Bioefcacy of Soil Actinomycetes and An Isolated Molecule

Applied Biochemistry and Biotechnology (2022) 194:4765–4782
https://doi.org/10.1007/s12010-021-03766-8
ORIGINAL ARTICLE
Bio‑efficacy of Soil Actinomycetes and an Isolated Molecule

1,2‑Benzenedicarboxylic Acid from Nonomuraea sp. Against
Culex quinquefasciatus Say and Aedes aegypti L. Mosquitoes
(Diptera: Culicidae)
Pachaiyappan Saravana Kumar1,2 · Appadurai Daniel Reegan3 ·

Karunakaran Rajakumari4 · Antony Cruz Asharaja4 · Kedike Balakrishna5 ·
Savarimuthu Ignacimuthu2
Received: 9 September 2021 / Accepted: 8 November 2021 / Published online: 22 November 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
Vector-borne diseases such as filariasis and dengue that contribute significantly to disease
burden, death, poverty, and social frailty are still a major public healthcare problem world-
wide. Currently, synthetic chemicals have been used in mosquito control programs. How-
ever, repeated use of chemical insecticides causes environmental pollution and harmful
effects on non-target organisms. Therefore, alternative ecofriendly sources from biological
source are urgently needed to manage mosquitoes. In this respect, the present study was
aimed to evaluate mosquito larvicidal and pupicidal activities of 22 crude extracts of soil
actinomycetes on Culex quinquefasciatus and Aedes aegypti and to identify the active mol-
ecule. Briefly, the crude ethyl acetate extract and fractions were tested at 62.5, 125, 250,
and 500 ppm and 2.5, 5.0, 7.5, and 10.0 ppm concentrations on larval and pupal stages of
Cx. quinquefasciatus and Ae. aegypti. The larval and pupal mortality was assessed after
24 h of treatment. Among the 22 isolates screened, Nonomuraea sp. VAS-16 exhibited
significant larvicidal and pupicidal activities against the tested mosquito species. Among
the 18 fractions screened, fraction-6 showed strong larvicidal and pupicidal activities with
the LC50 and L
C90 values of 9.1, 18.7, 9.82, and 22.85 ppm against the larvae and LC50 and
LC90 values of 10.5, 23.1, 12.3, and 24.13 ppm against the pupae of Cx. quinquefasciatus
and Ae. aegypti, respectively. Fascinatingly, the isolated compound 1,2-benzenedicarbox-
ylic acid from fraction-6 at 0.5, 1.0, 1.5, and 2.0 ppm concentration recorded lower L C50
and LC90 values of 4.27, 14.90, 4.67, and 11.90 ppm against the larvae and LC50 and LC90
values of 4.58, 12.06, 5.36, and 13.07 ppm against the pupae of Cx. quinquefasciatus and
Ae. aegypti, respectively. On the other hand, the compound recorded less ovicidal activity
of 11.0% and 10.3% at 2 ppm against the eggs of Cx. quinquefasciatus and Ae. aegypti,
respectively. The present study clearly shows that the crude extract and the compound from
Nonomuraea sp. VAS-16 can be used as an effective biopesticide in integrated mosquito
management program.
Pachaiyappan Saravana Kumar and Appadurai Daniel Reegan contributed equally to this paper.
Extended author information available on the last page of the article
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4766 Applied Biochemistry and Biotechnology (2022) 194:4765–4782
Keywords Culex quinquefasciatus · Aedes aegypti · Nonomuraea sp. VAS-16 ·

1,2-Benzenedicarboxylic acid · Larvicidal activity · Pupicidal activity
Introduction
Mosquitoes are blood-sucking insects responsible for transmitting disease-causing patho-

