**3. Plants toxic to leaf–cutting ants and their symbiotic fungi**

Plants exhibit several mechanisms to prevent herbivory, including producing alcohols, aldehydes, esters, phenols, and hydrocarbons, among other substances, which can be classified as secondary metabolites and can be toxic to leaf-cutting ants and/or their symbiotic fungi. As a result, many studies have been conducted in this field using different plant families, as listed in Table 1; some of these studies are described in more detail below.

**Plant species Family Identified substance Reference**

*Simarouba versicolor* Simaroubaceae 4,5-dimethoxy-canthin-6-one, 5-

*Spiranthera odoratissima*Rutaceae Furoquinolines, 2-arylquinolin-4-one,

*Virola sebifera and*

isolated substances.

**3.1.** *Sesamum indicum*

by Pagnocca et al. (1990; 1996) [

compounds with antifungal proprieties (Tables 2 and 3).

*Virola sp.* Myristicaceae

*Ageratum conyzoides* Asteraceae - Ribeiro et al., 2008[88] *Mentha piperita* Lamiaceae - Ribeiro et al., 2008[88]

*Citrus* sp. Rutaceae Xanthyletin Cazal et al., 2009[85]

*Tithonia diversifolia* Asteraceae - Valderrama-Eslava et al.,

kusunokinin

**Table 1.** Plant species with toxic effects against leaf-cutting ants and/or their symbiotic fungi and the associated

Crude extracts of the leaves, fruits, and seeds of sesame, *Sesamum indicum* L. (Pedaliaceae), were tested *in vitro* against the symbiotic fungus (*L. gongylophorus*) of *A. sexdens*, isolated from previously established nests. Bioassays were performed according to methodology developed

by Pagnocca et al. (1990) until reaching final concentrations between 7.5 and 60 mg/mL. Ten test tubes were used for each sample, with three replicates for leaf extracts and two replicates for the other extracts. Fungal growth was estimated macroscopically based on the surface area and density of the mycelium after 30-35 days of incubation. The control sample received the same amount of solvent, and the relative growth observed was characterised as follows: 5 + = growth equal to the control; 4 + = growth equivalent to 80% of the control; 3 + = growth equivalent to 60% of the control; 2 + = growth equivalent to 40% of the control; and 1 + = growth equivalent to 20% or less of the control. The crude extracts of sesame leaves, fruits, and seeds inhibited the growth of the symbiotic fungus, which suggests that this species produces

Sesamin and Epigalgavrin; (+) sesamin, (-)-hinoquinin, (-)-

*Tabebuia vellosoi* Bignoniaceae - Souza et al., 2010[90] *Magonia pubescens* Sapindaceae - Souza et al., 2010[90] *Annona reticulata* Annonaceae - Souza et al., 2010[90] *Amburana acreana* Leguminosae - Souza et al., 2010[90]

methoxy-canthin-6-one Peñaflor et al., 2009[22]

limonexic acid, limonin Terezan et al., 2010[89]

49,50]. The extracts were added to the culture medium described

2009[23]

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263

Bicalho et al., 2012[54]; Pagnocca et al., 1996[91]



**Table 1.** Plant species with toxic effects against leaf-cutting ants and/or their symbiotic fungi and the associated isolated substances.

#### **3.1.** *Sesamum indicum*

**3. Plants toxic to leaf–cutting ants and their symbiotic fungi**

262 Insecticides - Development of Safer and More Effective Technologies

in Table 1; some of these studies are described in more detail below.

*Sesamum indicum* Pedaliaceae Sesamin and/or sesamolin

*Eucalyptus maculata* Myrtaceae elemol, β-eudesmol

*Ricinus communis* Euphorbiaceae Palmitic acid; ricin

*Helietta puberula* Rutaceae Kokusagine, anthranilic acid,

*Cedrela fissilis* Meliaceae

**Plant species Family Identified substance Reference**

*Hymenaea courbaril* Fabaceae Caryophyllene epoxide Hubbel et al., 1983[80]

*Canavalia ensiformes* Fabaceae Fatty acids; canavalin, canatoxin Monteiro et al., 1998[55];

*Pilocarpus grandiflorus* Rutaceae Vanillic acid, syringaldehyde Godoy et al., 2002[82]

*Dimorphandra mollis* Fabaceae Astilbin Cintra et al., 2005[86]

*Raulinoa echinata* Rutaceae Limonoid, limonexic acid Biavatti et al., 2005[57]

*Carapa guianensis* Meliaceae 6α-acetoxygedunin Ambrozin et al., 2006[87]

*Azadirachta indica* Meliaceae Azadirachtin Santos-Oliveira et al., 2006[62];

3β-acetoxicarapine limonoid, oleanolic acid, oleanic acid, cipadesin A, ruageanin A, cipadesin, khayasin T, febrifugin, mexicanolide Pagnocca et al., 1990[49]; Pagnocca et al; 1996[50]; Ribeiro et al., 1998[51]; Morini et al., 2005[52]; Peres Filho and

Dorval, 2003[53]

Hebling et al., 2000[81]

Marsaro Junior et al., 2004[18]; Marinho et al., 2005[19]; Marinho et al., 2008[20]; Marinho et al., 2006[83]

Bigi et al., 2004[15]; Caffarini et al., 2008[84]; Cazal et al.,

Bueno et al., 2005[60]; Leite et

Brugger et al., 2008[63]

2009[85]

al., 2005[61]

dictamnine Almeida et al., 2007[20]

Plants exhibit several mechanisms to prevent herbivory, including producing alcohols, aldehydes, esters, phenols, and hydrocarbons, among other substances, which can be classified as secondary metabolites and can be toxic to leaf-cutting ants and/or their symbiotic fungi. As a result, many studies have been conducted in this field using different plant families, as listed

> Crude extracts of the leaves, fruits, and seeds of sesame, *Sesamum indicum* L. (Pedaliaceae), were tested *in vitro* against the symbiotic fungus (*L. gongylophorus*) of *A. sexdens*, isolated from previously established nests. Bioassays were performed according to methodology developed by Pagnocca et al. (1990; 1996) [ 49,50]. The extracts were added to the culture medium described by Pagnocca et al. (1990) until reaching final concentrations between 7.5 and 60 mg/mL. Ten test tubes were used for each sample, with three replicates for leaf extracts and two replicates for the other extracts. Fungal growth was estimated macroscopically based on the surface area and density of the mycelium after 30-35 days of incubation. The control sample received the same amount of solvent, and the relative growth observed was characterised as follows: 5 + = growth equal to the control; 4 + = growth equivalent to 80% of the control; 3 + = growth equivalent to 60% of the control; 2 + = growth equivalent to 40% of the control; and 1 + = growth equivalent to 20% or less of the control. The crude extracts of sesame leaves, fruits, and seeds inhibited the growth of the symbiotic fungus, which suggests that this species produces compounds with antifungal proprieties (Tables 2 and 3).


through transparent tubes (1.5 cm of diameter), were used in these bioassays. In this setup, one chamber was used to supply leaves, a second chamber housed the fungal garden (sponge), and the third chamber contained the residues (waste) from the ants. Fresh leaves (10 to 20 grams) were offered at 48-h intervals after removal of the waste from the previous treatment. In the older sponges in nests treated with *Eucalyptus*, 1.4×105 bacterial colony forming units/g (CFU/g) were recorded, while the average in the waste deposits reached 7.3×107 CFU/g. The most

MPN/g for older sponges and waste deposits, respectively, while in ant colonies treated with *S. indicum* leaves, these values were 3.3×107 CFU/g and 6.7×105 MPN/g. This increase in the numbers of bacteria and yeast led to visible changes in the colouration and humidity of the

The application of fractions of the extracts from sesame leaves at a 2.5 mg/mL concentration completely inhibited the development of the symbiotic fungus of the leaf-cutting ants, and 50% inhibition of fungal development was observed for some fractions at a 1.25 mg/mL concentration [51] (Table 4). Chromatographic analysis of the hexanic extracts of leaves revealed the presence of a mixture of tetradecanoic, hexadecanoic, octadecanoic, icosanoic, docosanoic, and 9,12,15-octadecatrienoic acids. Separation of the compounds in the mixture by fractionation resulted in a loss of or decrease in inhibitory activity against the fungus, indicating that the observed inhibition may be a consequence of the joint action of several

Hexane - [1.25]/(90) [1.25]/(60)

Ethyl Acetate [1.25]/(50) [1.25]/(50) [1.25]/(70)2

Methanol [1.25]/(90) [1.25]/(70) [1.25]/(50)2

Acetic Acid [1.25]/(100) [1.25]/(100) [1.25]/(1002)

**Table 4.** Fungal growth (%) of *Leucoagaricus gongylophorus* in culture medium containing different concentrations [mg/mL] of the hexane, dichloromethane, and methanol extracts from sesame. Control= (100). Source: Ribeiro et al.,

Dichloromethane - [1.25]/(50) -

**Extracts**

**Hexane Dichloromethane Methanol**


[5.00]/(90) [5.00]/(NG) [5.00]/(10)

[2.50]/(NG) [2.50]/(NG) [2.50]/(NG) [5.00]/(NG) [5.00]/(NG) [5.00]/(NG)

[2.50]/(NG) [2.50]/(NG) [2.50]/(NG) [5.00]/(NG) [5.00]/(NG) [5.00]/(NG)

[2.50]/(40) [2.50]/(NG) [2.50]/(NG) [5.00]/(10) [5.00]/(NG) [5.00]/(NG)


and 2.2×104

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probable numbers (MPNs) of yeast per gram of the material analysed were 1.3×105

fungal sponges of nests treated with sesame, which resulted in fungal death.

compounds in the leaves, rather than of a single substance.

1Thirty days of incubation/25°C; 2dry mix; NG=no growth

**Fractions**

1998 [51].

5+=growth identical to control; 4+=growth equivalent to 80% of control; 3+=growth equivalent to 60% of control; 2+=growth equivalent to 40% to control; 1+=growth equivalent to 20% of control of less. Source: Pagnocca et al., 1990 [49].

**Table 2.** Antifungal activity of sesame leaf extracts


**Table 3.** Antifungal activity of chloroform extracts of sesame leaves, fruits, and seeds at different stages of development. Source: Pagnocca et al., 1990 [49].

In another study, Pagnocca et al. (1996) determined the number of bacteria and yeast in the organicmatterwithinant colonies rearedinthe laboratorywith*Eucalyptus alba*Reinw. exBlume (control) or *S. indicum* (experiment). Transparent plastic pots (2.5 L), connected to each other through transparent tubes (1.5 cm of diameter), were used in these bioassays. In this setup, one chamber was used to supply leaves, a second chamber housed the fungal garden (sponge), and the third chamber contained the residues (waste) from the ants. Fresh leaves (10 to 20 grams) were offered at 48-h intervals after removal of the waste from the previous treatment. In the older sponges in nests treated with *Eucalyptus*, 1.4×105 bacterial colony forming units/g (CFU/g) were recorded, while the average in the waste deposits reached 7.3×107 CFU/g. The most probable numbers (MPNs) of yeast per gram of the material analysed were 1.3×105 and 2.2×104 MPN/g for older sponges and waste deposits, respectively, while in ant colonies treated with *S. indicum* leaves, these values were 3.3×107 CFU/g and 6.7×105 MPN/g. This increase in the numbers of bacteria and yeast led to visible changes in the colouration and humidity of the fungal sponges of nests treated with sesame, which resulted in fungal death.

**Solvent Final concentration (mg leaf dry weigh/mL) Relative fungal growth**

Chloroform 7.5 5+

Methanol 7.5 5+

Chloroform+Methanol 30.0+30.0 2+

Water 60.0 >5+

[49].

