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Insecticidal prospects of algal and cyanobacterial extracts against the cotton

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Vie et milieu - Life and environment, 2018, 68 (4): 199-212

Insecticidal prospects of algal and cyanobacterial

extracts against the cotton leafworm Spodoptera
littoralis

A. A. Saber 1*, S. M. Hamed 2, E. F. M. Abdel-Rahim 3, M. Cantonati 4

Botany Department, Faculty of Science, Ain Shams University, Abbassia Square-11566, Cairo, Egypt
1

2
Soil Microbiology Department, Soils, Water & Environment Research Institute, Agricultural Research Center, Giza, Egypt
3
Sids Station, Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt
4
MUSE - Museo delle Scienze, Limnology and Phycology Section, Corso del Lavoro e della Scienza 3, I-38123 Trento, Italy
* Corresponding author: abdullah_elattar@sci.asu.edu.eg

ALGAE ABSTRACT. – Information available on the utilization of algae and cyanobacteria, with mani-
CYANOBACTERIA
SPODOPTERA LITTORALIS
fold potential applications in agriculture and biotechnology, for commercial biopesticide pro-
BIOPESTICIDES duction is still scarce. We used the algal/cyanobacterial extract-larval contact method to assay in
FATTY ACIDS vitro against different developmental life cycle stages (the 2nd and 4th instars) of the cotton leaf-
BIOCONTROL MANAGEMENT worm Spodoptera littoralis crude ethanolic extracts of five different algal and cyanobacterial
species: Chara vulgaris (stonewort), Parachlorella kessleri, Ulva intestinalis, Cladophora
glomerata (green algae), and the heterocytous cyanobacterium Nostoc carneum. All the algal
and cyanobacterial treatments showed insecticidal activities against larvae of S. littoralis, with
2nd larval instars being more susceptible. In general, P. kessleri and N. carneum exhibited the
highest insecticidal toxic effects while the stonewort C. vulgaris showed the lowest insecticidal
activity. Effects of these algal and cyanobacterial treatments on larval duration, % pupal forma-
tion, pupal duration and weight, % moth emergence, adults’ fecundity and longevity, eggs’
hatching, sex ratios, and larval-pupal-moth malformations were discussed. Gas chromatogra-
phy-mass spectrometry (GC-MS) analyses of the crude algal and cyanobacterial extracts
revealed 29 compounds, mainly containing an array of saturated and unsaturated fatty acids,
some essential oils, and terpene compounds like phytol. Insecticidal activities of the algal and
cyanobacterial specimens can be attributed to the synergistic effects of the abovementioned
phytochemical constituents, particularly fatty acids such as the polyunsaturated ω-6 linoleic,
palmitic, oleic, myristic, α-linolenic, and 7, 10-hexadecadienoic acids. The coccoid green alga
P. kessleri and the cyanobacterium N. carneum can thus be recommended as ecofriendly biopes-
ticides for the integrated pest management of the cotton leafworm and other agricultural pests.

Introduction growth regulators (IGRs) have also been considered in

several studies (Pineda et al. 2009, Nasr et al. 2010, El-
Agriculture, including cotton cultivation, is consid- Sheikh & Aamir 2011). In addition, the development of
ered one of the main resources for the Egyptian economy. entomopathogenic nematodes and bacterial strains as
However, this crop with relevant economic value suffers biocontrol agents were discussed (Abdel-Razek 2006,
seriously from the infection by harmful pests, includ- Fernando et al. 2009, Azzouz et al. 2014). However, the
ing the Egyptian cotton leafworm Spodoptera littoralis cotton leafworm S. littoralis showed resistance capacity
Biosd. (Lepidoptera: Noctuidae), mainly affecting its net to several of the aforementioned insecticides, and this
productivity and quality (Amin & Gergis 2006). The cot- is likely to be due to enzymes inactivating or degrad-
ton leafworm is considered one of the most deleterious ing these toxic insecticides into non-toxic metabolites
pests infecting a wide spectrum of crops including cotton, (Mosallanejad & Smagghe 2009, Abd El-Mageed & Sha-
tobacco, corn, and several vegetables and mainly distrib- laby 2011).
uted in African, Mediterranean, and Middle-East coun- Sustainable agriculture not only aims at minimizing
tries (e.g., Salama et al. 1990, El-Aswad 2007, Azzouz et incidence of diseases to such a degree that they do not
al. 2014). cause extensive damages but it also favors increasing
During the last decades, different protocols to manage the yield and crop quality with an integrated pest man-
and control the cotton leafworm have been applied and agement (Isman 2006, Chandler et al. 2011, Ladhari et
evaluated, in Egypt and worldwide. Some synthetic chem- al. 2013, Hamed et al. 2018). Chemical pesticides are
ical compounds, with distinctly toxic effects on humans capable of killing a wide range of agricultural pests but
and the environment, have already been recommended as their overuses are unintentionally responsible for many
S. littoralis insecticides (Pluschkell et al. 1998, Abd El- troubles including human safety risks, environmental
Mageed & Shalaby 2011, Ghoneim et al. 2012). Insect contamination, remarkable decrease in biodiversity, and
200 A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