gens like dengue virus, chikungunya virus, Japanese encephalitis virus, filarial nematode,
and malarial parasites to humans [13, 19, 23]. Mosquito-borne diseases show increas-
ing trend in several tropical and subtropical countries with greater economic loss [21].
Many species of the mosquitoes serve as a vector, which is distributed in different geo-
graphical localities and responsible for 700,000,000 cases every year around the globe and
40,000,000 cases in India [14].
Culex quinquefasciatus Say is a major night-biting mosquito, which transmits filarial
nematode Wuchereria bancrofti in India [2]. A recent estimate shows that 859 million peo-
ple in 50 countries worldwide remain threatened by lymphatic filariasis [33]. Aedes aegypti
Linn. is a day-biting mosquito, which is highly anthropophilic in nature and transmits arbo-
viruses like dengue virus and chikungunya virus to human [15, 16]. There are 3900 million
people in 128 countries at risk of dengue infection alone [6].
On one hand, several efforts are taken by providing preventive chemotherapy to stop
the spread of infection, and on the other hand, chemical insecticides are used enormously
to control vector mosquitoes [4, 24]. Considering the ill effects caused by the chemical
insecticides, safe mosquito control measures are need of the hour in integrated vector con-
trol programme. In particular, the chemical larvicide temephos is reported to be toxic and
affects the normal functioning of different beneficial organisms [12, 22]. Furthermore,
the development of resistance to temephos and azadirachtin were also reported [7, 9, 31].
Keeping this in mind, we isolated several soil actinomycetes and tested them for mosqui-
tocidal activities, since actinomycete produces a variety of compounds that are medically
and industrially useful. In the present study, mosquitocidal activities of crude ethyl acetate
extracts of soil actinomycetes were evaluated, and the most active isolate Nonomuraea sp.
was further fractionated and isolated an active compound 1,2-benzenedicarboxylic acid for
target-specific mosquito management against Culex quinquefasciatus and Aedes aegypti
vector mosquitoes.
Materials and Methods
General Experimental Procedures
Analytical TLC was carried out on 0.2 mm pre-coated aluminum TLC Silica gel 60 F 254
plates, and acme’s 100–200 mesh silica gel was used for gravity column chromatography.
The IR spectra were taken on Perkin-Elmer Spectrum FT-IR grating Spectrophotometer in
the range 4000–450 cm−1 in KBr disc. The EI-MS was run on Q-Tof-Mass Spectometer
at 70 eV by direct inlet method. 1H NMR and 13C NMR were taken on Bruker instrument
(Germany) at 400 MHz and 100 MHz using DMSO-d6, respectively. Tetramethyl silane
(TMS) was used as internal standard, and the chemical shifts were given in delta scale (δ,
ppm). All the media were procured from HiMedia, and solvents used were of analytical
grade.
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Applied Biochemistry and Biotechnology (2022) 194:4765–4782 4767
Isolation and Identification of Terrestrial Actinomycetes
The schematic representation of collection site and isolation of the soil actinomycetes are
given as supplement (Fig. S1). The most active isolate VAS-16 was identified by growing
the cells on the various media to study its morphological characteristic features, and the
16S rRNA partial gene sequence was submitted in the GenBank, NCBI (Accession num-
ber: JN588559.1). It can also be found in our earlier publication [28] and supplementary
section of this article.
Bioassay‑Guided Isolation of Active Principle
The detailed procedures of mass production, extraction, fractionation, isolation, and identi-
fication of active compound 1,2-benzenedicarboxylic acid by various spectroscopic analy-
ses, viz., EI-MS, FT-IR, 1H NMR and 13C NMR with DEPT of active isolate Nonomuraea
sp., are given in the supplementary section of this article (Fig. S2a, S2b, S3-S7), and it also
can be found in our earlier publication [28].
Insect Rearing
Cyclic culture of two mosquito species, namely Cx. quinquefasciatus and Ae. aegypti, were
maintained in the laboratory. The rearing and experimental conditions for the laboratory
were 27–28 °C temperature, 70–75% relative humidity, and 11 ± 1 h photo period. Mos-
quito larvae were nurtured on powdered yeasts and dog biscuits (2:3 ratios). Similarly,
wet raisins and 10% sucrose solution were provided for adult mosquitoes. Female mosqui-
toes were periodically blood-fed on restrained albino rats principally for egg production.