Control=5+

**Table 2.** Antifungal activity of sesame leaf extracts

264 Insecticides - Development of Safer and More Effective Technologies

development. Source: Pagnocca et al., 1990 [49].

5+=growth identical to control; 4+=growth equivalent to 80% of control; 3+=growth equivalent to 60% of control; 2+=growth equivalent to 40% to control; 1+=growth equivalent to 20% of control of less. Source: Pagnocca et al., 1990

**Material and developmental stage Final concentration mg dry weight/mL Relative fungal growth**

L1 (leaves 30 days old) 60.0 2+ L2 (leaves 60 days old) 60.0 2+ L3a (green leaves 90 days old) 60.0 3+ L3b (yellow leaves 90 days old) 60.0 2+ Gfr (green fruit) 30.0 3+ RFr (ripe fruit) 30.0 2+ GS (green seed) 30.0 2+ RS (ripe seed) 30.0 2+

**Table 3.** Antifungal activity of chloroform extracts of sesame leaves, fruits, and seeds at different stages of

In another study, Pagnocca et al. (1996) determined the number of bacteria and yeast in the organicmatterwithinant colonies rearedinthe laboratorywith*Eucalyptus alba*Reinw. exBlume (control) or *S. indicum* (experiment). Transparent plastic pots (2.5 L), connected to each other

**(30-35 days)**

**(30-35 days)**

15.0 5+ 30.0 4+ 60.0 2+

15.0 5+ 30.0 4+ 60.0 2+

60.0+60.0 <1+

The application of fractions of the extracts from sesame leaves at a 2.5 mg/mL concentration completely inhibited the development of the symbiotic fungus of the leaf-cutting ants, and 50% inhibition of fungal development was observed for some fractions at a 1.25 mg/mL concentration [51] (Table 4). Chromatographic analysis of the hexanic extracts of leaves revealed the presence of a mixture of tetradecanoic, hexadecanoic, octadecanoic, icosanoic, docosanoic, and 9,12,15-octadecatrienoic acids. Separation of the compounds in the mixture by fractionation resulted in a loss of or decrease in inhibitory activity against the fungus, indicating that the observed inhibition may be a consequence of the joint action of several compounds in the leaves, rather than of a single substance.


1Thirty days of incubation/25°C; 2dry mix; NG=no growth

**Table 4.** Fungal growth (%) of *Leucoagaricus gongylophorus* in culture medium containing different concentrations [mg/mL] of the hexane, dichloromethane, and methanol extracts from sesame. Control= (100). Source: Ribeiro et al., 1998 [51].

Extracts from ripe sesame seeds were tested to investigate their toxicity through contact with *A. sexdens* workers. Ripe seeds of *Sesamum indicum* L. (Pedaliaceae) were triturated and pressed, yielding sesame butter. A known mass of this sesame butter was macerated for three days three times at room temperature and then extracted with solvents of increasing polarity (dichloromethane and methanol), resulting in a dichloromethane crude extract (SD) and a methanol crude extract (SM). The SD crude extract was subjected to liquid chromatography in a vacuum syntherised plate funnel with silica gel as the stationary phase and eluents of increasing polarity, which yielded the following fractions: hexane (SD-H), dichloromethane (SD-D), ethyl acetate (SD-E), and methanol (SD-M). The SD-E fraction was produced through successive chromatographic columns, with silica as the stationary phase and hexane/dichloro‐ methane/methanol as the eluent, in gradient mode. A total of 11 sub-fractions were obtained from this process, only four of which (A, B, C, D) contained a sufficient amount of material to be tested. At the tested concentrations, the same proportion as in the original SD-E fraction was maintained in the sub-fractions, and samples at double these concentrations were also tested (Figure 3). The SD-E sub-fractions were combined in amounts necessary to equal that of the original fraction. The seven sub-fractions (E-K) that were isolated in only small amounts were not tested. Tests were also performed in which the concentration of each sub-fraction was reduced by 50% in two combinations: A+B+C+D and A+B+C. To identify the compounds present in SD-E, hydrogen nuclear magnetic resonance (H NMR) and gas chromatographymass spectrometry (GC-MS) were used. The results demonstrated that *A. sexdens* workers that received the crude dichloromethane extract from sesame seeds (SD) on their *pronoto* exhibited high mortality. This crude extract was then fractionated, and the ethyl acetate fraction (SD-E) was found to be responsible for the toxic effect. However, no toxicity was observed when the SD-E sub-fractions (A, B, C, and D) were tested in the same proportions as found in the original fraction (Table 5). These results could be explained by three hypotheses: 1) each isolated subfraction is only toxic at concentrations above the concentration found in the ethyl acetate fraction; 2) the sub-fractions are only toxic when combined through a synergistic effect between their components; and 3) toxic compounds are be present in the untested sub-fractions (E-K), which corresponded to 26.77% of the ethyl acetate fraction. Experiments were conducted to determine why the formicidal activity was lost. First, the authors doubled the concentration of each sub-fraction, and only one fraction, composed of triglycerides, was found to be toxic (Table 5). Then, when sub-fractions A, B, C, and D were combined, the formicidal effect reappeared, even at concentrations reduced to 50% of the original concentration (Table 6). A mixture containing 73.23% (A + B + C + D) of the ethyl acetate fraction contains chemical compounds that reduce the survival of *A. sexdens*. [52]

**Material**

D\* 24

concentration reduced to 50%

**Concentration (mg mL-1)**

**% Mortality/Day**

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1 2 3 6 8 10 14 17 21 25

**Control D** - 0 0 2 10 35 53 72 90 93 100 10a

SD 200 2 7 18 47 73 90 93 95 97 100 7c

SM 200 3 10 17 45 67 75 87 90 97 100 7c

**Control H** - 0 5 7 23 32 57 75 78 85 100 9a

SD H 200 8 53 63 77 85 88 88 92 97 100 2c

**Control D** - 5 5 15 37 62 78 85 97 97 100 7b

SD-D 200 0 5 5 27 53 63 78 82 92 100 8a

**Control E** - 2 3 5 27 58 68 88 95 97 100 8a

SD-E 200 0 5 7 23 32 57 75 78 85 100 9a

**Control M** - 27 47 57 67 70 75 88 93 100 - 3c

SD-M 200 0 5 5 27 53 63 78 82 92 100 8a

**Control E** - 0 5 5 20 37 72 88 92 98 100 9a

A 57 2 10 10 37 62 65 77 82 92 100 8a

A 14 0 5 7 23 32 57 75 78 85 100 9a

**Control E** - 23 38 53 83 87 87 97 98 98 100 3c

B 53 0 5 8 18 35 53 70 80 85 100 10a

B 106 0 5 5 27 53 63 78 82 92 100 10a

C 25 2 2 2 28 65 83 95 97 100 - 8b

C 50 0 2 8 25 42 60 83 88 90 100 10a

**Control E** - 0 5 7 23 32 57 75 78 85 100 9a

D\* 12 0 2 3 18 30 63 85 93 98 100 9a

S50= Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test (b=0.01>p<0.05; c=p<0.01). Concentrations: A= 57 mg mL-1; B=53 mg mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*=

**Table 5.** Toxicity of SD-E sub-fraction combinations in *Atta sexdens* workers. Source: Morini et al., 2005 [52].

**S50**

267

The results shown in Table 6 indicate that five of the 11 possible combinations of the SD-E sub-fractions were toxic to leaf-cutting ants (A + B + C + D; A + B + C; A + C + D; A + C; B + C), and all of the toxic combinations contained sub-fraction C, which was composed of di‐ glycerides and furfuranic lignans (sesamin and sesamolin). The observed effects are likely due to the presence of lignin furfuranic, which is used as a synergistic factor in insecticides. However, sesamolin exhibited a biological activity that was five times stronger than that of sesamin. Moreover, sub-fraction D, which was composed only of sesamin, either had an in‐


Extracts from ripe sesame seeds were tested to investigate their toxicity through contact with *A. sexdens* workers. Ripe seeds of *Sesamum indicum* L. (Pedaliaceae) were triturated and pressed, yielding sesame butter. A known mass of this sesame butter was macerated for three days three times at room temperature and then extracted with solvents of increasing polarity (dichloromethane and methanol), resulting in a dichloromethane crude extract (SD) and a methanol crude extract (SM). The SD crude extract was subjected to liquid chromatography in a vacuum syntherised plate funnel with silica gel as the stationary phase and eluents of increasing polarity, which yielded the following fractions: hexane (SD-H), dichloromethane (SD-D), ethyl acetate (SD-E), and methanol (SD-M). The SD-E fraction was produced through successive chromatographic columns, with silica as the stationary phase and hexane/dichloro‐ methane/methanol as the eluent, in gradient mode. A total of 11 sub-fractions were obtained from this process, only four of which (A, B, C, D) contained a sufficient amount of material to be tested. At the tested concentrations, the same proportion as in the original SD-E fraction was maintained in the sub-fractions, and samples at double these concentrations were also tested (Figure 3). The SD-E sub-fractions were combined in amounts necessary to equal that of the original fraction. The seven sub-fractions (E-K) that were isolated in only small amounts were not tested. Tests were also performed in which the concentration of each sub-fraction was reduced by 50% in two combinations: A+B+C+D and A+B+C. To identify the compounds present in SD-E, hydrogen nuclear magnetic resonance (H NMR) and gas chromatographymass spectrometry (GC-MS) were used. The results demonstrated that *A. sexdens* workers that received the crude dichloromethane extract from sesame seeds (SD) on their *pronoto* exhibited high mortality. This crude extract was then fractionated, and the ethyl acetate fraction (SD-E) was found to be responsible for the toxic effect. However, no toxicity was observed when the SD-E sub-fractions (A, B, C, and D) were tested in the same proportions as found in the original fraction (Table 5). These results could be explained by three hypotheses: 1) each isolated subfraction is only toxic at concentrations above the concentration found in the ethyl acetate fraction; 2) the sub-fractions are only toxic when combined through a synergistic effect between their components; and 3) toxic compounds are be present in the untested sub-fractions (E-K), which corresponded to 26.77% of the ethyl acetate fraction. Experiments were conducted to determine why the formicidal activity was lost. First, the authors doubled the concentration of each sub-fraction, and only one fraction, composed of triglycerides, was found to be toxic (Table 5). Then, when sub-fractions A, B, C, and D were combined, the formicidal effect reappeared, even at concentrations reduced to 50% of the original concentration (Table 6). A mixture containing 73.23% (A + B + C + D) of the ethyl acetate fraction contains chemical

266 Insecticides - Development of Safer and More Effective Technologies

compounds that reduce the survival of *A. sexdens*. [52]

The results shown in Table 6 indicate that five of the 11 possible combinations of the SD-E sub-fractions were toxic to leaf-cutting ants (A + B + C + D; A + B + C; A + C + D; A + C; B + C), and all of the toxic combinations contained sub-fraction C, which was composed of di‐ glycerides and furfuranic lignans (sesamin and sesamolin). The observed effects are likely due to the presence of lignin furfuranic, which is used as a synergistic factor in insecticides. However, sesamolin exhibited a biological activity that was five times stronger than that of sesamin. Moreover, sub-fraction D, which was composed only of sesamin, either had an in‐

S50= Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test (b=0.01>p<0.05; c=p<0.01). Concentrations: A= 57 mg mL-1; B=53 mg mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*= concentration reduced to 50%

**Table 5.** Toxicity of SD-E sub-fraction combinations in *Atta sexdens* workers. Source: Morini et al., 2005 [52].

hibitory effect on the action of other sub-fractions (B + C + D; C + D) or was unable to modify their actions (A + D; B + D), showing that the factor responsible for the synergistic toxic ef‐ fect of sesame seeds is either sesamolin or the combination of sesamin + sesamolin, rather than sesamin alone [52] (Table 6).

