosymbiontsFisher’s in whitefly. LSD test). inWh whitefly.iteflies were Whiteflies treatment were with Kathon via spraying,treatment and 48 withh later Kathon the density via spraying, of Portiera and (A 48), Hamiltonella h later the density (B) and of RickettsiaPortiera ( (AC),) Hamiltonellawere analyzed.(B) Different and letters above barsRickettsia indicate significant(C) were analyzed. difference Different(p < 0.05, lettersone-way above analysis bars of indicate variance significant and Fisher’s difference LSD test). (p < 0.05, one-way analysis of variance and Fisher’s LSD test).

Antibiotics 2021, 10, 436 10 of 12

4. Discussion In this study, we accidentally observed that a commercial formulated tetramycin solution can effectively kill whiteflies (Figure2). In the process of tracing the insecticidal components in the tetramycin solution, we identified two isothiazoline biocides that exhib- ited insecticidal activity against whitefly. As isothiazoline is widely used as preservatives in many solutions [26], we presumed that it may have been added to the tetramycin solution during production. We next tested the effects of the most widely used isothiazoline biocide Kathon and its two constituents CMIT and MIT on the survival of five species of hemipteran insects. In our bioassay, we first used high concentrations (up to 600 mg/L) and then used lower concentrations by sequentially diluting the higher ones. Finally we chose 9.38 and 18.75 mg/L for bioassay as the European Union regulations on cosmetic products states that the upper limit of isothiazolinones such as Kathon in leave-on and rinse-off cosmetics is 15 mg/L [27]. Our results indicate that Kathon and its two constituents can cause considerable levels of mortality to three species of whiteflies from the B. tabaci complex and the green peach aphid when applied at the concentration close to, or lower than, the upper limit of these chemicals, permitted by the European Union regulations on cosmetic products (Figures3–5). Interestingly, Kathon seemed to be more potent against whitefly, suggesting a synergistic effect between CMIT and MIT (Figures3 and4). Additionally, brown planthoppers seem to be less susceptible to Kathon than the other insects (Figure6). The low susceptibility of brown planthoppers may be due to many reasons, for example the insect may degrade Kathon efficiently, among other. Nevertheless, further investigations are warranted to verify these hypotheses. The results also show that the two other isothiazolinones BIT and OIT were less toxic to the MEAM1 whitefly and the green peach aphid than Kathon and its two constituents (Figure7). Overall, the data indicate that some isothiazolines can exert considerable levels of mortality on some hemipteran insects, such as whiteflies and aphids. In recent decades, neonicotinoid insecticides have been widely used in the control of phloem-feeding insect pests, such as whiteflies, aphids and planthoppers [13,14]. The ex- tensive application of neonicotinoids in the field has seen rapid development of insect resistance to insecticides of this class, such as imidacloprid and thiamethoxam [13,28]. Rotated application of insecticides with different modes of action has proven to be an effective strategy for managing resistance [28]. To implement this strategy, the development of new insecticides, with novel modes of action that are benign to the environment, is necessary. Isothiazolones inhibit microbial growth and metabolism by inhibiting critical physiological processes, such as respiration and energy generation [29–31]. Further, isothiazolones may react with many thiol-containing compounds such as cysteine, thereby forming disulfide derivatives and in turn promoting a series of reactions that impair key cellular functions [29,31,32]. While mechanisms underlying the toxicity of isothiazolones to hemipteran insects are unknown, disruption of critical cellular processes and interaction with thiol-containing compounds may both be possible. Nevertheless, the mechanisms of action are likely to differ significantly from those of neonicotinoids that act selectively on the insect central nerve system as agonists of the postsynaptic nicotinic acetylcholine receptors [14]. With regard to the mechanism of action, here we observed that isothiazolone treatment marginally affected the density of endosymbionts in whitefly. However, elimination of endosymbionts in hemipteran insects such as whitefly does not affect the survival of treated insects [25,33,34]. Hence, the microbicidal property of isothiazolones may contribute little, if any, to the insecticidal activity of these compounds. Isothiazolinones are effective, fast-acting chemicals for the inhibition of microbial growth and metabolism and biofilm development. They are widely used for many in- dustrial purposes and as a preservative in cosmetics including lipsticks and shampoo; under ambient conditions, isothiazolinones are quite stable as their estimated half-life is six months [26]. While isothiazolinones, such as Kathon have been reported to cause skin sensitization in humans and may induce apoptosis in human keratinocytes [35]. A review Antibiotics 2021, 10, 436 11 of 12

of case studies indicates that they are safe to human when the concentration is below 15 mg/L [18]. When plants were sprayed with isothiazolinones at the concentrations used in our experiments (up to 150 mg/L), the plants did not suffer any appreciable sign of toxicity (data not shown). These results suggest that use of isothiazolinones as insecticides at a concentration lower than 15mg/L may have limited risks to humans and crops. However, as broad spectrum biocides, isothiazolinones may significantly impact organisms other than insect pests. Therefore, investigations on the effects of isothiazolinones to non-target organisms are necessary in evaluating their potential use as insecticides. Taken together, this study has identified the insecticidal properties of commonly used biocidal isothiazolinones including Kathon, BIT and OIT. Our findings indicate that isothiazolinones are promising candidates for the development of a new class of insecticides for the control of hemipteran pests.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10040436/s1, Figure S1: MS ion chromatograms of the substance peaked at 0.8396 min in LC analysis and Figure S2: MS ion chromatograms of the substance peaked at 1.9542 min in LC analysis. Author Contributions: X.W. and W.H. (Wenze He) conceived and designed research. W.H. (Wenze He), W.H. (Wenhao Han) and L.P. conducted experiments. W.H. (Wenze He) and L.P. analyzed data. W.H. (Wenze He) and L.P. wrote the manuscript. X.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: Financial support for this study was provided by the National Natural Science Foundation of China (31925033) and the earmarked fund for China Agricultural Research System (grant number: CARS-23-D07). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data in this study is available upon request. Acknowledgments: We thank Shu-Sheng Liu of our laboratory for advice and support throughout this study. We thank Zhi-Wei Ge from the Analysis Center of Agrobiology and Environmental Sciences of our university for assistance in LC-MS. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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