resurgence of insecticidal-resistant strains (Konstantinou Ulvales); and the heterocytous cyanobacterium Nostoc car-
et al. 2006, Reinecke & Reinecke 2007, Bretveld et al. neum (Cyanobacteria: Nostocales). Chara vulgaris specimens
2008, Czeher et al. 2008, Yadouleton et al. 2010, Desai (Fig. 1A-B) were obtained from the algal culture collection of
et al. 2016). Biopesticides can, in general, provide impor- the Phycology Unit No. 341 at the Botany Department, Faculty
tant contributions to sustainable agriculture and do help of Science, Ain Shams University, Cairo, Egypt. The original
to reduce the reliance on harmful chemical pesticides thalli were collected from the Springs of Moses (South Sinai,
(Saxena & Pandey 2001, Dubey et al. 2010, Chandler Egypt) (29°52’21.38”N, 32°39’00.41”E). The morphology and
et al. 2011, Abdel-Rahim & Hamed 2013b). Biopesti- life cycle in nature and laboratory, and the matK molecular phy-
cides are pest management agents mainly extracted from logeny of these C. vulgaris specimens have been the subject of a
microorganisms like bacteria, fungi, algae, nematodes, or previous study (Saber et al. 2018). All details about the culturing
natural products such as plant-derived pesticides, ‘botani- of these stoneworts are available in Saber et al. (2018). New-
cals’ and semiochemicals, known as ‘insect pheromones’ ly-formed thalli were cut, collected, deprived of reproductive
(Copping & Menn 2000, Gašić & Tanović 2013, Guerrero structures, then dried at room temperature and stored at –20 ºC
et al. 2014). Application of biopesticides is considered until the following experimental step. Freshly-sampled speci-
nowadays as an important prerequisite to continuously mens of the branched green filaments of C. glomerata (Fig. 1C)
find out novel structures and eco-environmentally safe were collected by hand from the bedrock of the Rosetta Branch
compounds with conventional activities (e.g., Mazid et of the River Nile at El-Qanater El-Khayreya (30°11’32.56”N, E
al. 2011, Ansari et al. 2012, Glare et al. 2016). 31°6’9.82”E) in sterile clean plastic bags, washed in the field to
Although there are considerable data published about remove any epiphytes, and then transported on ice to the labora-
prospects of some algal and cyanobacterial taxa as insec- tory for further analyses. In the lab, the specimens were cleaned
ticidal biopesticides in general (Kiviranta & Abdel- again using distilled water to completely remove epiphytes
Hameed 1994, Becher et al. 2007, Mori et al. 2016, Vieira and debris. This step was verified by microscopic examination
et al. 2017), very little information has hitherto been pro- (Fig. 1D). The specimens were dried at room temperature in
vided about the feasibility of using them to manage and the dark and stored at –20 ºC. Specimens of C. glomerata were
control the cotton leafworm (Sharaby et al. 1993, Dela- morphologically identified following John (2011). The fresh
ney & Wilkins 1995, Aboutabl et al. 2002, Abdel-Rahim monostromatic green thalli of U. intestinalis (Fig. 1E) were col-
& Hamed 2013a). The main aim of the present study was lected from the rocks of the Mediterranean coast in Alexandria
to investigate the different pesticidal effects of crude eth- (31°17’25.94”N, 30°1’21.64”E) (Al-Montazah district, Egypt).
anolic extracts of five algal and cyanobacterial species The macroalgal specimens were washed, collected in sterile
against different life stages of the cotton leafworm S. lit- clean plastic bottles, transported on ice to the laboratory, and
toralis (2nd and 4th larval instars) using the contact meth- then cleaned again in the lab using distilled water to completely
od, along with a gas chromatography-mass spectrometry remove epiphytes and debris. The cosmopolitan U. intestinalis
(GC-MS) analysis of their potentially bioactive constitu- materials were morphologically identified following the descrip-
ents, focusing on fatty acid profiles. tive classification adopted by Bliding (1963). The coccoid green
alga P. kessleri (Figs 1F-G) and the nostocalean cyanobacteri-
um N. carneum (Figs 1H-I) were obtained from the algal cul-
Materials and methods ture collection of the Agriculture and Microbiology Research
Department at the Soils, Water and Environment Research
Insect rearing: The cotton leafworm S. littoralis was reared Institute, the Agricultural Research Center, Giza, Egypt. The
in the laboratory for several generations at room temperature 26 original specimens were isolated from the Egyptian agricultural
± 2 °C and relative air humidity 60 ± 5 % (Shalaby et al. 2013). lands and phylogenetically identified using 16S and SSU rRNA
Larvae were fed on castor-oil-plant (Ricinus communis L.) gene sequences (Hamed et al. 2017). The specimens were mor-
leaves in large glass jars until pupation and adults’ emergence. phologically identified following the current taxonomic litera-
The newly emerged adults were allowed to mate inside glass ture: Komárek (2013) for the cyanobacterium N. carneum, and
jars supplied with a piece of cotton wetted with 10 % sugar solu- Krienitz et al. (2004) for the coccoid green alga P. kessleri. The
tion as a feeding source for emerged moths, and leaves of the cyanobacterium N. carneum was cultured in 1-L Erlenmeyer
castor oil plant as the oviposition site (El-Defrawi et al. 1964). flasks, each containing 500 ml BG-110 (N-free) medium (Rippka
Egg masses were kept in plastic jars until hatching. The 2nd and et al. 1979), while the unialgal strain P. kessleri was cultivated
4th larval instars generated were used for bioactivity tests. in Bold’s Basal Medium (BBM, Bold 1949, Bischoff & Bold
1963). The inoculated flasks were kept in a growth chamber at
Algal and cyanobacterial specimens: origin, identification, 28 ± 1 °C and 16:8 h L/D photoperiod using 20-W cool white
and culturing: Five different algal and cyanobacterial species fluorescent lamps at an irradiance of 100 μmol photons m –2
were considered in this study: the stonewort Chara vulgaris s–1 and shaken daily. The biomass growths of N. carneum and
(Streptophyta: Charales); the green algae Cladophora glom- P. kessleri were harvested in the late exponential growth phase
erata (Chlorophyta: Cladophorales), Parachlorella kessleri (ca. 3 weeks) by filtration on Whattman® filter papers, and then
(Chlorophyta: Chlorellales), Ulva intestinalis (Chlorophyta: dried at room temperature in the dark. Bright-field photomi-