**Sub–fraction combination** **% Mortality/Day**

1 2 3 6 8 10 14 17 21 25

**Control** 0 0 2 10 35 53 72 90 93 100 10a

A+B+C+D\* 2 7 18 47 73 90 93 95 97 100 7c

(A+B+C+D\*)\* 3 10 17 45 67 75 87 90 97 100 7c

**Control** 0 5 7 23 32 57 75 78 85 100 9a

A+B+C 8 53 63 77 85 88 88 92 97 100 2c

(A+B+C)\* 5 5 15 37 62 78 85 97 97 100 7b

**Control** 0 5 5 27 53 63 78 82 92 100 8a

A+B+D\* 2 3 5 27 58 68 88 95 97 100 8a

**Control** 0 5 7 23 32 57 75 78 85 100 9a

A+C+D\* 27 47 57 67 70 75 88 93 100 - 3c

**Control** 0 5 5 27 53 63 78 82 92 100 8a

B+C+D\* 0 5 5 20 37 72 88 92 98 100 9a

A+B 2 10 10 37 62 65 77 82 92 100 8a

**Control** 0 5 7 23 32 57 75 78 85 100 9a

A+C 23 38 53 83 87 87 97 98 98 100 3c

A+D\* 0 5 8 18 35 53 70 80 85 100 10a

**Control** 0 5 5 27 53 63 78 82 92 100 10a

B+C 2 2 2 28 65 83 95 97 100 - 8b

B+D\* 0 2 8 25 42 60 83 88 90 100 10a

Control 0 5 7 23 32 57 75 78 85 100 9a

C+D\* 0 2 3 18 30 63 85 93 98 100 9a

mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*= concentration reduced to 50%

B=53 mg mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*= concentration reduced to 50%

**Table 6.** Toxicity of SD-E sub-fraction combinations in *Atta sexdens* workers. Source: Morini et al., 2005 [52].

S50= Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test (b=0.01>p<0.05; c=p<0.01). Concentrations: A= 57 mg mL-1; B=53 mg

S50

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**Figure 3.** Diagram showing the procedure for obtaining crude extracts, fractions, and sub-fractions from sesame seeds (*S.indicum*) and the sequence of topical application on *A. sexdens rubropilosa* workers. Source: Morini et al., 2005 [52].

The efficiency of different commercial chlorpyrifos- sulfluramid- and fipronil-based formicidal baits as well as others that are manually manufactured using the leaves (15%) and seeds (10%, 20% and 30%) of *S. indicum* against *A. sexdens* Forel. control were assessed in the field. The nest activity was monitored at 30, 60, 90, and 150 days after treatment. The most efficient baits were sulfluramid- and fipronil-based, followed by the formulation derived from sesame leaves (15%). The sulfluramid- and fipronil-based baits caused colony activity to cease at 30 days, while the sesame leaf-based baits (15%) resulted in an 80% inhibition of activity at 90 days, confirming that *S. indicum* has great potential for the development of new products to control leaf-cutting ants [53].


hibitory effect on the action of other sub-fractions (B + C + D; C + D) or was unable to modify their actions (A + D; B + D), showing that the factor responsible for the synergistic toxic ef‐ fect of sesame seeds is either sesamolin or the combination of sesamin + sesamolin, rather

**Figure 3.** Diagram showing the procedure for obtaining crude extracts, fractions, and sub-fractions from sesame seeds (*S.indicum*) and the sequence of topical application on *A. sexdens rubropilosa* workers. Source: Morini et al.,

The efficiency of different commercial chlorpyrifos- sulfluramid- and fipronil-based formicidal baits as well as others that are manually manufactured using the leaves (15%) and seeds (10%, 20% and 30%) of *S. indicum* against *A. sexdens* Forel. control were assessed in the field. The nest activity was monitored at 30, 60, 90, and 150 days after treatment. The most efficient baits were sulfluramid- and fipronil-based, followed by the formulation derived from sesame leaves (15%). The sulfluramid- and fipronil-based baits caused colony activity to cease at 30 days, while the sesame leaf-based baits (15%) resulted in an 80% inhibition of activity at 90 days, confirming that *S. indicum* has great potential for the development of new products to control

than sesamin alone [52] (Table 6).

268 Insecticides - Development of Safer and More Effective Technologies

2005 [52].

leaf-cutting ants [53].

S50= Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test (b=0.01>p<0.05; c=p<0.01). Concentrations: A= 57 mg mL-1; B=53 mg mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*= concentration reduced to 50%

B=53 mg mL-1; C=25 mg mL-1; D\*= 12 mg mL-1; ( )\*= concentration reduced to 50%

**Table 6.** Toxicity of SD-E sub-fraction combinations in *Atta sexdens* workers. Source: Morini et al., 2005 [52].

#### **3.2.** *Virola sebifera*

Phytochemical analysis of the leaves of *Virola sebifera* Aubl. (Myristicaceae) resulted in the isolation of three lignans, (+)-sesamin (1), (-)-hinoquinin (2), and (-) – kusunokinin (3) (Figure 4), and three flavonoids, quercetin-3-O-α-L-rhamnoside, quercetin-3-O-β-D-glucoside, and quercetin-3-methoxy-7-O-β-D-glucoside. Techniques such as high-speed counter-current chromatography and high-performance liquid chromatography were employed in this process. The isolated substances were added to the artificial diet and tested against *A. sexdens* leaf-cutting ants at a concentration of 200 or 400 µg mL-1. Diets (0.4-0.5 g per dish) treated with the compounds (experimental treatment) or without (control) were offered daily in a small plastic cap. The percentage of survival was plotted as a function of time in a survival curve that was then used to calculate the median survival time (S50, the time at which 50% of the ants in each experiment remained alive). The lignin (-) - kusunokinin (3) resulted in 90% mortality of *A. sexdens* workers after 25 days of monitoring compared to the controls fed with an untreated diet. Although the other substances did not show biological activity against the ants, the (+)-sesamin (1), (-)-hinoquinin (2) and (-)-kusunokinin (3) lignans inhibited the growth of the symbiotic fungus by 74%, 72%, and 100%, respectively [54] (Figure 4).

one fraction (fraction 9) was active; all fractions were esterified with diazomethane and analysed by gas chromatography-mass spectrometry (GC-MS) to identify the active compo‐ nents. The main compounds identified in the active fraction were long-chain saturated fatty acids. In these experiments, it was not possible to identify which of the fatty acids was responsible for the fungicidal action. However, comparison of the different fractions showed that the fatty acids with chains containing 11, 17, 19, 22, and 23 carbon atoms were likely the most active (Table 7), as the fractions in which these fatty acids were not among the major

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**Fraction Major compounds Minor compounds**

3 C16, C18 C10, C14, C15 4 C16, C20 C18, C21, C22

5 C16 C12

**Table 7.** Fatty acids found in *Canavalia ensiformis*. Source: Monteiro et al., 1998 [55].

9\* C11, C16, C17, C18, C19, C20, C22, C23, C24, C26 C8, C14, C15, C21, C25, C27, C28, C30 2 C10, C16, C18, C24, C26 C14, C15, C17, C22, C23, C25, C28, C29

Phytochemical analyses of the roots of *Raulinoa echinata* R.S.Cowan (Rutaceae) resulted in the isolation and identification of the following limonoids: fraxinellone, fraxinellonone, and epoxy-fraxinellone. Limonexic acid was isolated from the stem of the plant. The toxicity of the compounds against *A. sexdens* was determined in ingestion bioassays according to the protocol described by Bueno et al. (1997) [56]. The ants in the treatment groups received a diet enriched with epoxy-fraxinellone or limonexic acid at a concentration of 200 µg mL-1. Control ants were fed with a component-free diet. Over 25 days, the number of dead ants in each Petri dish was counted, the survival curve of the leaf-cutting ants in each treatment was estimated, and their average longevity was calculated. Limonexic acid (4) (Figure 5) reduced the longevity of *A. sexdens* considerably (11 days) compared to the control (22 days) [57]. *R. echinata* was also able to produce substances that were active against the symbiotic fungus of the leaf-cutting ants; several furoquinoline alkaloids (skimmianine (5), kokusaginine (6), maculine (7) and flinder‐ siamine (8)) and quinolones (2-n-Nonyl-4-quinolone (9), 1-Methyl-2-n-nonyl-4-quinolone (10), 1-Methyl-2-phenyl-4-quinolone (11)) (Figure 6; Table 8) that exhibited fungicidal activity

against *L. gongylophorus* were isolated from extracts of its stems and leaves [58].

Methanolic, hexanic, and dichloromethane extracts obtained from the stems, leaves, and branches of *Helietta puberula* R. E. Fr. (Rutaceae) were tested against *A. sexdens* workers and the symbiotic fungus of this ant species. Experimental diets were prepared by mixing plant

components showed no fungicidal activity [55].

\*Active fraction

**3.4.** *Raulinoa echinata*

**3.5.** *Helietta puberula*

**Figure 4.** Chemical structures of the compounds isolated from *Virola sebifera,* (+)-sesamin (1), (-)-hinoquinin (2), and (-) – kusunokinin (3). Source: Bicalho et al., 2012 [54].

#### **3.3.** *Canavalia ensiformis*

*In vitro* tests showed inhibitory effect on the symbiotic fungus of a hexanic extract of *Canavalia ensiformis* (L.) DC. (Fabaceae) leaves, applied at a 1,000 µg mL-1 concentration. This extract was fractionated by column chromatography using silica gel as the stationary phase. A total of 11 fractions were obtained and used in fungal bioassays at a concentration of 500 µg mL-1. Only one fraction (fraction 9) was active; all fractions were esterified with diazomethane and analysed by gas chromatography-mass spectrometry (GC-MS) to identify the active compo‐ nents. The main compounds identified in the active fraction were long-chain saturated fatty acids. In these experiments, it was not possible to identify which of the fatty acids was responsible for the fungicidal action. However, comparison of the different fractions showed that the fatty acids with chains containing 11, 17, 19, 22, and 23 carbon atoms were likely the most active (Table 7), as the fractions in which these fatty acids were not among the major components showed no fungicidal activity [55].


**Table 7.** Fatty acids found in *Canavalia ensiformis*. Source: Monteiro et al., 1998 [55].

### **3.4.** *Raulinoa echinata*

**3.2.** *Virola sebifera*

270 Insecticides - Development of Safer and More Effective Technologies

Phytochemical analysis of the leaves of *Virola sebifera* Aubl. (Myristicaceae) resulted in the isolation of three lignans, (+)-sesamin (1), (-)-hinoquinin (2), and (-) – kusunokinin (3) (Figure 4), and three flavonoids, quercetin-3-O-α-L-rhamnoside, quercetin-3-O-β-D-glucoside, and quercetin-3-methoxy-7-O-β-D-glucoside. Techniques such as high-speed counter-current chromatography and high-performance liquid chromatography were employed in this process. The isolated substances were added to the artificial diet and tested against *A. sexdens* leaf-cutting ants at a concentration of 200 or 400 µg mL-1. Diets (0.4-0.5 g per dish) treated with the compounds (experimental treatment) or without (control) were offered daily in a small plastic cap. The percentage of survival was plotted as a function of time in a survival curve that was then used to calculate the median survival time (S50, the time at which 50% of the ants in each experiment remained alive). The lignin (-) - kusunokinin (3) resulted in 90% mortality of *A. sexdens* workers after 25 days of monitoring compared to the controls fed with an untreated diet. Although the other substances did not show biological activity against the ants, the (+)-sesamin (1), (-)-hinoquinin (2) and (-)-kusunokinin (3) lignans inhibited the growth

of the symbiotic fungus by 74%, 72%, and 100%, respectively [54] (Figure 4).

