Talaromyces sp. Ethyl Acetate Crude Extract as Potential Mosquitocide to Control Culex pipiens quinquefasciatus

Vector control is considered an effective approach to controlling diseases spread by mosquito bites. Entomopathogenic fungi are widely used in agriculture to control insect pests, and fungal metabolites can potentially be developed as effective mosquitocides. In this study, a high-throughput screening method was used to search for potential mosquitocides in the Global Fungal Extract Library (GFEL). We tested the larvicidal activity of 264 fungal ethyl acetate crude extracts against Culex pipiens quinquefasciatus. Nine fungal extracts caused moderate to high mortality rates (>50%), with two fungal extracts (58A7 and 101H12) causing a 100% mortality rate. The lethal concentrations for 50% of the population (LC50) were 44.27 mg/L and 31.90 mg/L, respectively. Fraction 14 had a high mortality rate, with an LC50 value of 12.13 mg/L, and was isolated from 58A7 (Fractions 1–11) and 101H12 (Fractions 12–15). Further analyses showed that Fraction 14 was made up of vermistatin and dihydrovermistatin. In a Cx. p. quinquefasciatus larvicidal bioassay, vermistatin (LC50 = 28.13 mg/L) was more toxic than dihydrovermistatin (LC50 = 83.87 mg/L). Our findings suggested that the active fungal extract 101H12 from Talaromyces sp. and its compound vermistatin could be developed as mosquitocides.


Introduction
Mosquitoes are important vectors for transmitting a wide range of serious human and animal diseases (e.g., malaria, dengue), posing a threat to public health.Malaria killed more than 400,000 people in 2019 [1].Culex pipiens quinquefasciatus, known as the 'southern house mosquito', is distributed throughout southern China.Cx. p. quinquefasciatus has been identified as a vector for the transmission of St. Louis encephalitis and the West Nile virus in North America [2,3], as well as Japanese encephalitis and the Zika virus in China [4].
Due to the lack of vaccines, vector control has been widely used in the prevention of vector-borne diseases and has proven to be a successful strategy in reducing death cases globally [5].In Africa, insecticide-treated nets are the most important contributor to indoor residual spraying and artemisinin-based combination therapy in reducing clinical malaria cases [6].However, the extensive use of chemicals has resulted in environmental pollution and an increase in resistance.In Réunion Island, Cx. p. quinquefasciatus has developed a 233-fold resistance against permethrin, deltamethrin, temephos, chlorpyrifos, malathion, and dieldrin.In addition, the vector population could retain high dieldrinresistant Rdl R frequencies for decades [7], implying that new vector control strategies should be implemented.
The Global Fungal Extract Library (GFEL) contains over 10,000 fungal isolates including Penicillium spp., Aspergillus spp., Fusarium spp., Podospora spp., Mucor spp., Cladosporium spp., and Stoloniferum spp.[14].Among these fungal metabolites, asperaculane B, extracted from Aspergillus aculeatus, has been widely used in malaria control as it inhibits malaria infection and transmission [15].Jin et al. [16] discovered six fungal extracts with high toxicity against the An.gambiae Sua5B cell line after screening 192 fungal extracts for effective An.gambiae insecticide discovery.Among these extracts, Penicillium toxicarium 76F6 extract showed high larvicidal and adulticidal activity against An.gambiae.The GFEL is an essential resource for novel insecticide discovery in the control of mosquitoes.
To discover new insecticides against Cx.p. quinquefasciatus with a friendly profile for humans and the environment, we used conventional bioassays on mosquito larvae to screen 264 candidate fungal extracts for the most effective fungal metabolites.Following the discovery of these candidate metabolites, we then isolated the active compounds and validated their larvicidal activities.

Discussion
The screening of active compounds from fungal extracts has received more and more interest and the GFEL has proven to be a successful resource.Niu et al. [14] screened 460 fungal extracts from the GFEL, found that four fungal extracts showed more than 90%