Vie Milieu, 2018, 68 (4)

 ALGAL AND CYANOBACTERIAL BIOPESTICIDES 201

Fig. 1. – Light microscopy (LM) of algal and cyanobacterial specimens included in this study. A: Macroscopic habitus of the stonewort
Chara vulgaris. B: Close-up view on the terminal part of C. vulgaris thallus showing lateral branchlets completely free from reproduc-
tive structures. C: Mass growth of the filamentous green alga Cladophora glomerata. D: LM photomicrograph of C. glomerata con-
firming complete absence of epiphytes. E: Macroscopic habitus of the monostromatic green alga Ulva intestinalis. F-G: The coccoid
green alga Parachlorella kessleri showing spherical cells, cup-shaped chloroplasts and a four-celled mother cell (Fig. G; arrowhead).
H-I: LM images of the heterocytous cyanobacterium Nostoc carneum showing flexuous filaments (Fig. H), barrel-shaped to short
cylindrical vegetative cells, heterocyte (arrowhead), and ellipsoidal to oval-cylindrical akinetes (Fig. I; double arrowheads). Scale
bars = 1 cm (Figs A-C); 50 μm (Fig. D); 5 cm (Fig. E); 10 μm (Figs F-I).

Vie Milieu, 2018, 68 (4)

202 A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

crographs of algal and cyanobacterial specimens studied were Gas chromatography-mass spectrometry (GC/MS) analysis:
examined using a BEL® photonics biological microscope (BEL® Fatty acid methyl esters and other phytochemical compounds
Engineering, Monza, Italy) fitted with a Canon Powershot G12 were analyzed by GC-MS using a DB-1 capillary column (15 m
digital camera. × 0.25 mm ID, 0.25 μm film thickness) in a TRACE™ 1310 gas
chromatograph (ThermoFisher Scientific Co., Paisley, UK) cou-
Extraction of algal and cyanobacterial materials: The eth-
pled to ISQ™ LT single quadrupole mass spectrometer. The car-
anolic algal and cyanobacterial extractions were carried out
rier gas was helium with a constant flow rate of 1.5 mL.min–1.
following the method described by Abdel-Rahim & Hamed
The oven temperature was initially kept at 115 ºC for 1 min and
(2013a) with a slight modification. The dried algal biomass
for each specimen was crushed well, weighted, then soaked then ramped at 7.5 ºC.min–1 to 280 ºC for 3 min. 1.0 μL of each
in 99 % ethanol overnight and finally agitated in an orbital sample solution was injected. Mass spectra were obtained by
shaker (MaxQTM 2000 open-air platform shaker, ThermoFisher electron ionization (EI) at 70 eV. The compounds were identi-
Scientific, Paisley, UK) at 120 rpm for 2 h. Briefly, three dif- fied by comparison of their mass spectra with those of the Wiley
ferent concentrations were prepared by soaking 500, 750, and and NIST 05 L mass spectral databases.
1000 mg of each dried algal/cyanobacterial biomass in 10 ml of
99 % (v/v) ethanol. The algal and cyanobacterial extracts were Statistical analysis: The data were statistically analyzed using
separated by centrifugation. The effects of LC50 of each etha- SPSS version 22.0 (IBM SPSS Statistics for Windows, version
nolic algal/cyanobacterial extract on the main biological char- 22.0, Armonk, NY: IBM Corp 2013). Student’s independent
acterizations and activities of the cotton leafworm S. littoralis,
t-test and Waller-Duncan test were used to analyse the statistical
including larval and pupal durations, percentages of pupation
significance of differences between the algal and cyanobacte-
and adults’ emergence, pupal weights, pupal and moth malfor-
rial treatments and those of the control at p < 0.05 and 0.01. All
mations, fecundity, eggs’ hatchability, adults’ longevity and sex
ratios, were investigated using the contact application method as the results were expressed as mean values ± standard deviation
described later. (SD). Percentages of the larval mortality were recorded after
24 h and corrected following Abbott’s formula (Abbott 1925).
Algal/cyanobacterial-insect interaction ‘contact method’: The data were analyzed using Probit analysis (Finney 1971) and
2 ml of each algal and cyanobacterial extract was gently dis- the LC50 values were estimated for the 2nd and 4th larval instars.
tributed on the inner surface of the cleaned and sterilized 9-cm
Petri dish, and then kept at room temperature (ca. 12 h) until
ethanol evaporation and formation of thin films of the algal Results
and cyanobacterial extracts in the Petri dishes (Abdel-Rahim &
Hamed 2013a). Free ethanol (99 %) was used as the control. Ten Insecticidal activity of the algal and cyanobacterial
larval individuals of each instar were kept for about 24 h in each extracts
algal and cyanobacterial extract-containing Petri dish. After that,
the larval instars were transferred to clean glass containers and Larval instars of the cotton leafworm S. littoralis
fed by fresh castor oil plant leaves until pupation. Forty 2nd and
showed a distinct sensitivity to algal and cyanobacterial
4th larval instars were used in each treatment and the control.
ethanolic extracts with a concentration-dependent pattern.
Peculiar malformations were recorded and photographed.
Furthermore, resistance of the instars to the crude algal
Lipid extraction and transmethylation: Total lipids were extracts significantly depended on their ages. Percentages
extracted from the algal and cyanobacterial specimens follow- of larval mortality of the 2nd larval instars of S. littoralis
ing the method adopted by Folch et al. (1957). Briefly, the crude (Fig. 2A) were distinctly more susceptible to all the crude
extracts were re-suspended in 10 ml of chloroform/methanol 2:1 ethanolic algal and cyanobacterial extracts than the 4th lar-
(v/v), sonicated for 15 min in an ultrasonic bath (Sonorex Super, val instars (Fig. 2B). The ethanolic extract of the coccoid
Bandelin electronics, Berlin, Germany), and centrifuged at green alga P. kessleri at the lower concentrations (50 and
3000x g for 10 min at room temperature to separate the organic 75 mg.mL–1) exhibited significantly (p < 0.01) the high-
phase (bottom layer). The procedure was repeated two times and est insecticidal toxic effect both on the 2nd and 4th larval
the organic phases were collected, washed with 20 mL of a 5 %
instars. On the contrary, the ethanolic extracts of the green
NaCl solution, filtered by using glass filters under vacuum, and
macroalga U. intestinalis (50 mg.mL–1) and of the stone-
reduced to dryness by rotary evaporation (Büchi Labortechnik
wort C. vulgaris (75 and 100 mg.mL–1) showed a non-
AG, Flawil, Switzerland). Fatty acids methyl esters were pre-
pared following a modified version of the procedure described significant insecticidal efficacy on the 2nd larval instars
by Mason & Waller (1964). 250 μL methanolic HCl (3M) was (Fig. 2A) as compared to other algal and cyanobacterial
added into 1 ml of each lipidomic extract. The tubes were then ethanolic extracts. However, the C. vulgaris 100 mg.mL–1
capped tightly with a Teflon-lined cap and subsequently incu- extract showed a significant (p < 0.05) insecticidal toxic
bated at 60 °C for 20 min. effect on the 4th larval instars (Fig. 2B).

Vie Milieu, 2018, 68 (4)