**Figure 4.** Chemical structures of the compounds isolated from *Virola sebifera,* (+)-sesamin (1), (-)-hinoquinin (2), and

*In vitro* tests showed inhibitory effect on the symbiotic fungus of a hexanic extract of *Canavalia ensiformis* (L.) DC. (Fabaceae) leaves, applied at a 1,000 µg mL-1 concentration. This extract was fractionated by column chromatography using silica gel as the stationary phase. A total of 11 fractions were obtained and used in fungal bioassays at a concentration of 500 µg mL-1. Only

(-) – kusunokinin (3). Source: Bicalho et al., 2012 [54].

**3.3.** *Canavalia ensiformis*

Phytochemical analyses of the roots of *Raulinoa echinata* R.S.Cowan (Rutaceae) resulted in the isolation and identification of the following limonoids: fraxinellone, fraxinellonone, and epoxy-fraxinellone. Limonexic acid was isolated from the stem of the plant. The toxicity of the compounds against *A. sexdens* was determined in ingestion bioassays according to the protocol described by Bueno et al. (1997) [56]. The ants in the treatment groups received a diet enriched with epoxy-fraxinellone or limonexic acid at a concentration of 200 µg mL-1. Control ants were fed with a component-free diet. Over 25 days, the number of dead ants in each Petri dish was counted, the survival curve of the leaf-cutting ants in each treatment was estimated, and their average longevity was calculated. Limonexic acid (4) (Figure 5) reduced the longevity of *A. sexdens* considerably (11 days) compared to the control (22 days) [57]. *R. echinata* was also able to produce substances that were active against the symbiotic fungus of the leaf-cutting ants; several furoquinoline alkaloids (skimmianine (5), kokusaginine (6), maculine (7) and flinder‐ siamine (8)) and quinolones (2-n-Nonyl-4-quinolone (9), 1-Methyl-2-n-nonyl-4-quinolone (10), 1-Methyl-2-phenyl-4-quinolone (11)) (Figure 6; Table 8) that exhibited fungicidal activity against *L. gongylophorus* were isolated from extracts of its stems and leaves [58].

#### **3.5.** *Helietta puberula*

Methanolic, hexanic, and dichloromethane extracts obtained from the stems, leaves, and branches of *Helietta puberula* R. E. Fr. (Rutaceae) were tested against *A. sexdens* workers and the symbiotic fungus of this ant species. Experimental diets were prepared by mixing plant

**Figure 5.** Limonexic acid isolated from *Raulinoa echinata* stems. Source: Biavatti et al., 2005 [57]

H2O, R<sup>3</sup>

=OMe

 **(8)** R<sup>1</sup> =R<sup>2</sup> =OCH

material (crude extract, partially purified extract, or pure compound) and the basic formula described by Bueno et al. (1997) [56]. The final concentrations of crude extracts, fractions, and isolated substances from *H. puberula* in the diet were 2.0, 1.6, and 0.3 mg mL-1, respectively. Blocks of 0.4 g of the experimental diets per plate (control or experimental) were offered daily to the workers. Evaluations were conducted over 25 days, and the number of dead ants was recorded daily. The following substances were isolated from *H. puberula:* anthranilic acid (12), flindersiamine (13), dictamnine (14), kokusaginin (15), maculine (16), and sitosterol. The anthranilic acid, kokusaginine, and dictamnine resulted in 90%, 86%, and 88% mortality, respectively, compared with 68% mortality in the control. The substances anthranilic acid, kokusaginine, masculine, and dictamnine caused fungal inhibition (≥80%) at a concentration

**Table 8.** Evaluation of the growth inhibitory activity of crude extracts, fractions, and compounds of *Raulinoa echinata*

MLE: methanol leaf extract

**% growth inhibition of** *L. gongylophorus*

MSEa - - - - 80 Skimmianine (1) 60 80 NT NT NT Kokusagine (2) 20 100 NT NT NT Maculine (3) 10 50 NT NT NT Flindersiamine (4) - 50 NT NT NT MLEc - - - - 80 MLE (hexane fraction) 80 100 2-n-Nonyl-4-quinolone (5) 20 50 NT NT NT 1-Methyl-2-n-nonyl-4-quinolone (6) - - - - - 1-methyl-2-phenyl-4-quinolone (7) NT NT NT NT NT

**µg mL-1 50 100 250 500 1000**

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Leaf-cutting ants may exhibit behavioural changes when exposed to plant extracts; Anjos and Santana (1994) [59] observed bites and mutilations among *A. sexdens* and *A. laevigata* nestmates subjected to contact with leaves of four *Eucalyptus* sp. belonging to the family Myrtaceae. With the aim of isolating and identifying the compounds responsible for these changes, *E. macu‐ late* leaves were subjected to extraction with hexane, followed by chromatographic fractiona‐ tion, resulting in the isolation of six active sesquiterpenes (elemol, β-eudesmol, α-eudesmol,

Fragments of filter paper in a rectangle, square, or triangle shape were prepared and (a) impregnated with solvent alone as a control (square), (b) left blank (rectangle), or (c) impregnat‐ ed with the treatment to be tested using 100 µL of the extract solution or pure compound (triangle).

of 0.1 mg mL-1 [21] (Figure 7).

MSE: Methanol stem extract, bNT: not tested, c

**Extract/fraction/compound**

guaiol, hinesol, and γ-eudesmol).

**3.6.** *Eucalyptus* **sp.**

a

 **(9)** R  **(10) (11)** 1 =H, R<sup>2</sup> =*n*-non R1 =Me, R<sup>2</sup> =*n*-n R1 =Me, R2 =phe nyl nonyl nyl

**Figure 6.** Compounds identified as skimmianine (5), kokusagine (6), masculine (7), flindersiamine (8), 2-n-Nonyl-4-qui‐ nolone (9), 1-Methyl-2-n-nonyl-4-quinolone (10), and 1-Methyl-2-phenyl-4-quinolone (11) based on comparison with spectral data presented in the literature. Source: Biavatti et al., 2002 [58]


**Table 8.** Evaluation of the growth inhibitory activity of crude extracts, fractions, and compounds of *Raulinoa echinata*

material (crude extract, partially purified extract, or pure compound) and the basic formula described by Bueno et al. (1997) [56]. The final concentrations of crude extracts, fractions, and isolated substances from *H. puberula* in the diet were 2.0, 1.6, and 0.3 mg mL-1, respectively. Blocks of 0.4 g of the experimental diets per plate (control or experimental) were offered daily to the workers. Evaluations were conducted over 25 days, and the number of dead ants was recorded daily. The following substances were isolated from *H. puberula:* anthranilic acid (12), flindersiamine (13), dictamnine (14), kokusaginin (15), maculine (16), and sitosterol. The anthranilic acid, kokusaginine, and dictamnine resulted in 90%, 86%, and 88% mortality, respectively, compared with 68% mortality in the control. The substances anthranilic acid, kokusaginine, masculine, and dictamnine caused fungal inhibition (≥80%) at a concentration of 0.1 mg mL-1 [21] (Figure 7).

#### **3.6.** *Eucalyptus* **sp.**

**Figure 5.** Limonexic acid isolated from *Raulinoa echinata* stems. Source: Biavatti et al., 2005 [57]

  **(5)** R<sup>1</sup>

 **(6)** R<sup>1</sup> =R<sup>2</sup> =OM

 **(7)** R<sup>1</sup> =R<sup>2</sup> =OCH

 **(8)** R<sup>1</sup> =R<sup>2</sup> =OCH

=H, R2 =R

R3 =OMe Me, R<sup>3</sup> =H H2O, R<sup>3</sup> =H

272 Insecticides - Development of Safer and More Effective Technologies

H2O, R<sup>3</sup>

spectral data presented in the literature. Source: Biavatti et al., 2002 [58]

=OMe

**Figure 6.** Compounds identified as skimmianine (5), kokusagine (6), masculine (7), flindersiamine (8), 2-n-Nonyl-4-qui‐ nolone (9), 1-Methyl-2-n-nonyl-4-quinolone (10), and 1-Methyl-2-phenyl-4-quinolone (11) based on comparison with

 **(9)** R  **(10) (11)**

1 =H, R<sup>2</sup>

R1 =Me, R<sup>2</sup>

R1 =Me, R2

=*n*-non

=*n*-n

nyl nonyl nyl

=phe

Leaf-cutting ants may exhibit behavioural changes when exposed to plant extracts; Anjos and Santana (1994) [59] observed bites and mutilations among *A. sexdens* and *A. laevigata* nestmates subjected to contact with leaves of four *Eucalyptus* sp. belonging to the family Myrtaceae. With the aim of isolating and identifying the compounds responsible for these changes, *E. macu‐ late* leaves were subjected to extraction with hexane, followed by chromatographic fractiona‐ tion, resulting in the isolation of six active sesquiterpenes (elemol, β-eudesmol, α-eudesmol, guaiol, hinesol, and γ-eudesmol).

Fragments of filter paper in a rectangle, square, or triangle shape were prepared and (a) impregnated with solvent alone as a control (square), (b) left blank (rectangle), or (c) impregnat‐ ed with the treatment to be tested using 100 µL of the extract solution or pure compound (triangle).

Upon analysis, the composition of the chemical profile of the cuticles of the workers that had contact with *β*-eudesmol was different than that found in the other workers. (E)-*β*-farnesene, busenol, and (E,E)-farnesol were present in the cuticles of ants exposed to *β*-eudesmol [20]. The changes in the composition of the cuticle interfered in the process of recognition between nestmates. The ants triggered an alarm behaviour when they did not recognise the workers

The survival of *A. sexdens* workers was significantly reduced when they were fed diets containing hexane or dichloromethane-soluble extracts of the root and leaves of *Cedrela fissilis* Vell. (Meliaceae). These extracts and those derived from fruits and branches, which were hexane- or dichloromethane-soluble, respectively, also inhibited the growth of the *L. gongylo‐*

The limonoid 3*β*-acetoxicarapin and the triterpenes oleanoic and oleanonic acid were isolated from roots of *C. fissilis*. These compounds and six other mexicanolide-type limonoids (cipa‐ desin A, ruageanin A, cipadesin, khayasin T, febrifugin, and mexicanolide) that were previ‐ ously isolated from *Cipadessa fruticosa* Blume exhibited insecticidal activity against *A. sexdens* leaf-cutting ants. The median survival period (S50) was significantly different from that of the

**Days**

**1 2 3 6 8 10 14 17 21 25**

1- cipadesin B 0 0 6 22 40 58 76 88 92 98 9ª 2- swietemahonolide 0 0 4 40 54 58 82 90 96 98 8ª 3- 3β-acetoxycarapin 0 2 12 34 50 76 98 100 - - 8ª 4- oleanolic acid 0 0 10 52 70 90 100 - - - 6ª 5c- oleanonic acid 0 0 8 38 60 86 100 - - - 8ª 6- cipadesin A 0 2 8 34 46 72 100 - - - 9ª 7- ruageanin A 0 4 18 50 62 74 96 98 98 100 6ª 8- cipadesin 0 8 12 46 68 76 100 - - - 7ª 9- khayasin T 0 4 10 54 72 86 98 100 - - 6ª 10- febrifugin 0 2 6 38 58 70 88 94 98 100 7ª 11- mexicanolide 0 4 16 50 60 70 100 - - - 6ª

Control (ethyl acetate) 0 0 6 22 36 50 74 90 94 96 10

**Table 9.** Mortality (%) of *Atta sexdens* workers fed on compounds 1-11 at a concentration of 100 µg mL-1. Source:

Significant difference according to the log-rank test (p<0.05).