Freshly laid eggs and third stage larvae and pupae of these mosquito species were used for
the present investigation.
Larvicidal and Pupicidal Assay
Larvicidal and pupicidal activities were assessed as per the standard method [26, 32]. The
larvicidal and pupicidal activities of crude ethyl acetate extract of all the isolated actinomy-
cetes were checked at 62.5, 125, 250, and 500 ppm concentrations with five replicates on
third stage larvae/pupae of Cx. quinquefasciatus and Ae. aegypti mosquitoes. Twenty lar-
vae/pupae were exposed in each replication. The fractions were pooled based on the TLC
profile, and finally a total of 18 fractions were obtained. The fractions of the active crude
extract were studied at 2.5, 5.0, 7.5, and 10.0 ppm concentrations, and the compounds were
studied at 0.5, 1.0, 1.5, and 2.0 ppm concentrations with five replicates. One milliliter of
DMSO was used to dissolve each fraction and compound for each replication, which was
added in experimental cups containing 249 mL of tap water. In solvent control, 1 mL of
DMSO was added without any bacterial material and water control was maintained sepa-
rately. Larval and pupal mortality was assessed at 24 h post experiment period.
Formula (1) was used to calculate the percent mortality, and Abbott’s [1] Formula
(2) was used to carry out corrections. The results were compared with azadirachtin and
temephos.
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Percentage of mortality:
No. of Dead larvae
× 100 (1)
No. of Larvae introduced
Abbott’s formula for corrections:
(1 − n in T after treatment × 100)
× 100 (2)
n in C after treatment
where n is the number of larvae, T is the treated, and C is the control. The corrected per-
centage mortality value for each concentration was considered to estimate L C50 and LC90 val-
ues using US EPA probit analysis software (version 1.5).
Ovicidal Assay
Ovicidal activity was carried out following the method of Reegan et al. [26]. Freshly laid eggs
of Cx. quinquefasciatus and Ae. aegypti were subjected to different concentrations of com-
pound, viz., 0.5, 1.0, 1.5, and 2.0 ppm. Five replications were maintained, and ten eggs were
used in each replication. Water control and solvent control (DMSO in water) were maintained
separately. The egg hatchability was evaluated at 120 h post-treatment, and percent egg mor-
tality was calculated using Formula (3), and the results were compared with azadirachtin and
temephos.
Number of unhatched eggs
× 100 (3)
Total number of eggs exposed
Sub‑lethal Effects
Sub-lethal effects of the active compound 1,2-benzenedicarboxylic acid were studied at the
concentration of 0.5 and 1.0 ppm. Growth disruption and morphological deformities were
observed till the adult emergence according to the established method [25]. Five replicates of
treated and control were maintained with twenty (1st instar) larvae in each. The larvae were
fed with powdered yeast and dog biscuits (2:3 ratios).
Statistical Analysis
Larvicidal and pupicidal data were subjected to probit analysis (US EPA probit; version 1.5),
and the differences were considered significant at p ≤ 0.05. The significant level of chi-square
test was 0.05. The calculated ovicidal rate was subjected to one-way analysis of variance to
find out the significance of the treatments, and means were separated by Tukey’s test of multi-
ple comparison using SPSS software (version 11.5).
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Results
Isolation of Soil Actinomycetes
A total of 22 different actinobacteria designated as VAS (1–22) were isolated from ant
mound soil using Actinomyces Isolation Agar (AIA) on the basis of colony morphology,
viz., powered, chalky, colored, and pigmented colonies, from the isolation plates. The iso-
lates were given three letters alphabetic and a number code based on the location and col-
lection site. Briefly, first alphabet indicates the area from where it was collected Tiruvanna-
malai district, “Vengodu” (V); second and third alphabet indicates the exact location of the
sample collection site “ant mound soil” (AS). The crude ethyl acetate extracts of all the 22
isolates were tested against two mosquito stages. However, based on the preliminary derep-
lication by colony morphology and stability in subculture, 20 isolates were photographed.
Among them 4 isolates were pure white (VAS-6, 17, 18, and 21), 4 isolates were silk gray
(VAS-7, 12, 14, and 15), 2 isolates were umbra gray (VAS-9 and 11) and a pastel orange
(VAS-1), sun yellow (VAS-2), yellow-orange (VAS-3), pastel yellow (VAS-4), beige red