 ALGAL AND CYANOBACTERIAL BIOPESTICIDES 203

Effects of the algal and cyanobacterial extracts on

different developmental stages of S. littoralis

Potential toxic activities of the crude algal and

cyanobacterial ethanolic extracts on the larval instars of
the cotton leafworm S. littoralis were clearly observed on
their main morphological features and different life stag-
es. All the five algal and cyanobacterial extracts extend-
ed in general the larval duration, in particular at the 2nd
larval instars, to 13 days as compared to 12 days in the
control. The ethanolic algal extracts of U. intestinalis and
C. glomerata expanded the durations of 4th larval instars
to 13 days as compared to 11.8 days in the case of the
control (p < 0.01; Fig. 3A). With reference to the reduc-
tion in pupation, P. kessleri, C. glomerata and N. carneum
distinctly reduced the formation of pupae from the 2nd lar-
val instars, as compared to the control, up to 60, 60, and
50 %, respectively, and to 50, 50, and 45 %, respectively,
from the 4th larval instars (Fig. 3B). The crude ethanolic
extracts of C. vulgaris and U. intestinalis only reduced the
pupation process from the 2nd and 4th larval instars up to
40 % as compared to the control. The ethanolic extract of
the nostocalean cyanobacterium N. carneum significantly
(p < 0.01) prolonged the pupal duration of the 2nd larval
instars to an average 11.8 days as compared to 9.9 days
in the control (Fig. 3C), but the other algal crude extracts
shortened the pupal durations to less than the mean peri-
od recorded for the control. Nevertheless, the ethanolic
extract of the unicellular green alga P. kess­leri had the
most distinctive effect on pupal duration of the 4th larval
instars and mainly limited it to only 6.8 days (p < 0.01).
Marked reductions in the pupal weights were observed
using all the five extracts (Fig. 3D). In particular, N. car-
Fig. 2. – Percent of larvae mortality of the cotton leafworm neum and P. kessleri significantly (p < 0.01) decreased the
S. littoralis using different concentrations of the ethanolic algal pupal weights of the 2nd larval instars by 30.4 and 28.8 %,
and cyanobacterial extracts. A: Treatments of the 2 nd larval respectively, as compared to the control. The ethanolic
instars. B: Treatments of the 4th larval instars. Data are expressed
as mean ± SD. * significant difference p < 0.05; ** significant extracts of C. vulgaris and P. kessleri displayed the most
difference p < 0.01. toxic activities on the pupal weights of the 4th larval instars
and significantly (p < 0.01) reduced them by 24 % and
23.4 %, respectively. Gener-
ally, all extracts affected moth
Table I. – Insecticidal effects of the crude ethanolic algal and cyanobacterial extracts on adults’ emergence from the 2 nd and
fecundity, longevity, eggs hatching, and sex ratio produced from the 4 larval instars at LC50
th 4 th
larval instars (Fig. 3E).
values. Data are expressed as mean ± SD. * difference is significant at p < 0.05; ** difference is The P. kessleri extract, in par-
significant at p < 0.01.
ticular, was most effective in
Fecundity Eggs hatching Longevity Adult sex ratio (%) reducing adults’ emergence
Treatments
(eggs /f) (%) (days) Female Male from the 2 nd larval instars
Nostoc carneum 73.3 ± 3.3 **
78 7.5 ± 1.7 **
48.1 51.9 (46.7 %; p < 0.01), whilst
Parachlorella kessleri 65 ± 4.2 **
75 8.7 ± 1.4 56.5 43.5 N. carneum, U. intestinalis,
Ulva intestinalis 60 ± 10** 75 7.1 ± 2** 47.6 52.4 and C. glomerata caused the
highest decreases in adults’
Cladophora glomerata 75 ± 5** 70 9.1 ± 2.4 57.1 42.9
emergence from the 4th larval
Chara vulgaris 101 ± 8.5** 88 7.2 ± 3** 50 50
instars with similar percent-
Control 452 ± 271 100 9.4 ± 2 28 72
ages (76.4, 75, and 76.2). The
L.S.D. at 0.05 82 12 stonewort C. vulgaris had the
0.01 189 1.8 lowest insecticidal effects in

Vie Milieu, 2018, 68 (4)

204 A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

Fig. 3. – Insecticidal activities of the crude ethanolic algal and

cyanobacterial extracts on the 2nd and 4th larval instars of S. lit-
toralis. A: Larval period. B: Percent of pupation. C: Pupal dura-
tion. D: Pupal weight. E: Percent of moth emergence. Data are
expressed as mean ± SD. * significant difference p < 0.05; **
significant difference p < 0.01.

reducing the moths’ emergence, as compared to the con- nificant decline (p < 0.01) in the adults’ longevity (ca. 7
trol, from the 2nd and 4th larval instars (see Fig. 3E). days) was also recorded using N. carneum, U. ����������
intestina-
Treatments of the 4 th larval instars with the crude lis, C. vulgaris as compared to 9.4 ± 2 days in the control.
ethanolic algal and cyanobacterial extracts significantly Treatments of the 4th larval instars with all the algal and
(p < 0.01) reduced the adults’ fecundity where U. ������ intes- cyanobacterial extracts also distinctly modified the sex
tinalis followed by P. kessleri, N. carneum and C. ���� glo- ratio of males and females with respect to the control (see
merata mainly decreased the fecundity to average values Table I for more details).
of 60, 65, 73.3 and 75 eggs, respectively, as compared to
452 eggs in the control, while C. vulgaris had the weak- Insect malformations
est insecticidal effect (Table I). The same observation was
also detected on hatching (only 70-78 %) using the four Pupal malformations and distortions, produced from
aforementioned algal and cyanobacterial extracts. A sig- the 2nd and 4th larval instars, were observed in all treat-

Vie Milieu, 2018, 68 (4)

 ALGAL AND CYANOBACTERIAL BIOPESTICIDES 205

Fig. 4. – Malformations in pupae, larvae and moth adults observed in this study. A-B: Pre-pupae with complete blackening of the body.
C-D: Larval-pupal intermediates with larval cuticle patches, head capsule and thoracic legs, and posterior half of the body with pupal
properties. E: Undersized pupa showing body shrinkage. F-G: Pupal-moth intermediates maintaining the old pupal skin. H-J: Mal-
formed moth adults with abnormal body and slightly-twisted wings. K-L: Normal pupa and moth adult.