**Survival median (S50)/days**

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control, confirming activity against *A. sexdens* [61] (Table 9).

exposed to *β*-eudesmol.

**3.7.** *Cedrela fissilis*

*phorus* fungus [60,61].

**Compounds**

a

Leite et al., 2005 [61].

**Figure 7.** Molecular structures of substances from *Helietta puberula*: anthranilic acid (12), flindersiamine (13), dictam‐ nine (14), kokusaginine (15), and maculine (16). Source: Almeida et al., 2007 [21].

After solvent evaporation, two of the filter paper fragments of each of the three different geometric shapes were placed on three glass slides, which were then transferred to the colonies. Monitor‐ ing was performed for 30 minutes after placement of the filter paper, and the number of groups of attackers, the number of ants in each group, and the number of mutilated ants in each group were counted. Elemol (17) and *β*-eudesmol (18) (Figure 8) were the most active ingredients, and the latter substance was associated with greater numbers of groups of attackers (84.2) and mutilated ants (285.8). After contact with the filter paper impregnated with *β-*eudesmol, the ants exhibited alarm behaviour and held their mandibles open. When encountering nestmates that had previously contacted the filter paper, they touched their antennas and then attacked each other, frequently on the legs, but also on other parts of the body [18].

**Figure 8.** Chemical structures of the sesquiterpenes identified in the most active fraction of the *Eucalyptus maculate* leaf extract. Source: Marsaro Junior et al., 2004 [18].

Upon analysis, the composition of the chemical profile of the cuticles of the workers that had contact with *β*-eudesmol was different than that found in the other workers. (E)-*β*-farnesene, busenol, and (E,E)-farnesol were present in the cuticles of ants exposed to *β*-eudesmol [20]. The changes in the composition of the cuticle interfered in the process of recognition between nestmates. The ants triggered an alarm behaviour when they did not recognise the workers exposed to *β*-eudesmol.

### **3.7.** *Cedrela fissilis*

After solvent evaporation, two of the filter paper fragments of each of the three different geometric shapes were placed on three glass slides, which were then transferred to the colonies. Monitor‐ ing was performed for 30 minutes after placement of the filter paper, and the number of groups of attackers, the number of ants in each group, and the number of mutilated ants in each group were counted. Elemol (17) and *β*-eudesmol (18) (Figure 8) were the most active ingredients, and the latter substance was associated with greater numbers of groups of attackers (84.2) and mutilated ants (285.8). After contact with the filter paper impregnated with *β-*eudesmol, the ants exhibited alarm behaviour and held their mandibles open. When encountering nestmates that had previously contacted the filter paper, they touched their antennas and then attacked each

**Figure 7.** Molecular structures of substances from *Helietta puberula*: anthranilic acid (12), flindersiamine (13), dictam‐

**Figure 8.** Chemical structures of the sesquiterpenes identified in the most active fraction of the *Eucalyptus maculate*

other, frequently on the legs, but also on other parts of the body [18].

nine (14), kokusaginine (15), and maculine (16). Source: Almeida et al., 2007 [21].

274 Insecticides - Development of Safer and More Effective Technologies

leaf extract. Source: Marsaro Junior et al., 2004 [18].

The survival of *A. sexdens* workers was significantly reduced when they were fed diets containing hexane or dichloromethane-soluble extracts of the root and leaves of *Cedrela fissilis* Vell. (Meliaceae). These extracts and those derived from fruits and branches, which were hexane- or dichloromethane-soluble, respectively, also inhibited the growth of the *L. gongylo‐ phorus* fungus [60,61].

The limonoid 3*β*-acetoxicarapin and the triterpenes oleanoic and oleanonic acid were isolated from roots of *C. fissilis*. These compounds and six other mexicanolide-type limonoids (cipa‐ desin A, ruageanin A, cipadesin, khayasin T, febrifugin, and mexicanolide) that were previ‐ ously isolated from *Cipadessa fruticosa* Blume exhibited insecticidal activity against *A. sexdens* leaf-cutting ants. The median survival period (S50) was significantly different from that of the control, confirming activity against *A. sexdens* [61] (Table 9).


a Significant difference according to the log-rank test (p<0.05).

**Table 9.** Mortality (%) of *Atta sexdens* workers fed on compounds 1-11 at a concentration of 100 µg mL-1. Source: Leite et al., 2005 [61].

#### **3.8.** *Azadirachta indica*

Seeds of *Azadirachta indica* were tritured and pressed, yielding a neem paste. After one week, the floating material was isolated, which was referred to as crude extract of neem oil. A known mass of the remaining material, referred to as crude extract of seed neem paste, was macerated for three days three times at room temperature and extracted with solvents of increasing polarity (hexane, dichloromethane and methanol), resulting in three crude extracts. When incorporated in an artificial diet, the crude extract of neem seed oil caused significant toxicity to *A. sexdens* workers at all of the concentrations tested. The survival of the ants was signifi‐ cantly reduced in the diets containing the neem seed paste hexane extract at concentrations of 10 and 20 µg mL-1, the dichloromethane extract at all concentrations tested (2, 10, and 20 µg mL-1), and the methanol extract at concentrations of 10 and 20 µg mL-1.

**3.9.** *Simarouba versicolor*

The dichloromethane-soluble fraction of methanolic extracts of the leaves, stems, and branches of *Simarouba versicolor* St. Hill (Simaroubaceae) was tested *in vitro* on ants through ingestion bioassays and with the symbiotic fungus in culture medium. The median survival period for workers was significantly reduced (S50=4 days) compared to the control (S50=16 days), and 100% inhibition of *L. gongylophorus* growth was observed. From these fractions, two alkaloids were isolated, 4,5-dimethoxy-canthin-6-one (19) and 5-methoxy-canthin-6-one (20) (Figure 9), both of which were toxic to the symbiotic fungus and completely inhibited growth at a concentration of 0.1 mg mL-1. However, only the alkaloid 5-methoxy-canthin-6-one reduced the median survival period of the workers from 14 days (control) to seven days at a 0.3 mg mL-1 concen‐ tration (Table 10). The triterpenes isolated from the other extracts of the plant (lupenone and lupeol) showed no deleterious effects on the leaf-cutting ants of the symbiotic fungus [22].

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**Figure 9.** Chemical structures of substances isolated from *Simarouba vesicolor*: 4,5-dimethoxycanthin-6-one (19) and

Control 0 2 8 22 30 38 52 62 74 76 14a Lupenone 4 4 6 12 24 30 46 54 64 76 16a 4,5-dimethoxycanthin-6-one 2 2 8 34 36 48 56 58 68 82 13a Lupenol 2 2 10 26 26 28 34 36 44 54 19a 5-methoxycanthin-6-one 0 0 10 46 52 56 76 78 80 88 7b

2S50=Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test. Different letters after the S50 values compared to the respective control indicate a significant difference according

**Table 10.** Toxicity (% mortality and S50) of substances isolated from *Simarouba versicolor* in *Atta sexdens* workers.

**Day of experiment 1 2 3 6 8 10 14 17 21 25 S50 <sup>2</sup>**

5 methoxycanthin-6-one (20). Source: Peñaflor et al., 2009 [22].

1Isolated substances at a concentration of 0.3 mg mL-1;

to the log-rank test (P<0.05). Source: Peñaflor et al., 2009 [22].

**Treatment1**

There was a negative relationship between the neem oil concentration and the frequency of contact of ants with the artificial diet. The lowest frequency of contact was obtained with the highest concentration tested (30 µg mL-1). Moreover, the initial contact with the diet was dependent on the presence of neem. Thus, the period required for the ants to feed on the artificial diet for the first time was 8 seconds in the control, 4 minutes and 36 seconds at a concentration of 5 µg mL-1, 19 minutes at a concentration of 10 µg mL-1, and 55 minutes at a concentration of 30 µg mL-1. Some changes in the behaviours of the ants were observed when the workers contacted the diets containing neem seed oil. Contact between the antenna or legs and the diet caused instantaneous retraction of these body parts. The ants positioned them‐ selves offensively with open mandibles and performed self-grooming. The workers that cleaned themselves by licking showed symptoms of intoxication, such as slow movements, disorientation, and prostration [62].

The hexanic extract of *A. indica* neem was tested against *Acromyrmex rugosus* F. Smith (Formi‐ cidae) workers. Two colonies of *A. rugosus* were used, and from each colony, 30 groups of 20 workers each were isolated. A citrus pulp containing neem at concentrations of 0.1, 1.0, and 10% was offered to these groups. In the treatments, pastes composed of hexanic extracts of neem (from leaves, branches and seeds) were prepared with the following composition: pure glucose (10%), citrus pulp powder and soybean oil (10%). The sulfluramid treatment was offered in the form of a paste, in which 0.3% sulfluramid was dissolved in 10% soybean oil and mixed with citrus pulp powder and 10% pure glucose. The positive control was prepared in the same manner, but the active sulfluramid was not added to the paste. Treatments were performed with 5 g of paste per replicate, which was removed from the jars after 48 hours. Monitoring lasted five minutes and was performed immediately, 30 minutes, and 24 hours after treatment. The relative frequencies of each of the workers' behaviours and the number of deaths were recorded. High mortality was observed within the first 24 h in the treatments with neem (>20 workers) compared to the control (5 workers), which is not the slow type of action desired for formicides. The delayed action of the active ingredients in formicide formulations is an essential feature because colonies of leaf-cutting ants are very populous, and the control of their nests depends on contamination of all individuals. If ants detect that the presented substrate is not adequate, they will stop carrying it and can even remove parts of the symbiotic fungus contaminated with this substrate and isolate it in waste chambers [63].

#### **3.9.** *Simarouba versicolor*

**3.8.** *Azadirachta indica*

276 Insecticides - Development of Safer and More Effective Technologies

disorientation, and prostration [62].

Seeds of *Azadirachta indica* were tritured and pressed, yielding a neem paste. After one week, the floating material was isolated, which was referred to as crude extract of neem oil. A known mass of the remaining material, referred to as crude extract of seed neem paste, was macerated for three days three times at room temperature and extracted with solvents of increasing polarity (hexane, dichloromethane and methanol), resulting in three crude extracts. When incorporated in an artificial diet, the crude extract of neem seed oil caused significant toxicity to *A. sexdens* workers at all of the concentrations tested. The survival of the ants was signifi‐ cantly reduced in the diets containing the neem seed paste hexane extract at concentrations of 10 and 20 µg mL-1, the dichloromethane extract at all concentrations tested (2, 10, and 20 µg

There was a negative relationship between the neem oil concentration and the frequency of contact of ants with the artificial diet. The lowest frequency of contact was obtained with the highest concentration tested (30 µg mL-1). Moreover, the initial contact with the diet was dependent on the presence of neem. Thus, the period required for the ants to feed on the artificial diet for the first time was 8 seconds in the control, 4 minutes and 36 seconds at a concentration of 5 µg mL-1, 19 minutes at a concentration of 10 µg mL-1, and 55 minutes at a concentration of 30 µg mL-1. Some changes in the behaviours of the ants were observed when the workers contacted the diets containing neem seed oil. Contact between the antenna or legs and the diet caused instantaneous retraction of these body parts. The ants positioned them‐ selves offensively with open mandibles and performed self-grooming. The workers that cleaned themselves by licking showed symptoms of intoxication, such as slow movements,

The hexanic extract of *A. indica* neem was tested against *Acromyrmex rugosus* F. Smith (Formi‐ cidae) workers. Two colonies of *A. rugosus* were used, and from each colony, 30 groups of 20 workers each were isolated. A citrus pulp containing neem at concentrations of 0.1, 1.0, and 10% was offered to these groups. In the treatments, pastes composed of hexanic extracts of neem (from leaves, branches and seeds) were prepared with the following composition: pure glucose (10%), citrus pulp powder and soybean oil (10%). The sulfluramid treatment was offered in the form of a paste, in which 0.3% sulfluramid was dissolved in 10% soybean oil and mixed with citrus pulp powder and 10% pure glucose. The positive control was prepared in the same manner, but the active sulfluramid was not added to the paste. Treatments were performed with 5 g of paste per replicate, which was removed from the jars after 48 hours. Monitoring lasted five minutes and was performed immediately, 30 minutes, and 24 hours after treatment. The relative frequencies of each of the workers' behaviours and the number of deaths were recorded. High mortality was observed within the first 24 h in the treatments with neem (>20 workers) compared to the control (5 workers), which is not the slow type of action desired for formicides. The delayed action of the active ingredients in formicide formulations is an essential feature because colonies of leaf-cutting ants are very populous, and the control of their nests depends on contamination of all individuals. If ants detect that the presented substrate is not adequate, they will stop carrying it and can even remove parts of the symbiotic fungus contaminated with this substrate and isolate it in waste chambers [63].

mL-1), and the methanol extract at concentrations of 10 and 20 µg mL-1.