(VAS-8), dusty gray (VAS-10), bright red (VAS-16), concrete gray (VAS-19), light gray
(VAS-20), and cream (VAS-22) (Fig. 1).
VAS-1 VAS-2 VAS-3 VAS-4 VAS-6
Fig. 1 Macroscopic images of VAS isolates on Actinomyces Isolation Agar (AIA)
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Larvicidal and Pupicidal Activities
Initially, all the isolates were screened for larvicidal and pupicidal activities against
Cx. quinquefasciatus and Ae. aegypti. Among the 22 isolates, 4.6% (1 isolate) showed
good activity, 13.6% (3 isolate) showed weak activity, and 81.8% (18 isolate) showed
no activity against the larvae and pupae of the two tested mosquito species (Tables 1
and 2). Interestingly, the crude ethyl acetate extract of VAS-16 recorded higher larval
and pupal mortality against Cx. quinquefasciatus and Ae. aegyptimosquitoes. On the
other hand, the lower LC50 and L C90 values of VAS-16 were recorded to be 73.7 and
147.2 ppm and 84.7 and 225.2 ppm against the third instar larvae of Cx. quinquefascia-
tus and Ae. aegypti, respectively (Table 1). Similarly, L C50 and L
C90 results of crude
ethyl acetate extract of VAS-16 were 101.7 and 299.1 ppm and 111.7 and 330.7 ppm
against the pupae of Cx. quinquefasciatus and Ae. aegypti, respectively (Table 2). These
combined significant larvicidal and pupicidal activities further encouraged us to inves-
tigate its chemical constituents and compound isolation. Furthermore, the active iso-
late VAS-16 was identified morphologically (Fig. 2) and confirmed as Nonomuraea
sp. by 16S rRNA sequencing analysis, and the sequence was deposited at GenBank
(JN588559.1).
Bioassay‑Guided Fractionation
The larvicidal and pupicidal activities of different fractions eluted from ethyl acetate
extract of Nonomuraea sp. on Cx. quinquefasciatus and Ae. aegypti larvae and pupae are
depicted in Tables 3 and 4, respectively. Only five fractions showed larvicidal and pupi-
cidal activities. Among the 18 fractions screened, fraction-6 recorded highest larvicidal
activity with LC50 and L
C90 result of 9.1 and 18.7 ppm and 9.8 and 22.8 ppm against the
third stage larvae of Cx. quinquefasciatus and Ae. aegypti, respectively (Table 3). The
LC50 and L C90 results in pupicidal activity of fraction-6 were 10.5 and 23.1 ppm and
12.3 and 24.1 ppm against the pupae of Cx. quinquefasciatus and Ae. aegypti, respec-
tively (Table 4).
Characterization of the Active Principle
The active compound was obtained as colorless solid from aq.MeOH (mp. 289, yield
100 mg). It answered for carboxylic acid by giving yellow spot on a blue background
on treating the TLC plate with bromocresol green reagent (Rf = 0.53). EI-MS (m/z) 149
[phthalic anhydride + H]+, 128, 104, 77 and 57 (Fig. S3). IR ʋmax: 3089–2526 cm−1
(OH str. of COOH), 1695 cm−1 (C = O str. of COOH), 1583, 1494, 1402 cm−1 (aromatic
C = C), 904, 792 and 734 cm−1 (aromatic CH bending) (Fig. S4). 1H NMR (δ, DMSO-
d6, 400 MHz): 7.51–7.69 (4H, m, ArH) (Fig. S5). 13C NMR (δ, DMSO-d6, 100 MHz):
131.26 (C-1 and C-6), 128.21 (C-2 and C-5), 133.26 (C-3 and C-4) and 169.20 (COOH)
(Fig. S6). The 13C NMR-DEPT is given in Fig. S7. The structure of the compound was
confirmed to be 1,2-benzenedicarboxylic acid (Fig. 3). Finally, the identity was con-
firmed by comparison with authentic sample (MP. mmp, co-TLC, and superimposable
IR).
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Table 1 Lethal concentration (in ppm) of crude ethyl acetate extract of different actinomycetes against the third instar larvae of Cx. quinquefasciatus and Ae. aegypti
Mosquito species Treatment LC50 (ppm) 95% confidence limit LC90 (ppm) 95% confidence limit Slope ± SE Intercept ± SE χ2
LL UL LL UL
Culex quinquefasciatus VAS-2 161.6 139.7 186.1 609.3 477.4 859.5 2.2 ± 0.2 0.1 ± 0.4 3.2*
VAS-12 268.5 236.5 309.6 825.9 650.3 1153.0 − 1.3 ± 0.5 2.6 ± 0.2 3.0*
VAS-16 73.7 65.1 81.6 147.2 129.7 175.9 4.2 ± 0.4 − 2.9 ± 0.9 2.3*
VAS-18 405.9 332.0 536.1 1999.6 1262.8 4179.9 1.8 ± 0.2 0.1 ± 0.5 1.7*
Aedes aegypti VAS-2 170.74 137.49 211.18 684.06 480.71 1234.94 0.2 ± 0.6 2.1 ± 0.3 0.01*
VAS-12 319.7 279.6 374.8 992.1 762.0 1447.1 − 1.5 ± 0.6 2.6 ± 0.2 5.9*
VAS-16 84.7 72.8 96.1 225.2 192.5 278.2 3.0 ± 0.3 − 0.8 ± 0.6 1.7*
VAS-18 436.5 366.7 552.2 1576.1 1091.8 2777.7 − 1.0 ± 0.6 2.2 ± 0.2 3.9*
LC50 lethal concentration that kills 50% of the exposed larvae, LC90 lethal concentration that kills 90% of the exposed larvae, LL lower limit (95% confidence limit), UL upper
limit (95% confidence limit). *p ≤ 0.05, level of significance of chi-square values