ments using the five crude ethanolic algal and cyanobacte- with pupal properties (Figs 4C-D), (3) undersized pupae
rial extracts by the contact method. The following abnor- with reduced bodies (Fig. 4E), (4) pupal-moth interme-
mal life stages were detected (Figs 4A-L): (1) pre-pupae diates with the old pupal skin (Fig. 4F), (5) malformed
with a complete blackening of the body ending by the moth adults with abnormal bodies and wings (Figs 4G-I),
death or failing to cast the old cuticle (Figs 4A-B), (2) lar- or slightly twisted wings (Fig. 4J) as compared to the nor-
val-pupal intermediates with larval cuticle patches, head mal pupae (Fig. 4K) and adults (Fig. 4L).
capsule, thoracic legs and the posterior half of the body

Vie Milieu, 2018, 68 (4)

 Table II. – GC-MS analysis of the crude extracts of algal and cyanobacterial specimens investigated in this study. Peak area (or percentage composition) of algal and cyanobacterial com-
206

pounds identified are relative to other constituents within the same extract of each species.
Peak area (%)
RT
(min) Nostoc Parachlorella Ulva Cladophora Chara
Compounds identified carneum kessleri intestinalis glomerata vulgaris
5,5-dimethylcyclopentadiene 5.30 2.74
Levulinic acid, methyl ester (4-oxo-pentanoic acid, methyl ester) 5.56 5.07 3.34 3.81
Lauric acid, methyl ester (dodecanoic acid, methyl ester) 14.65 2.21 0.97 3.95 1.23
Azelaic acid, dimethyl ester (nonanedioic acid, dimethyl ester) 15.15 1.73 4.69 2.21
6-phenyldodecane 15.78 2.76
3-phenylundecane 16.51 2.20
8-heptadecene 17.00 0.91
1-heptadecane 17.30 3.33 1.00
2-phenylundecane 17.45 1.35
Myristic acid, methyl ester (tetradecanoic acid, methyl ester) 17.73 3.44 2.31 4.65 26.25 3.84
5-phenyldodecane 17.83 2.43
3-phenyldodecane 18.26 1.61
2-phenyldodecane 18.94 4.00
6-phenyltridecane 19.16 3.05 1.68 1.91 0.94
4-phenyltridecane 19.45 1.39
Neophytadiene (7,11,15-trimethyl,3-methylene-1-hexadecene) 19.61 1.53 2.53 1.11 2.76
Phytol (3,7,11,15-tetramethyl-2-hexadecen-1-ol) 19.68 1.05 1.26 2.60 2.10 3.47

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3-phenyltridecane 19.81 1.60
7,10-hexadecadienoic acid, methyl ester 20.14 11.42 3.72 6.21
Palmitoleic acid, methyl ester (9-hexadecenoic acid, methyl ester) 20.27 6.70 1.95 5.76 5.68 2.29
2-phenyltridecane 20.38 2.27
Palmitic acid, methyl ester (hexadecanoic acid, methyl ester) 20.62 22.19 16.94 29.60 33.20 26.67
5-methylhexane-1,5-diol 20.74 1.15
Linoleic Acid, (Z,Z)-isomer (cis-9,cis-12-octadecadienoic acid) 21.55 1.15
A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

Oleic acid, methyl ester (9-octadecenoic acid, methyl ester) 22.78 11.43 11.87 15.90
Linoleic acid, methyl ester (9,12-octadecadienoic acid, methyl ester) 22.82 9.16 29.95 11.85 7.15
α-Linolenic acid, methyl ester (9,12,15-octadecatrienoic acid, methyl ester) 22.91 5.36
Stearic acid, methyl ester (octadecanoic acid, methyl ester) 23.08 1.58 2.00 3.60 0.96 2.40
Arachidonic acid, methyl ester (5,8,11,14-eicosatetraenoic acid, methyl ester) 24.69 1.24
 ALGAL AND CYANOBACTERIAL BIOPESTICIDES 207

Bioorganic compounds (with special attention to fatty and unsaturated fatty acids (11 different species), some
acids) in the algal and cyanobacterial extracts essential oils (alkane derivatives), and terpene compounds
such as phytol and neophytadiene (Table II). In each algal
GC-MS analyses of the algal and cyanobacterial and cyanobacterial species, fatty acids had the highest
extracts investigated in this study revealed 29 different relative concentrations (peak areas) with respect to other
chemical compounds, that mainly represented saturated phytochemical constituents. The heterocytous cyanobac-

Fig. 5. – GC-MS chromatographs

of fatty acids and other phy-
tochemical constituents (as %
relative areas to other constitu-
ents within the same lipidomic
extract of each species) shown in
this study. A: Nostoc carneum.
B : P a r a c h l o re l l a k e s s l e r i .
C: Ulva intestinalis.