The dichloromethane-soluble fraction of methanolic extracts of the leaves, stems, and branches of *Simarouba versicolor* St. Hill (Simaroubaceae) was tested *in vitro* on ants through ingestion bioassays and with the symbiotic fungus in culture medium. The median survival period for workers was significantly reduced (S50=4 days) compared to the control (S50=16 days), and 100% inhibition of *L. gongylophorus* growth was observed. From these fractions, two alkaloids were isolated, 4,5-dimethoxy-canthin-6-one (19) and 5-methoxy-canthin-6-one (20) (Figure 9), both of which were toxic to the symbiotic fungus and completely inhibited growth at a concentration of 0.1 mg mL-1. However, only the alkaloid 5-methoxy-canthin-6-one reduced the median survival period of the workers from 14 days (control) to seven days at a 0.3 mg mL-1 concen‐ tration (Table 10). The triterpenes isolated from the other extracts of the plant (lupenone and lupeol) showed no deleterious effects on the leaf-cutting ants of the symbiotic fungus [22].

**Figure 9.** Chemical structures of substances isolated from *Simarouba vesicolor*: 4,5-dimethoxycanthin-6-one (19) and 5 methoxycanthin-6-one (20). Source: Peñaflor et al., 2009 [22].


1Isolated substances at a concentration of 0.3 mg mL-1;

2S50=Survival median 50%. Different letters after the S50 values indicate a significant difference according to the log-rank test. Different letters after the S50 values compared to the respective control indicate a significant difference according to the log-rank test (P<0.05). Source: Peñaflor et al., 2009 [22].

**Table 10.** Toxicity (% mortality and S50) of substances isolated from *Simarouba versicolor* in *Atta sexdens* workers.

#### **3.10.** *Ageratum conyzoides*

An assessment of the formicidal activity of a hexanic extract from the leaves of goatweed, *Ageratum conyzoides* L. (Asteraceae), against leaf-cutting ants was performed using the acetonediluted extract at a concentration of 1.0 mg mL-1. Each worker was topically treated with 1.0 µL of this solution, which was applied on the pronoto of the insect. In the control treatment, the insects were treated with an equal volume of pure acetone. The numbers of living and dead individuals were counted 24 and 48 hours after treatment. The crude extract of goatweed caused increased mortality of *Atta laevigata* F. Smith (Hymenoptera: Formicidae) and *Atta subterraneus subterraneus* Forel (Hymenoptera: Formicidae) workers. The goatweed extract was then fractionated, resulting in the isolation of the compound coumarin. Coumarin was tested against ants at different concentrations (0.5, 4.0, 7.0, 16.0, 50.0, and 100.0 mg mL-1 in acetone) to determine its toxicity among the two species of leaf-cutting ants. The median lethal concentration (LC50) decreased (10.9-fold) with increased application time for *A. subterraneus subterraneus*. The LC50 was 55.42 mg mL-1 at 24 hours and decreased to 5.07 mg mL-1 at 48 hours. For *A. laevigata*, the LC50 decreased 1.8-fold, from 23.20 mg mL-1 at 24 hours to 12.70 mg mL-1 at 48 hours. Thus, coumarin is a potential agent for ant control in the form of granulated attractive baits because it has a delayed insecticidal effect [64].

> dose dependent. Symptoms of intoxication could be perceived after 24 hours and consisted of a reduction or cessation of movement, followed by disorientation, lack of coordination, and

**Table 11.** Activity of fatty acids present in the methanol fraction of hexane extracts from *Ricinus communis* leaves

**Acid %** Decanoic 1.2 Myristic 0.5 Pentadecanoic 6.4 Palmitic 81.0 Heptadecanoic 0.3 Estearic 6.6 Eicosanoic 1.1 Docosanoic 0.2 Tricosanoic 0.7 Tetracosanoic 0.2

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The development of the symbiotic fungus *L. gongylophorus* is inhibited *in vitro* by synthetic compounds containing a piperonyl group: 1-(3,4-methylenedioxybenzyloxy)methane (22); 1- (3,4-methylenedioxybenzyloxy)ethane (23); 1-(3,4-methylenedioxybenzyloxy)butane (24); 1- (3,4-methylenedioxybenzyloxy)hexane (25); 1-(3,4-methylenedioxybenzyloxy)octane (26); 1- (3,4-methylenedioxybenzyloxy)decane (27); and 1-(3,4-methylenedioxybenzyloxy)dodecane (28) (Figure 11). Moreover, *A. sexdens* workers fed daily with an artificial diet containing these compounds showed high mortality compared to controls. The inhibition of fungal growth increased with the number of carbon atoms in the lateral chain, which varied from 1 to 8

**Figure 10.** Ricinin isolated from leaf extracts of *Ricinus communis.* Bigi et al., 2004 [15].

(MFHE) against *Leucoagaricus gongylophorus*. Source: Bigi et al., 2004 [15].

**3.12. Synthetic analogues of plant origin**

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death [15].

#### **3.11.** *Ricinus communis*

Dry *R. communis* leaves (2 kg) were ground in a Willey mill, and crude extracts were prepared via sequential maceration (3 litres for 7 days for each solvent) with hexane (24.8 g of extract), dichloromethane (32.8 g), ethyl acetate (18.8 g), methanol (54.0 g), and water. With the exception of the water extract, all extracts were subjected to chromatography on silica gel 60 as the stationary phase under vacuum (0.040-0.063 mm, 400 g; column with a sinterised filter in the bottom, internal diameter 10 cm, length 25 cm) with hexane, dichloromethane, ethyl acetate, and methanol (1 litre each) as eluents, yielding four fractions for each extract. The water extract was not fractionated. A portion of the methanol fraction of the hexane extract was refractionated, yielding 12 fractions (MFHE 1-12). These extracts were tested against the symbiotic fungus according to the methodology of Pagnocca et al. (1990) [49]. The sub-fractions MFHE-6, MFHE-9, and MFHE-10 inhibited fungal growth by 80% at a concentration of 0.5 mg mL-1. The same result was observed for the MFHE-11 sub-fraction at a 1.0 mg mL-1 concentra‐ tion. Sub-fraction MFHE-9 contained a mixture of two glycosidic steroids (β-sitosterol-3-*O*-*β*-D-glucoside and stigmasterol-3-*O*-*β*-D-glucoside) and fatty acids (decanoic, myristic, pentadecanoic, palmitic, heptadecanoic, estearic, eicosanoic, docosanoic, tricosanoic, and tetracosanoic acids). Among the above-mentioned compounds, only palmitic acid exhibited antifungal activity and inhibited the growth of the symbiotic fungus by 80% (Table 11).

The methanolic fraction of the dichloromethane-soluble extract of *R. communis* leaves was also re-fractionated, resulting in the isolation of ricin (21) (Figure 10) and monoglyceride (1-palmitic acid glycerol ester). Ricin caused significant death of *A. sexdens* workers when added to their artificial diets. The median survival periods (S50) were 6.93 and 5.27 days at 0.2 and 0.4 mg mL-1, respectively, compared to 10.82 days in the control. However, the effect on mortality was


**Table 11.** Activity of fatty acids present in the methanol fraction of hexane extracts from *Ricinus communis* leaves (MFHE) against *Leucoagaricus gongylophorus*. Source: Bigi et al., 2004 [15].

dose dependent. Symptoms of intoxication could be perceived after 24 hours and consisted of a reduction or cessation of movement, followed by disorientation, lack of coordination, and death [15].

**Figure 10.** Ricinin isolated from leaf extracts of *Ricinus communis.* Bigi et al., 2004 [15].

#### **3.12. Synthetic analogues of plant origin**

**3.10.** *Ageratum conyzoides*

278 Insecticides - Development of Safer and More Effective Technologies

**3.11.** *Ricinus communis*

An assessment of the formicidal activity of a hexanic extract from the leaves of goatweed, *Ageratum conyzoides* L. (Asteraceae), against leaf-cutting ants was performed using the acetonediluted extract at a concentration of 1.0 mg mL-1. Each worker was topically treated with 1.0 µL of this solution, which was applied on the pronoto of the insect. In the control treatment, the insects were treated with an equal volume of pure acetone. The numbers of living and dead individuals were counted 24 and 48 hours after treatment. The crude extract of goatweed caused increased mortality of *Atta laevigata* F. Smith (Hymenoptera: Formicidae) and *Atta subterraneus subterraneus* Forel (Hymenoptera: Formicidae) workers. The goatweed extract was then fractionated, resulting in the isolation of the compound coumarin. Coumarin was tested against ants at different concentrations (0.5, 4.0, 7.0, 16.0, 50.0, and 100.0 mg mL-1 in acetone) to determine its toxicity among the two species of leaf-cutting ants. The median lethal concentration (LC50) decreased (10.9-fold) with increased application time for *A. subterraneus subterraneus*. The LC50 was 55.42 mg mL-1 at 24 hours and decreased to 5.07 mg mL-1 at 48 hours. For *A. laevigata*, the LC50 decreased 1.8-fold, from 23.20 mg mL-1 at 24 hours to 12.70 mg mL-1 at 48 hours. Thus, coumarin is a potential agent for ant control in the form of granulated

Dry *R. communis* leaves (2 kg) were ground in a Willey mill, and crude extracts were prepared via sequential maceration (3 litres for 7 days for each solvent) with hexane (24.8 g of extract), dichloromethane (32.8 g), ethyl acetate (18.8 g), methanol (54.0 g), and water. With the exception of the water extract, all extracts were subjected to chromatography on silica gel 60 as the stationary phase under vacuum (0.040-0.063 mm, 400 g; column with a sinterised filter in the bottom, internal diameter 10 cm, length 25 cm) with hexane, dichloromethane, ethyl acetate, and methanol (1 litre each) as eluents, yielding four fractions for each extract. The water extract was not fractionated. A portion of the methanol fraction of the hexane extract was refractionated, yielding 12 fractions (MFHE 1-12). These extracts were tested against the symbiotic fungus according to the methodology of Pagnocca et al. (1990) [49]. The sub-fractions MFHE-6, MFHE-9, and MFHE-10 inhibited fungal growth by 80% at a concentration of 0.5 mg mL-1. The same result was observed for the MFHE-11 sub-fraction at a 1.0 mg mL-1 concentra‐ tion. Sub-fraction MFHE-9 contained a mixture of two glycosidic steroids (β-sitosterol-3-*O*-*β*-D-glucoside and stigmasterol-3-*O*-*β*-D-glucoside) and fatty acids (decanoic, myristic, pentadecanoic, palmitic, heptadecanoic, estearic, eicosanoic, docosanoic, tricosanoic, and tetracosanoic acids). Among the above-mentioned compounds, only palmitic acid exhibited antifungal activity and inhibited the growth of the symbiotic fungus by 80% (Table 11).