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Table 2 Lethal concentration (in ppm) of crude ethyl acetate extract of different actinomycetes against the pupae of Cx. quinquefasciatus and Ae. aegypti
LL UL LL UL
Culex quinquefasciatus VAS-2 204.41 160.46 267.03 1059.19 658.38 2555.57 0.8 ± 0.6 1.7 ± 0.2 3.7*
VAS-12 344.7 212.6 1169.7 951.2 487.5 4655.9 − 2.3 ± 1.2 2.9 ± 0.5 5.9*
VAS-16 101.7 87.8 115.5 299.1 251.6 377.3 2.7 ± 0.2 − 0.4 ± 0.5 5.8*
VAS-18 647.3 505.9 953.6 2619.3 1569.7 6295.5 − 0.9 ± 0.7 2.1 ± 0.2 3.6*
Aedes aegypti VAS-2 226.01 178.13 299.14 1155.82 709.33 2861.81 0.7 ± 0.6 1.8 ± 0.2 0.8*
VAS-12 359.1 316.1 418.8 983.4 768.9 1401.3 − 2.4 ± 0.7 2.9 ± 0.2 4.9*
VAS-16 111.77 90.82 133.12 330.71 260.71 475.23 − 0.5 ± 0.7 2.7 ± 0.3 2.1*
VAS-18 1013.9 654.2 2362.5 8186.1 3179.9 56,624.0 1.4 ± 0.2 0.7 ± 0.6 0.6*
LC50 lethal concentration that kills 50% of the exposed pupae, LC90 lethal concentration that kills 90% of the exposed pupae, LL lower limit (95% confidence limit), UL upper
MNGA SKM AIA ISP-1
ISP-2 ISP-3 ISP-4 ISP-5
ISP-6 ISP-7 SCA PTA
Fig. 2 Macroscopic images of active isolate Nonomuraea sp. VAS-16 on Modified Nutrient Glucose
(MNGA), Skim Milk Agar (SKM), Actinomyces Isolation Agar (AIA), International Streptomyces Agar
(ISP) – 1–7, Starch Casein Agar (SCA), and Peptone tween agar (PTA)
Bioassay Results of 1,2‑Benzenedicarboxylic Acid
The isolated compound 1,2-benzenedicarboxylic acid showed good larvicidal and pupi-
cidal activities against Cx. quinquefasciatus and Ae. aegypti. The observed LC50 and
LC90 results were 4.27 and 14.90 ppm and 4.67 and 11.90 ppm against the third stage
larvae of Cx. quinquefasciatus and Ae. aegypti, respectively (Table 5). The observed
LC50 and L C90 results against the pupae of Cx. quinquefasciatus and Ae. aegypti were
4.58 and 12.06 ppm and 5.36 and 13.07 ppm, respectively (Table 6). Furthermore, all
the chi-square values were significant in these results. The larvicidal and pupicidal
results of 1,2-benzenedicarboxylic acid were compared to positive controls azadirachtin
and temephos (Tables 5 and 6).
The isolated compound 1,2-benzenedicarboxylic acid recorded less ovicidal activity
against the eggs of both Cx. quinquefasciatus and Ae. aegypti mosquitoes. The percent
egg mortality at 2 ppm concentration was recorded to be 11.0% and 10.3% against the
eggs of Cx. quinquefasciatus and Ae. aegypti, respectively, after 120 h post-treatment
(Fig. 4a and b). The positive control azadirachtin recorded the highest egg mortality of
68.0% and 60.8% against the eggs of Cx. quinquefasciatus and Ae. aegypti, respectively.
Temephos recorded egg mortality of 32.0% and 22.8% against the eggs of Cx. quinque-
fasciatus and Ae. aegypti, respectively (Fig. 4a and b).
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Table 3 Lethal concentration (in ppm) of different fractions against the third instar larvae of Cx. quinquefasciatus and Ae. aegypti
LL UL LL UL
Culex quinquefasciatus Fraction 3 24.7 15.55 273.17 58.9 26.27 4323.09 3.3 ± 1.1 0.2 ± 1.1 0.1*
Fraction 6 9.1 8.15 10.48 18.7 15.19 26.06 4.0 ± 0.4 1.0 ± 0.4 1.3*
Fraction 7 21.3 14.64 99.17 47.6 24.25 800.09 3.6 ± 1.1 0.1 ± 1.1 0.1*
Fraction 11 26.4 16.51 148.07 76.8 32.73 1871.80 2.7 ± 0.8 1.0 ± 0.7 0.5*
Fraction 15 18.5 13.79 44.64 41.2 23.62 225.68 3.7 ± 0.9 0.3 ± 0.8 0.1*
Aedes aegypti Fraction 3 25.2 15.98 162.27 66.5 29.37 1993.5 3.0 ± 0.9 0.7 ± 0.8 0.2*
Fraction 6 9.82 8.59 11.78 22.85 17.55 34.76 3.4 ± 0.4 1.5 ± 0.3 4.9*
Fraction 7 21.8 14.97 76.65 54.0 27.12 577.22 3.2 ± 0.9 0.6 ± 0.8 0.1*
Fraction 11 27.6 16.65 256.77 77.0 31.43 4350.9 2.8 ± 0.9 0.8 ± 0.8 0.2*
Fraction 15 19.5 14.14 52.03 44.7 24.64 294.85 3.5 ± 0.9 0.4 ± 0.8 0.1*
Table 4 Lethal concentration (in ppm) of different fractions against the pupae of Cx. quinquefasciatus and Ae. aegypti
LL UL LL UL
Culex quinquefasciatus Fraction 3 25.2 16.02 146.71 67.5 29.93 1721.1 2.9 ± 0.9 0.7 ± 0.8 0.1*
Fraction 6 10.5 9.26 12.85 23.1 17.74 36.23 3.7 ± 0.5 1.1 ± 0.4 2.3*
Fraction 7 22.6 14.96 156.15 51.1 24.69 1682.08 3.6 ± 1.2 0.1 ± 1.1 0.1*
Fraction 11 30.4 16.80 3460.32 80.0 29.15 293,272.1 3.0 ± 1.2 0.4 ± 1.1 0.1*
Fraction 15 20.0 14.20 73.43 42.8 23.06 479.05 3.8 ± 1.1 − 0.1 ± 1.1 0.1*
Aedes aegypti Fraction 3 27.7 16.59 301.26 75.6 30.73 5545.75 2.9 ± 0.9 0.7 ± 0.8 0.4*
Fraction 6 12.3 10.76 15.77 24.13 18.07 42.70 4.3 ± 0.7 0.2 ± 0.6 0.3*
Fraction 7 23.3 15.45 105.49 59.5 28.18 978.46 3.1 ± 0.9 0.6 ± 0.8 0.1*
Fraction 11 35.1 18.35 1627.13 110.8 36.20 95,342.32 2.5 ± 0.9 1.0 ± 0.8 0.3*
Fraction 15 20.5 14.53 62.17 48.9 25.80 401.70 3.4 ± 0.9 0.5 ± 0.8 0.1*
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Fig. 3 Chemical structure of