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208 A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

terium N. carneum (Fig. 5A) had noticeable concentra- extract of the freshwater filamentous green alga C. glom-
tions of saturated palmitic acid, methyl ester (C16:0) erata revealed only 6 fatty acids with a total peak area of
(22.19 %), polyunsaturated ω-6 linoleic acid, methyl ester ca. 80 %. The saturated palmitic, methyl ester (33.20 %)
(C18:2) (9.16 %), monounsaturated ω-7 palmitoleic acid, and myristic, methyl ester (26.25 %) were the dominant
methyl ester (C16:1) (6.70 %), followed by myristic acid, species (Fig. 6A). The stonewort C. vulgaris contained 8
methyl ester (C14:0) (3.44 %), lauric acid, methyl ester fatty acids, and the prevailing species was the saturated
(C12:0) (2.21 %), and stearic acid, methyl ester (C18:0) palmitic acid, methyl ester (26.67 %) (Fig. 6B). Polyun-
(1.58 %). As regards the coccoid green alga P. kessleri, saturated ω-6 arachidonic acid, methyl ester (C20:4) was
9 fatty acids, representing ca. 72 % of the total constitu- only identified in this algal species but with a much lower
ents, were characterized and the main ones being: linoleic concentration (1.24 %) (Table II).
acid, methyl ester (ca. 30 %), palmitic acid, methyl ester
(ca. 17 %), and 7,10-hexadecadienoic acid, methyl ester
(11.42 %) (Fig. 5B). Additionally, both the polyunsaturat- Discussion
ed (ω-3) α-linolenic acid, methyl ester and linoleic acid,
(Z, Z)-isomer had only been found in this algal species This study revealed that the crude ethanolic extracts
with concentration values of 5.36 % and 1.15 %, respec- of the heterocytous cyanobacterium N. carneum, of the
tively. As for the green macroalga U. intestinalis, 8 fatty green algae P. kessleri, U. intestinalis, C. glomerata, and
acid methyl esters were identified with palmitic, linole- of the stonewort C. vulgaris have, in general, substantial
ic, and the monounsaturated ω-9 oleic fatty acid methyl insecticidal effects against larvae and other developmen-
ester being the most abundant (29.60, 11.85, and 11.43 %, tal life-history stages of the cotton leafworm S. littoralis,
respectively) (Fig. 5C). GC-MS analysis of the crude using the algal/cyanobacterial extract-larval contact meth-

Fig. 6. – GC-MS chromatographs

of fatty acids and other phy-
tochemical constituents (as %
relative areas to other constitu-
ents within the same lipidomic
extract of each species) shown in
this study. A: Cladophora glom-
erata. B: Chara vulgaris.

Vie Milieu, 2018, 68 (4)