The methanolic fraction of the dichloromethane-soluble extract of *R. communis* leaves was also re-fractionated, resulting in the isolation of ricin (21) (Figure 10) and monoglyceride (1-palmitic acid glycerol ester). Ricin caused significant death of *A. sexdens* workers when added to their artificial diets. The median survival periods (S50) were 6.93 and 5.27 days at 0.2 and 0.4 mg mL-1, respectively, compared to 10.82 days in the control. However, the effect on mortality was

attractive baits because it has a delayed insecticidal effect [64].

The development of the symbiotic fungus *L. gongylophorus* is inhibited *in vitro* by synthetic compounds containing a piperonyl group: 1-(3,4-methylenedioxybenzyloxy)methane (22); 1- (3,4-methylenedioxybenzyloxy)ethane (23); 1-(3,4-methylenedioxybenzyloxy)butane (24); 1- (3,4-methylenedioxybenzyloxy)hexane (25); 1-(3,4-methylenedioxybenzyloxy)octane (26); 1- (3,4-methylenedioxybenzyloxy)decane (27); and 1-(3,4-methylenedioxybenzyloxy)dodecane (28) (Figure 11). Moreover, *A. sexdens* workers fed daily with an artificial diet containing these compounds showed high mortality compared to controls. The inhibition of fungal growth increased with the number of carbon atoms in the lateral chain, which varied from 1 to 8 (substances 22 to 26). Compounds containing 10 or 12 carbon atoms in the lateral chain did not inhibit fungal growth (substances 27 and 28) (Figure 11). Compound 26, 1-(3,4-methyle‐ nedioxybenzyloxy)octane, was the most active and inhibited fungal development by 80% at 15 µg mL-1. In workers, a toxic effect was caused by compound 26 (C8); this effect increased with an increase in the number of carbon atoms in the lateral chains (C10 and C12). Thus, at the same concentration (100 µg mL-1), the mortality rates after eight days of ingestion were 82%, 66%, and 42% under treatment with 1-(3,4-methylenedioxybenzyloxy)decane (com‐ pound 28), 1-(3,4-methylenedioxybenzyloxy)dodecane (compound 27), and compound 26, respectively, while for piperonyl butoxide, the observed mortality was 68%. The last com‐ pound, which is known as a synergistic insecticide, inhibited the symbiotic fungus with an intensity that was statistically similar to that observed for synthetic compound 26. The results indicate that a formulation can be designed to attack both ants and their symbiotic fungus; such a formulation could represent an advantage over the chemical products used for leafcutting ant control, which are directed only towards the ants [65].

The 3-(3,4-methylenedioxyphenyl)-2-(E)-propenamide (30) portion was maintained, and only groups R1 and R2 linked to the nitrogen (Figure 12) were altered. Thus, nine amides were synthesised, and the yield varied between 36 and 86% (Table 12; Figure 13). Compounds 3 (S50= 11 days) and 8 (S50=7.5 days) significantly reduced the median survival period (S50) for workers compared to the control (S50= 14 days) at 100 µg mL-1 when added to the artificial diet offered daily. Compounds 1, 2, 4, 5, 6, 7, and 9 had no effect on the median survival period at any of the concentrations tested (25, 50, and 100 µg mL-1). At 100 µg mL-1, compounds 1, 2, and 3 completely inhibited fungal growth, and partial inhibition was observed for compounds 4 (80%), 5 (40%), and 6 (20%), while compounds 7, 8, and 9 had no effect on the growth of the

**Figure 12.** E)-3-(3,4-methylenedioxyphenyl)-2-propenamide group. Source: Pagnocca et al., 2006 [66].

NHCH2CH (CH3)CH2CH3

**Table 12.** Amines, the respective yields of the amides, and the eluents used in the chromatographic separation.

1 Piperidine 44 4:6

2 Diethylamine N(CH2CH3)2 42 6:4

3 Pyrrolidine 68 1:1

5 Morpholine 40 5.5:4.5

 Aniline NHC6H5 39 5.5:4.5 Disopropylamine N[CH(CH3)2]2 36 2:1 Benzylamine NHCH2C6H5 36 4:1 Dicyclohexylamine N(C6H11)2 86 7.5:2.5

**Amide Amine NR1R2 Yield (%) Eluent (hexane/ethyl acetate)**

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38 7:3

symbiotic fungus [66].

4 2-Methylbutylamine

Source: Pagnocca et al., 2006 [66].

**Figure 11.** Structures of the synthesised compounds 1-(3,4-methylenedioxybenzyloxy)methane (22); 1-(3,4-methyle‐ nedioxybenzyloxy)ethane (23); 1-(3,4-methylenedioxybenzyloxy)butane (24); 1-(3,4-methylenedioxybenzyloxy)hex‐ ane (25); 1-(3,4-methylenedioxybenzyloxy)octane (26); 1-(3,4-methylenedioxybenzyloxy)decane (27); 1-(3,4 methylenedioxybenzyloxy)dodecane (28); and the commercial compound piperonyl butoxide (29). Victor et al., 2001 [65].

Several studies have suggested that the amides found in species of the *Piper* genus show potential for insecticidal use due to their effectiveness and knockdown effects. Therefore, the natural amides N-pyrrolidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenamide and Npiperidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenamide, found in the roots of *Piper piresii* Yunck (Family: Piperaceae), were used as a model for the synthesis of analogous amides. The 3-(3,4-methylenedioxyphenyl)-2-(E)-propenamide (30) portion was maintained, and only groups R1 and R2 linked to the nitrogen (Figure 12) were altered. Thus, nine amides were synthesised, and the yield varied between 36 and 86% (Table 12; Figure 13). Compounds 3 (S50= 11 days) and 8 (S50=7.5 days) significantly reduced the median survival period (S50) for workers compared to the control (S50= 14 days) at 100 µg mL-1 when added to the artificial diet offered daily. Compounds 1, 2, 4, 5, 6, 7, and 9 had no effect on the median survival period at any of the concentrations tested (25, 50, and 100 µg mL-1). At 100 µg mL-1, compounds 1, 2, and 3 completely inhibited fungal growth, and partial inhibition was observed for compounds 4 (80%), 5 (40%), and 6 (20%), while compounds 7, 8, and 9 had no effect on the growth of the symbiotic fungus [66].

(substances 22 to 26). Compounds containing 10 or 12 carbon atoms in the lateral chain did not inhibit fungal growth (substances 27 and 28) (Figure 11). Compound 26, 1-(3,4-methyle‐ nedioxybenzyloxy)octane, was the most active and inhibited fungal development by 80% at 15 µg mL-1. In workers, a toxic effect was caused by compound 26 (C8); this effect increased with an increase in the number of carbon atoms in the lateral chains (C10 and C12). Thus, at the same concentration (100 µg mL-1), the mortality rates after eight days of ingestion were 82%, 66%, and 42% under treatment with 1-(3,4-methylenedioxybenzyloxy)decane (com‐ pound 28), 1-(3,4-methylenedioxybenzyloxy)dodecane (compound 27), and compound 26, respectively, while for piperonyl butoxide, the observed mortality was 68%. The last com‐ pound, which is known as a synergistic insecticide, inhibited the symbiotic fungus with an intensity that was statistically similar to that observed for synthetic compound 26. The results indicate that a formulation can be designed to attack both ants and their symbiotic fungus; such a formulation could represent an advantage over the chemical products used for leaf-

cutting ant control, which are directed only towards the ants [65].

280 Insecticides - Development of Safer and More Effective Technologies

22- R1= 23- R1= 24- R1= 25- R1= 26- R1= 27- R1= 28- R1= 29- R1= R2=

[65].

=CH2OCH3, R2= =CH2OCH2CH3, =CH2OCH2(CH2 =CH2OCH2(CH2 =CH2OCH2(CH2 =CH2OCH2(CH2 =CH2OCH2(CH2 =CH2(OCH2CH2 =CH2CH2CH3

=H , R2=H 2)2CH3, R2=H 2)4CH3, R2=H 2)6CH3, R2=H 2)8CH3, R2=H 2)10CH3, R2=H 2)2O(CH2)3CH3,

**Figure 11.** Structures of the synthesised compounds 1-(3,4-methylenedioxybenzyloxy)methane (22); 1-(3,4-methyle‐ nedioxybenzyloxy)ethane (23); 1-(3,4-methylenedioxybenzyloxy)butane (24); 1-(3,4-methylenedioxybenzyloxy)hex‐ ane (25); 1-(3,4-methylenedioxybenzyloxy)octane (26); 1-(3,4-methylenedioxybenzyloxy)decane (27); 1-(3,4 methylenedioxybenzyloxy)dodecane (28); and the commercial compound piperonyl butoxide (29). Victor et al., 2001

Several studies have suggested that the amides found in species of the *Piper* genus show potential for insecticidal use due to their effectiveness and knockdown effects. Therefore, the natural amides N-pyrrolidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenamide and Npiperidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenamide, found in the roots of *Piper piresii* Yunck (Family: Piperaceae), were used as a model for the synthesis of analogous amides.

**Figure 12.** E)-3-(3,4-methylenedioxyphenyl)-2-propenamide group. Source: Pagnocca et al., 2006 [66].


**Table 12.** Amines, the respective yields of the amides, and the eluents used in the chromatographic separation. Source: Pagnocca et al., 2006 [66].

chymotrypsinase) [69]. Interestingly, although the fungus (hyphal extracts) produces chiti‐ nase, the plants do not contain chitin. It has been suggested that the chitinases present in the rectal fluid of primitive genera (*Cyphomyrmex*, *Mycocepurus*, and *Myrmicocrysta*) are important in the degradation of substrates such as the carcasses of leaf-cutting ants, which can be used as a substrate for the growth of new hyphae from the symbiotic fungus. It has also been proposed that these enzymes may play an important role in the lysis of competitive funguses,

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The results from analysis of the labial glands of larvae and adults of *A. subterraneus* indicate that they secrete chitinases. Chromatographic tests to detect chitinolytic activity in the labial glands of the larvae revealed profiles similar to those observed for the glands of the workers, indicating that this enzymatic activity may have a fungal origin. Interestingly, the peak of chitinolytic activity in the middle intestine of the larvae does not coincide with the peak in the labial gland, indicating that the chitinase produced in the labial gland is not active in the middle

In addition to chitinases, pectinases, xylanases, and cellulases derived from the fungus have been detected in the faecal fluid of *Atta colobica tonsipes* [75]. Pectinases present in the faeces of *Acromyrmex echinatior* and *Atta colombica* have been suggested to be of fungal origin [7]. Interestingly, no pectinase, xylanase, or cellulase activity was detected in *A. subterraneus* adults. However, elevated α-glycosidase activity was detected in fractions from the middle intestine and rectum of adult leaf-cutting ants, indicating the importance of this enzyme in the assimi‐ lation of glucose and, most likely, of saccharose and maltose present in the plant material [73]. Pectinases and xylanases were detected in the middle intestine of the larvae, where their only

Febvay and Kermarrec (1984) suggested that digestion of the walls of the fungal cells occurs in the infrabuccal pocket of *Acromyrmex octospinosus*. It is possible that the adult garden ants regurgitate chitinases through their labial glands onto the fungal material to feed the larvae, initiating the partial digestion of fungal parts, without the adult benefiting from ingestion. The larvae can regurgitate their own secretions from their labial glands, providing the energetic

Other authors have reported that the secretions offered by the larvae originate in a liquid produced from the anal region, in a process referred to as proctodeal trophallaxis (Figure 14). The workers contact the anal region of the larvae, which may be of different sizes, and the larvae release a small transparent drop that is immediately ingested by the workers. This observation reveals that the larvae of the leaf-cutting ants play a fundamental role in the nutrient flux in the colony because, although adult individuals are incapable of feeding on the solid portions of the fungus, the larvae are able to feed on these portions, digest their walls, and transfer nutrients to the haemolymph, where they are absorbed by the Malpighian tubules, transferred to the posterior intestine, and offered to the workers, making them available to

needs of the adults, who receive these liquids through trophallaxis [76].

many of which have cellular walls made of chitin [72].

source of nutrients is the symbiotic fungus [74].

other individuals in the colony [77].

intestine [73, 74].