Discussion
The screening of active compounds from fungal extracts has received more and more interest and the GFEL has proven to be a successful resource.Niu et al. [14] screened 460 fungal extracts from the GFEL, found that four fungal extracts showed more than 90% inhibition activity in preventing fibrinogen-related protein 1 from binding to Plasmodium falciparum lysates, and eventually found that Penicillium thomii extract can block Plasmodium falciparum to transmit to An. gambiae.Jin et al. [16] screened 192 fungal extracts from the GFEL, established an MTT-based cytotoxicity screening approach, and found that Penicillium toxicarium extract exhibit high toxicity to mosquito larvae and adults of An. gambiae.Similarly, Aspergillus aculeatus extract completely inhibited Plasmodium falciparum transmission to An. gambiae.Asperaculane B was successfully purified and identified, with a half maximal inhibitory concentration (IC 50 ) of 7.89 µM for Penicillium falciparum transmission and an IC 50 of 3 µM for the development of asexual Penicillium falciparum [15].In addition, sterigmatocystin and pulixin, isolated from Penicillium janthinellum extract and Purpureocillium lilacinum extract, respectively, limited Plasmodium falciparum proliferation, transmission, or infection [14,15].In this study, we ultimately discovered that nine candidates were able to kill more than half of the larval mosquitoes at a concentration of 100 mg/L.Only 101H12 (Talaromyces sp.) and 58A7, causing 100% mortality, were chosen for further mosquito larvicidal bioassays.The other seven fungal extracts showed mediumto-high toxic activity, which also deserves a comprehensive study.Future investigations are required to determine the larvicidal effects of the remaining seven fungal extracts and identify their active compounds, assessing their potential use in mosquito control.
Vermistatin is often extracted together with derivatives.Dihydrovermistatin, one of its derivatives, is often extracted together with vermistatin from Alternaria alternate [25], Penicillium rubrum [28], and Phoma sp.[29].In addition to dihydrovermistatin, other derivatives such as 6-demethylvermistatin [30], methoxyvermistatin, and hydroxyvermistatin [23] have been separated.In our results, dihydrovermistatin exhibited low biological activity against Cx.p. quinquefasciatus.Similarly, dihydrovermistatin exhibited no activity against leukemia cell lines [28].The probable reason is that the structure of the propenyl tail in dihydrovermistatin causes biological inactivity [31].In comparison with the propenyl tail, the methoxyl group at the γ-pyrone ring exhibited a higher cytotoxic activity in methoxyvermistatin than in vermistatin against the KB tumor cell line, with IC 50 values of 20.0 and 90.2 mg/mL, respectively [23].
In our study, a mixture of vermistatin and dihydrovermistatin (Fraction 14, LC 50 = 12.13 mg/L) showed higher larvicidal activity than the crude fungal extract (LC 50 = 31.90mg/L) and the single compounds vermistatin (LC 50 = 28.13mg/L) and dihydrovermistatin (LC 50 = 83.87mg/L), indicating that the mixture may be more suitable for further mosquitocide development.In addition, the mechanism behind this deserves further study.
The efficacy of Talaromyces sp.extract or vermistatin needs to be improved for their further application.Synergists such as piperonyl butoxide, a well-known cytochrome P450 inhibitor, S,S,S-tributylphosphorotrithioate, an esterase-specific inhibitor, and diethyl maleate, a glutathione S-transferases inhibitor have been proved to be effective synergists in mosquito control [32,33].Nanoencapsulation has been employed to protect active compounds from external factors and to maximize their biological activity.For example, nanoliposomes of carvacrol showed a better larvicidal efficacy against An.stephensi and Cx.quinquefasciatus [34].Hence, a broad source of active compounds can be synergized and nanoencapsulated for potential mosquitocides.

Rearing Mosquitos
Cx. p. quinquefasciatus was obtained from the Nanchang Center for Disease Control and Prevention.Mosquitos had originally been collected in Nanchang, South China, and have been reared for more than 10 years.An artificial diet of yeast:lactose albumin (1:1) was used to rear Cx. p. quinquefasciatus larvae.Mosquitoes were kept in the insect room at 26 • C, 65% relative humidity (RH), and a photoperiod of 12 h light followed by 12 h dark.Larvae were fed with 0.1 mg ground fish food.Adults were fed with 8% sucrose and mouse blood for egg production.

Fungal Culture and Metabolite Extraction
Ethyl acetate crude extracts of fungi were acquired from the Global Fungal Extract Library (GFEL, Aulandin, Jiashan, China) [14].The detailed fermentation process was described previously [15].Five hundred grams of cereal (General Mills, Minneapolis, MN, USA) was sterilized at 121 • C for 20 min.When the cereal was dried, it was mixed with 1 L 0.3% sterile sucrose solution supplemented with 50 mg/L chloramphenicol (Life Tech, Grand Island, NY, USA).Fungal cultures were kept in a mushroom bag at 27 • C for 4 weeks.One liter of ethyl acetate was added to the culture twice to extract fungal metabolites.The ethyl acetate solution was filtered using a Büchne Flask with two layers of cheesecloth and the solid residue was discarded.A rotary evaporator (Heidolph, Elk Grove Village, IL, USA) was used to eliminate the resultant ethyl acetate supernatant and to acquire crude gums.

Larvicidal Bioassay
The mosquito larval bioassay was conducted based on the requirements of the WHO with slight modifications [36].For the initial screening, a 24-well plate was used, and each well contained 2 mL of distilled water.Ethyl acetate crude extracts of fungi were dissolved in dimethyl sulfoxide (DMSO, 99%AR, Hengxing company, Tianjin, China) and were pipetted into the wells to make final concentrations at 100 mg/L.Twelve 4th-instar larvae were transferred into the wells and mortality was recorded after 24 h.
The efficacy of the screened fungal extracts, namely 101H12 and 58A7, was evaluated across a range of concentrations (10,20,40,60,80, and 100 mg/L) in 100 mL of distilled water using glass beakers.Similarly, the isolated fractions 1-15 were initially tested at a concentration of 20 mg/L, and a series of concentrations (5, 10, 15, 20, and 30 mg/L) of Fraction 14 and of its components vermistatin and dihydrovermistatin (10, 20, 30, 40, 50 and 60 mg/L) were tested.Negative control was 1% DMSO without any fungal extract.Batches of 25 4th-instar larvae were tested and 4 replicates were conducted.Mortality was recorded after 24 h if no reaction was seen when the larvae were touched with a soft plastic dropper.No food was provided during the experiment.Every test was replicated three times on different days.