 ALGAL AND CYANOBACTERIAL BIOPESTICIDES 209

od. More specifically, the 2nd larval instars were more sus- acid, methyl ester, in addition to the diterpene compounds
ceptible than the 4th larval instars. This observation is in phytol and neophytadiene. In even better agreement with
good agreement with results of Abdel-Rahim & Hamed our results, Ramos-López et al. (2012) assessed the fatty
(2013a) who found that the insect-algal contact treatment acid components of the hexane leaf extract of Ricinus
using the cyanobacterium Anabaena flosaquae was more communis against Spodoptera frugiperda and identi-
effective than the feeding treatment, and that the 2nd larval fied four constituents (linolenic, linoleic, palmitic, and
instars of S. littoralis were more sensitive to this kind of stearic acids) characterized by remarkable insectistatic
biological treatment than the 4th larval instars. Moreover, and insecticidal activities. A recent investigation carried
a recent paper by Shalaby et al. (2013) supported our out by Ali et al. (2017) discussed and confirmed the lar-
observations as it showed that the ethanolic extract of the vicidal and biochemical activities of garlic and lemon oils
plant Strychnos nux-vomica had remarkable insecticidal on S. littoralis, however without characterizing the poten-
effects against S. littoralis, with 2nd instars being more tially active constituents. As regards cyanobacteria, little
sensitive than the 4th instars. Abd El-Mageed & Shalaby attention was hitherto given to their potential insecticidal
(2011) also reported the same conclusion using mixtures activities on the cotton leafworm S. littoralis (e.g., Abdel-
of several chemical insecticides including chlorosan, fer- Rahim & Hamed 2013a), whilst the toxic effects of some
oban, cygron, engeo, and kingbo. Adjalian et al. (2015) of their metabolites were investigated in relation to other
documented and discussed the insecticide-contact nature pests. Harada et al. (2000) linked the larval mortality of
of certain essential oils extracted from the higher plants the mosquito Aedes albopictus to the unsaturated fatty
Premna angolensis and P. quadrifolia on the grain moth acids present in the filamentous cyanobacterium Oscilla-
Sitotroga cerealella. toria agardhii. Becher et al. (2007) isolated and identified
In this study, the crude ethanolic extracts of the coc- four insecticidal alkaloids including 12-epi-Hapalindole
coid green alga P. kessleri and of the heterocytous J isonitrile, 12-epi-hapalindole C isonitrile, hapalindole
cyanobacterium N. carneum appeared, in general, to be L and 12-epi-hapalindole E isonitrile from the fresh-
more toxic and effective against the different develop- water cyanobacterium Fischerella ATCC 43239 (order
mental life-cycle stages (2nd and 4th larval instars) of the Nostocales) and showed that these metabolites could be
cotton leafworm S. littoralis, while C. vulgaris specimens considered as promising natural compounds for dipteran
had the lowest insecticidal effect. This observation is biocontrol. Biondi et al. (2004) also pointed out that the
likely to be related to the synergistic effects of saturated methanolic extract of the heterocytous cyanobacterium
and unsaturated fatty acids, of some essential oils (alkane Nostoc strain ATCC 53789, producing cryptophycins,
derivatives) like 2-phenyldodecane, 3-phenylundecane has a remarkable biopesticidal activity against the cotton
and 6-phenyltridecane, and of terpene compounds such bollworm Helicoverpa armigera. Moreover, Delaney &
as phytol and neophytadiene. With respect to fatty acids, Wilkins (1995) pointed to the efficacy of the cyanobac-
the species most likely to show substantial insecticidal terial cyclic metabolite hepatotoxin ‘microcystin-LR’ as