1,2-benzenedicarboxylic acid
Sub‑lethal Effects
In the present study, the compound 1,2-benzenedicarboxylic acid did not produce any
sub-lethal effects such as larval deformities, pupal deformities, or any other growth distur-
bances against Ae. aegypti and Cx. quinquefasciatus. It did not produce any intermediates
and larval longevity at the sub-lethal concentration against both the mosquito species.
Discussion
Newer methods and tools are warranted for safer and effective mosquito control, due to the
fact that mosquitoes develop resistance to conventional insecticides [18]. From the litera-
ture, it is evident that crude extract and the compounds isolated from actinomycetes have
proved to be effective against various species of mosquitoes [3, 8, 26, 30]. Furthermore,
microbial origin bioactive compounds are said to possess many advantages, particularly
it is target-specific and safe to environment and does not harm/kill co-existing beneficial
organisms [5, 26]. Hence, this would be an ideal eco-friendly approach for the control of
vector mosquitoes.
The present study revealed that the crude ethyl acetate extract of Nonomuraea sp.
and the isolated compound 1,2-benzenedicarboxylic acid is effective against the third
stage larvae and pupae of Cx. quinquefasciatus and Ae. aegypti mosquitoes. Similar to
our study, Saurav et al. [29] reported the larvicidal activity of an actinobacterial extract
and compound 5-(2,4-dimethylbenzyl) pyrrolidin-2-one against An. stephensi and Cx.
tritaeniorhynchus mosquitoes with LC50 values of 88.97 and 0.817 ppm and 74.95 and
0.781 ppm, respectively, against An. stephensi and Cx. tritaeniorhynchus mosquito lar-
vae. In another study, 30 different marine actinobacteria extracts were screened against
Ae. aegypti and An. stephensi mosquito larvae. Five isolates have shown the most signif-
icant mortality rate of the Ae. aegypti and An. stephensi mosquito larvae. Among them,
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Table 5 Lethal concentration (in ppm) of 1,2-benzenedicarboxylic acid isolated from Nonomuraea sp. against the third instar larvae of Cx. quinquefasciatus and Ae. aegypti
Mosquito species Treatment LC50 (ppm) 95% confidence LC90 (ppm) 95% confidence Slope ± SE Intercept ± SE χ2
limit limit
LL UL LL UL
Culex quinquefasciatus 1,2-Benzenedicarboxylic acid 4.27 2.70 25.49 14.90 5.98 631.57 3.1 ± 0.2 3.5 ± 0.1 0.5*
Azadirachtin 0.28 0.12 0.37 0.55 0.46 0.66 1.9 ± 0.3 7.4 ± 0.3 0.1*
Temephos 0.61 0.55 0.67 1.19 1.07 1.35 5.9 ± 0.1 4.4 ± 0.4 4.0*
Aedes aegypti 1,2-Benzenedicarboxylic acid 4.67 2.80 691.67 11.90 4.74 137.35 3.3 ± 0.4 2.8 ± 0.3 0.07*
Azadirachtin 0.34 0.22 0.43 1.04 0.90 1.27 2.4 ± 0.2 6.2 ± 0.1 3.7*
Temephos 0.63 0.57 0.70 1.25 1.13 1.43 5.8 ± 0.1 4.3 ± 0.4 3.6*
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Table 6 Lethal concentration (in ppm) of 1,2-benzenedicarboxylic acid isolated from Nonomuraea sp. against the pupae of Cx. quinquefasciatus and Ae. aegypti
Mosquito species Treatment LC50 (ppm) 95% confidence LC90 (ppm) 95% confidence Slope ± SE Intercept ± SE χ2
limit limit
LL UL LL UL
Culex quinquefasciatus 1,2-Benzenedicarboxylic acid 4.58 3.07 16.85 12.06 5.81 141.42 2.9 ± 0.2 3.0 ± 0.8 0.6*
Azadirachtin 0.58 0.52 0.64 1.10 1.00 1.25 6.0 ± 0.1 4.6 ± 0.4 5.1*
Temephos 0.65 0.56 0.73 1.62 1.42 1.93 6.1 ± 0.2 5.5 ± 0.1 1.7*
Aedes aegypti 1,2-Benzenedicarboxylic acid 5.36 3.19 158.47 13.07 5.37 5003.41 2.5 ± 0.3 3.3 ± 1.2 0.1*
Azadirachtin 0.60 0.54 0.66 1.15 1.04 1.31 5.9 ± 0.1 4.5 ± 0.4 4.6*
Temephos 0.92 0.11 1.66 1.82 1.17 646.1 6.3 ± 0.3 5.1 ± 0.1 2.4*
Fig. 4 Percent ovicidal activity of 1,2-benzenedicarboxylic acid isolated from Nonomuraea sp. (VAS-16)
against the eggs of (a) Cx. quinquefasciatus and (b) Ae. aegypti compared with azadirachtin and temephos.