Identification of Fungal Species
The conserved sequences of the internal transcribed spacer (ITS) region of 5.8S and 28S ribosomal DNA were used to identify individual fungal species.A small amount of mycelium (0.1-1 mg) was taken from fungus 101H12, rinsed in 400 µL sterilized water, and then collected by centrifugation at 15,000× g for 2 min.The mycelium was resuspended in 100 µL sterilized water, 1 µL of which was used for PCR.The DNA fragments were amplified using ITS1 (TCC GTAGGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TATTGA TAT GC) primers.The reaction was carried out under the following conditions: 94 • C for 2 min to denature DNA; 35 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 1 min; and 72 • C for 5 min to complete the reaction.The amplified PCR product was gel-extracted, purified, and sequenced (Eurofins Genomics, Louisville, KY, USA).The raw sequence data were analyzed, and low-quality sequence ends were removed.Subsequently, the edited sequences were compared with those in the National Center for Biotechnology Information database using BLAST to identify individual fungal species.The fungus was identified as Talaromyces sp. and found to be 100% identical to GenBank: ON427045.1.

Data Analysis
SPSS (version 19.0) was used to analyze the data.Larvicidal bioassay was conducted by standard probit analysis.The lethal concentrations for 50% and 90% mortality (LC 50 and LC 90 ), standard deviation, and 95% confidence intervals (CLs) of the means of LC 50 and LC 90 were calculated.GraphPad (version 8.0) was used to generate the figures.

Conclusions
In this study, we successfully discovered several potential mosquitocides by a highthroughput screening of fungal extracts from the GFEL.We tested 264 fungal crude extracts in ethyl acetate and found two extracts (101H12 and 58A7) that caused a 100% mortality rate.A total of 15 fractions (Fractions 1-11 from 58A7 and Fractions 12-15 from 101H12) were separated but only Fraction 14 caused a high mortality in the bioassay.Fraction 14 was constituted by vermistatin and dihydrovermistatin.Vermistatin showed strong biological activity in the larvae of Cx. p. quinquefasciatus.Our findings indicated that 101H12 Talaromyces sp. and its active compound vermistatin are suitable candidates for mosquito control, which will lay the foundations for further development of Talaromyces sp. and vermistatin as novel mosquitocides or pesticides.

Figure 1 .
Figure 1.Toxicity of 264 fungal crude extracts in ethyl acetate against fourth-instar larvae of Culex pipiens quinquefasciatus at 100 mg/L.Numbers indicate the percentage of mortality rate.Lighter red colors indicate a lower mortality rate, and darker red colors indicate a higher mortality rate.

Figure 2 .
Figure 2. Larvicidal bioassay of 101H12 and 58A7 against fourth-instar larvae of Culex pipiens quinquefasciatus.Batches of 25 fourth-instar larvae were tested and four replicates were conducted.Mortality was recorded after 24 h.Standard probit analysis was conducted.

Figure 1 .
Figure 1.Toxicity of 264 fungal crude extracts in ethyl acetate against fourth-instar larvae of Culex pipiens quinquefasciatus at 100 mg/L.Numbers indicate the percentage of mortality rate.Lighter red colors indicate a lower mortality rate, and darker red colors indicate a higher mortality rate.

Figure 1 .
Figure 1.Toxicity of 264 fungal crude extracts in ethyl acetate against fourth-instar larvae of Culex pipiens quinquefasciatus at 100 mg/L.Numbers indicate the percentage of mortality rate.Lighter red colors indicate a lower mortality rate, and darker red colors indicate a higher mortality rate.

Figure 2 .
Figure 2. Larvicidal bioassay of 101H12 and 58A7 against fourth-instar larvae of Culex pipiens quinquefasciatus.Batches of 25 fourth-instar larvae were tested and four replicates were conducted.Mortality was recorded after 24 h.Standard probit analysis was conducted.

Figure 2 .
Figure 2. Larvicidal bioassay of 101H12 and 58A7 against fourth-instar larvae of Culex pipiens quinquefasciatus.Batches of 25 fourth-instar larvae were tested and four replicates were conducted.Mortality was recorded after 24 h.Standard probit analysis was conducted.

Figure 3 .
Figure 3. Toxicity of 15 fractions of 101H12 and 58A7 against fourth-instar larvae of Culex pipiens quinquefasciatus at a concentration of 20 mg/L.

Figure 3 .
Figure 3. Toxicity of 15 fractions of 101H12 and 58A7 against fourth-instar larvae of Culex pipiens quinquefasciatus at a concentration of 20 mg/L.

Table 1 .
Effects of fungal crude extracts and compounds against Culex pipiens quinquefasciatus.LC 50 and LC 90 values (concentrations that caused mortality in 50% or 90% of a sample population) were determined at 24 h.
in Supplementary Materials).