properties are polyunsaturated ω-6 linoleic acid (the pre- a potent insecticide against the third instars of the cot-
dominant fatty acid species in P. kessleri), followed by ton leafworm S. littoralis, and confirmed that it yielded
palmitic acid (C16:0), oleic acid (C18:1), myristic acid 24 h LD50 values of 4.7 and 13.1 mg.kg–1, respectively.
(C14:0), α-linolenic acid (C18:3), and 7, 10-hexadecadi- Sathiyamoorthy & Shanmugasundaram (1996) also iso-
enoic acid. Accordingly, recent contributions by Manilal lated and purified a glycine-rich peptide toxin from the
et al. (2012) and Vieira et al. (2017) reviewed the state of cyanobacterium strain Scytonema MKU 106 and dis-
the art of the pesticidal activity of the brown macroalga cussed its potent effects as an insecticide against larvae of
Lobophora variegata (methanolic extract) and attribut- some cotton pests, not including S. littoralis. With regard
ed this property to the strong synergistic effects of fatty to charophytes, Jacobsen & Pedersen (1983) found insec-
acids such as α-linolenic, oleic, myristic, and hexadeca- ticidal properties in derivatives of propane-l, 3-dithiol
trienoic acids. Farag et al. (2011) found a bioactive array (analogues of the insecticidal dithiolane and trithiane) in
of fatty acids, including linoleic acid methyl ester, oleic the stonewort Chara globularis. With reference to insec-
acid methyl ester, free oleic acid, hexadecanol, palmitic ticidal-related oils, Daniewski et al. (2012) demonstrated
acid, methyl esters of stearic acid and myristic acids, in the insecticidal activity of some essential oils (alkane
the ripe fruits of the higher plant Melia azedarach. These derivatives), like those identified in the present study, in
compounds exhibited potent insecticidal and growth-inhi- the fruit bodies of the popular edible mushroom Canthar-
bition activity against the 2nd and 4th larval instars of the ellus cibarius (chanterelle), such as 2-phenylundecane,
cotton leafworm S. littoralis. Barakat (2011) also pointed 5-phenylundecane, 2-phenyldodecane, and 5- and 6-phe-
out that the acetonic extract of Casimiroa edulis leaf has a nyldodecane. Pavela (2011) also pointed out that various
promising insecticidal and antifeedant activity against the essential oils are documented to exhibit acute toxic effects
4th instar larvae of S. littoralis and attributed this influence against S. littoralis, and they could therefore be consid-
to the phytochemical compounds including fatty acids, ered promising for the development of novel botanical
mainly hexadecanoic acid, methyl ester and linolenic insecticides.

Vie Milieu, 2018, 68 (4)

210 A. A. SABER, S. M.HAMED, E. F. M. ABDEL-RAHIM, M. CANTONATI

The coccoid green alga P. kessleri and the cyanobac- rent trend towards increasing organic farming (e.g., Sola
terium N. carneum showed significant effects in delaying et al. 2014, Mehrotra et al. 2017).
pupae formation and particularly minimized the percent-
Acknowledgments. – Authors are thankful to Soils, Water
ages of their formation, prolonged their life durations, and
& Environment Research Institute and Plant Protection Research
reduced their weights and the moth emergence. Accord- Institute, Agricultural Research Center, and the Botany Depart-
ingly, Abdel-Rahim & Hamed (2013a) obtained the same ment, Faculty of Science, Ain Shams University, Cairo, Egypt
observations on the cotton leafworm S. littoralis using for providing all facilities to carry out this research work.
the blue-green alga Anabaena flosaquae. Aboutabl et
al. (2002) also stated that the crude polar extracts of the
brown seaweed Sargassum dentifolium exhibited poten- References
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