Each concentration represents mean of five replicates ± SD; bar graphs carrying different letters are statisti-
cally different by Tukey’s test at p = 0.05
one actinobacterial extract (S. alboflavus) showed significant larvicidal activity with
LC50 and LC90 result of 1.48 and 3.33 ppm against Ae. aegypti and 1.30 and 3.13 ppm
against An. stephensi mosquito larvae, respectively [5].
However, the compound 1,2-benzenedicarboxylic acid showed less ovicidal activity
against the tested mosquito eggs. Studying the mode of action of the effective com-
pound will provide us a greater advantage in developing an effective insecticidal prod-
uct. Literature reveals that the compounds isolated from plant and microbial origin show
their action in midgut cells and/or interact with insect hormones and prevent the larvae
to mature into the next stage [25, 27]. Some natural compounds disturb the enzyme/
endocrine regulation and produced deformities/malformation in the developing stage of
the mosquitoes [17, 20, 25, 26]. In our present investigation, the compound 1,2-ben-
zenedicarboxylic acid did not produce any sub-lethal effects on the developing stage of
the Cx. quinquefasciatus and Ae. aegypti mosquitoes. Since the compound 1,2-benzen-
edicarboxylic acid is an aromatic acid, it might have affected the respiratory system of
the mosquito larvae or acted on the calcium release channel and produced mortality as
specified earlier [10]. Previously, Feng et al. [10, 11] synthesized new amides from the
compound 1,2-benzenedicarboxylic acid (phthalic acid) and tested its efficacy on the
larvae of a moth species Plutella xylostella. In their study, the few compounds recorded
higher insecticidal activity up to 90% against P. xylostella. Nevertheless, further studies
on the efficacy of derivatives of this compound or structure–activity relationship need to
be established to enhance its mosquitocidal potential against vector mosquitoes.
Conclusion
In conclusion, actinomycetes are rich source for bioactive compounds. It is clear from
this study that the Nonomuraea sp. and the isolated compound 1,2-benzenedicarboxylic
acid were active against Cx. quinquefasciatus and Ae. aegypti mosquitoes. This can be
used as natural mosquitocide in integrated mosquito management program.
13
Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1007/s12010-021-03766-8.
Acknowledgements The authors are grateful to lab attendants, who are involved in maintaining cyclic col-
ony of mosquitoes and helped in conducting all the experiments.
Author Contribution All authors contributed to the study conception and design. Isolation of soil actinomy-
cetes, media preparation, isolation and identification of active compound, bio-assay experiments, data col-
lection, and data analysis were performed by Pachaiyappan Saravana Kumar and Appadurai Daniel Reegan.
Spectroscopic analysis of the compound was performed by Kedike Balakrishna. The first draft of the manu-
script was written by Appadurai Daniel Reegan. All authors read and approved the final manuscript.
Data Availability The datasets used and/or analyzed during the current study are available from the corre-
sponding author on reasonable request.
Declarations
Ethics Approval This research article does not contain any studies with human participants or animals per-
formed by any of the authors. Hence, no formal consent is required.
Consent to Participate The authors give their consent to publish this research article.
Consent for Publication The authors give their consent to participate in this research article.
Conflict of Interest The authors declare no competing interests.
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Authors and Affiliations
Pachaiyappan Saravana Kumar1,2 · Appadurai Daniel Reegan3 ·

Karunakaran Rajakumari4 · Antony Cruz Asharaja4 · Kedike Balakrishna5 ·
Savarimuthu Ignacimuthu2
* Pachaiyappan Saravana Kumar

savanah.kumar@gmail.com
* Appadurai Daniel Reegan
danielreegan85@gmail.com
1
CAS Key Laboratory of Tropical Marine Bio‑Resources and Ecology, Guangdong Key Laboratory
of Marine Materia Medica, Sea Institute of Oceanology, Chinese Academy of Sciences,
Guangzhou 510 301, China
2
Xavier Research Foundation, St. Xavier’s College, Affiliated to Manonmaniam Sundaranar
University, Palayamkottai 627 002, Tamil Nadu, India
3
National Centre for Disease Control, Bengaluru Branch, No:8, NTI Campus, Bellary Road,
Bengaluru 560 003, Karnataka, India
4
P.G. and Research Department of Zoology, Pasumpon Muthuramalinga Thevar College, Affiliated
to Manonmaniam Sundaranar University, Melaneelithanallur, Tenkasi 627 953, Tamil Nadu, India
5
Entomology Research Institute, Loyola College, Affiliated to University of Madras,
Chennai 600 034, Tamil Nadu, India
13

Bioefcacy of Soil Actinomycetes and An Isolated Molecule

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Bioefcacy of Soil Actinomycetes and An Isolated Molecule

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Applied Biochemistry and Biotechnology (2022) 194:4765–4782

Bio‑efficacy of Soil Actinomycetes and an Isolated Molecule

Pachaiyappan Saravana Kumar1,2 · Appadurai Daniel Reegan3 ·

Extended author information available on the last page of the article

Keywords Culex quinquefasciatus · Aedes aegypti · Nonomuraea sp. VAS-16 ·

Mosquitoes are blood-sucking insects responsible for transmitting disease-causing patho-

Materials and Methods

General Experimental Procedures

Isolation and Identification of Terrestrial Actinomycetes

Bioassay‑Guided Isolation of Active Principle

Larvicidal and Pupicidal Assay

Isolation of Soil Actinomycetes

VAS-1 VAS-2 VAS-3 VAS-4 VAS-6

VAS-7 VAS-8 VAS-9 VAS-10 VAS-11

VAS-12 VAS-14 VAS-15 VAS-16 VAS-17

VAS-18 VAS-19 VAS-20 VAS-21 VAS-22

Fig. 1 Macroscopic images of VAS isolates on Actinomyces Isolation Agar (AIA)

Larvicidal and Pupicidal Activities

Characterization of the Active Principle

MNGA SKM AIA ISP-1

ISP-2 ISP-3 ISP-4 ISP-5

ISP-6 ISP-7 SCA PTA

Bioassay Results of 1,2‑Benzenedicarboxylic Acid

Fig. 3 Chemical structure of

Conflict of Interest The authors declare no competing interests.

Authors and Affiliations

Pachaiyappan Saravana Kumar1,2 · Appadurai Daniel Reegan3 ·

* Pachaiyappan Saravana Kumar

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