**Figure 13.** Synthesis of amides 1-9. Source: Pagnocca et al., 2006 [66].

### **4. Control of leaf–cutting ants via enzymatic inhibition**

When the cut plant fragments reach the colony, a complex process of preparation of the plant substrate for its incorporation into the fungal garden begins. During this processing, the workers may ingest the plant's sap while cutting and pressing the borders of the plant fragment. By scraping the surface, they remove the epicuticular wax layer and facilitate the decomposition of the substrate by the fungus [42,67] indicating the importance of the hydro‐ lytic enzymes in this process. This behaviour is also related to the decontamination of the substrate [68].

Ultra-structural studies of the colonisation of the plant substrate by the fungus have demon‐ strated that the fungus can only use the portions that have had their border cut. In addition, the cuticular surface of the leaf at the time of colonisation appears to be intact, which suggests an absence of cutinases (enzymes that catalyse the hydrolysis of cutin, a structural component of the cuticle of the plant) in this process. Therefore, it is believed that this symbiotic fungus is a saprophyte that is unable to penetrate into plants that are not damaged [69] (Figure 13).

Therefore, maceration aids in the destruction of the physical barrier of the leaf cuticle, increasing permeability to allow fungal growth, which is assisted by enzymes present in the faecal fluid. [70] The symbiotic fungus is an important mediator involved in providing nutrition to the ants via the hydrolysis of polysaccharides from plant [71] as it produces large amount of enzymes, particularly pectinases, that are ingested by ants, concentrated in the intestine, returned to the fungal garden via faecal fluid, and utilised for the digestion of plant tissues [7]. Therefore, this association is also essential for fungal access to the nutrients in the plant material that is transported by the ants to the nest [71].

The profile of the hydrolytic enzymes involved in this relationship between leaf-cutting ants and fungi has been studied. The extracts from the fungal hyphae of garden fungi exhibit a wide range of activities involving carbohydratases (pectinase, laminarinase, α-glucosidase, βglucosidase, α-galactosidase), with the proteinase chitinase presenting the highest activity (Erthal et al., 2009).

Extracts from fungal gardens contain a wide variety of digestive enzymes, including carbo‐ hydratases (e.g., pectinase, laminarinase, and β-1,3 glucanase) and proteinases (trypsinase and chymotrypsinase) [69]. Interestingly, although the fungus (hyphal extracts) produces chiti‐ nase, the plants do not contain chitin. It has been suggested that the chitinases present in the rectal fluid of primitive genera (*Cyphomyrmex*, *Mycocepurus*, and *Myrmicocrysta*) are important in the degradation of substrates such as the carcasses of leaf-cutting ants, which can be used as a substrate for the growth of new hyphae from the symbiotic fungus. It has also been proposed that these enzymes may play an important role in the lysis of competitive funguses, many of which have cellular walls made of chitin [72].

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**Figure 13.** Synthesis of amides 1-9. Source: Pagnocca et al., 2006 [66].

282 Insecticides - Development of Safer and More Effective Technologies

substrate [68].

(Erthal et al., 2009).

**4. Control of leaf–cutting ants via enzymatic inhibition**

plant material that is transported by the ants to the nest [71].

When the cut plant fragments reach the colony, a complex process of preparation of the plant substrate for its incorporation into the fungal garden begins. During this processing, the workers may ingest the plant's sap while cutting and pressing the borders of the plant fragment. By scraping the surface, they remove the epicuticular wax layer and facilitate the decomposition of the substrate by the fungus [42,67] indicating the importance of the hydro‐ lytic enzymes in this process. This behaviour is also related to the decontamination of the

Ultra-structural studies of the colonisation of the plant substrate by the fungus have demon‐ strated that the fungus can only use the portions that have had their border cut. In addition, the cuticular surface of the leaf at the time of colonisation appears to be intact, which suggests an absence of cutinases (enzymes that catalyse the hydrolysis of cutin, a structural component of the cuticle of the plant) in this process. Therefore, it is believed that this symbiotic fungus is a saprophyte that is unable to penetrate into plants that are not damaged [69] (Figure 13). Therefore, maceration aids in the destruction of the physical barrier of the leaf cuticle, increasing permeability to allow fungal growth, which is assisted by enzymes present in the faecal fluid. [70] The symbiotic fungus is an important mediator involved in providing nutrition to the ants via the hydrolysis of polysaccharides from plant [71] as it produces large amount of enzymes, particularly pectinases, that are ingested by ants, concentrated in the intestine, returned to the fungal garden via faecal fluid, and utilised for the digestion of plant tissues [7]. Therefore, this association is also essential for fungal access to the nutrients in the

The profile of the hydrolytic enzymes involved in this relationship between leaf-cutting ants and fungi has been studied. The extracts from the fungal hyphae of garden fungi exhibit a wide range of activities involving carbohydratases (pectinase, laminarinase, α-glucosidase, βglucosidase, α-galactosidase), with the proteinase chitinase presenting the highest activity

Extracts from fungal gardens contain a wide variety of digestive enzymes, including carbo‐ hydratases (e.g., pectinase, laminarinase, and β-1,3 glucanase) and proteinases (trypsinase and

The results from analysis of the labial glands of larvae and adults of *A. subterraneus* indicate that they secrete chitinases. Chromatographic tests to detect chitinolytic activity in the labial glands of the larvae revealed profiles similar to those observed for the glands of the workers, indicating that this enzymatic activity may have a fungal origin. Interestingly, the peak of chitinolytic activity in the middle intestine of the larvae does not coincide with the peak in the labial gland, indicating that the chitinase produced in the labial gland is not active in the middle intestine [73, 74].

In addition to chitinases, pectinases, xylanases, and cellulases derived from the fungus have been detected in the faecal fluid of *Atta colobica tonsipes* [75]. Pectinases present in the faeces of *Acromyrmex echinatior* and *Atta colombica* have been suggested to be of fungal origin [7]. Interestingly, no pectinase, xylanase, or cellulase activity was detected in *A. subterraneus* adults. However, elevated α-glycosidase activity was detected in fractions from the middle intestine and rectum of adult leaf-cutting ants, indicating the importance of this enzyme in the assimi‐ lation of glucose and, most likely, of saccharose and maltose present in the plant material [73]. Pectinases and xylanases were detected in the middle intestine of the larvae, where their only source of nutrients is the symbiotic fungus [74].

Febvay and Kermarrec (1984) suggested that digestion of the walls of the fungal cells occurs in the infrabuccal pocket of *Acromyrmex octospinosus*. It is possible that the adult garden ants regurgitate chitinases through their labial glands onto the fungal material to feed the larvae, initiating the partial digestion of fungal parts, without the adult benefiting from ingestion. The larvae can regurgitate their own secretions from their labial glands, providing the energetic needs of the adults, who receive these liquids through trophallaxis [76].

Other authors have reported that the secretions offered by the larvae originate in a liquid produced from the anal region, in a process referred to as proctodeal trophallaxis (Figure 14). The workers contact the anal region of the larvae, which may be of different sizes, and the larvae release a small transparent drop that is immediately ingested by the workers. This observation reveals that the larvae of the leaf-cutting ants play a fundamental role in the nutrient flux in the colony because, although adult individuals are incapable of feeding on the solid portions of the fungus, the larvae are able to feed on these portions, digest their walls, and transfer nutrients to the haemolymph, where they are absorbed by the Malpighian tubules, transferred to the posterior intestine, and offered to the workers, making them available to other individuals in the colony [77].

being conducted with the purpose of isolating the substances associated with enzymatic

Data from the literature clearly demonstrate that several plants are capable of producing substances with direct action against leaf-cutting ants and/or their symbiotic fungi, such as ricinine (*Ricinus communis*; Euphorbiaceae); β-eudesmol (*Eucalyptus maculata*; Myrtaceae), the limonoid limonéxico acid (*Raulinoa echinata*; Rutaceae), sesamin and sesamoline (Sesamum indicum; Pedaliaceae), anthranilic acid, kokusaginine and dictamine (*Helietta puberula*; Rutaceae), 4.5- dimetoxicantin-6-one and 5-metoxicantin-6-one (*Simarouba versicolor*; Simar‐ oubaceae), (-)-hinokinin and (-) kusunokin (*Virola sebifera;* Myristicaceae), among others. The active substances extracted from these plants may provide the basis for studies aimed at the synthesis of organic molecules and the development of new commercial products that are stable and show low persistence in the environment. In Brazil, these studies intensified after the establishment of restrictive policies by government entities and certifying institutions regarding the use of the active ingredients that are currently available in the market. Several molecules have already been synthesised, although they are not yet available for use by farmers; however, expectations for the use of plant-derived products in the control of leaf-

To "Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)", "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)", "Fundação de Amparo à Pesquisa do Estado de Goiás (FAPEG)" and "Fundação de Amparo à Pesquisa do Estado de

1 Department of Entomology, Federal University of Lavras (Universidade Federal de Lavras

2 Department of Chemistry, Federal University of Lavras (Universidade Federal de Lavras -

and Dejane Santos Alves1

\*Address all correspondence to: ciencias\_biologicas@hotmail.com

, Denilson Ferreira de Oliveira2

,

Plant–Derived Products for Leaf–Cutting Ants Control

http://dx.doi.org/10.5772/55035

285

inhibition.

**5. Conclusions**

cutting ants are high.

**Acknowledgements**

Minas Gerais (FAPEMIG)".

Juliana Cristina dos Santos1\*, Ronald Zanetti1


UFLA/Lavras), Minas Gerais, Brazil

**Author details**

Giovanna Cardoso Gajo2

**Figure 14.** Colonisation of freshly cut leaves by *Leucoagaricus gongylophorus*. Panel A: LF—leaf fragment; HM—hy‐ phal mass. Magnification: ×100. Panel B: St—stomata; CL—cut leaf edge exposing mesophyll tissue. Magnification: ×200. Panel C: H—hyphae; LS—leaf surface. Magnification: ×1000. Panel D: CL—cut leaf edge; MS—exposed meso‐ phyll tissue. Magnification: ×200. Source: Erthal Jr. et al., 2009 [69].

Hydrolytic enzymes are directly involved in this energy transfer within the colony. Thus, studies that seek new insecticides have been conducted with an emphasis on plant extracts or pure substances that exhibit fungicidal, insecticidal, or enzyme inhibitory actions. The integrated application of these three types of functions should lead to the development of a new product with an effective control capacity. In this context, crude extracts of *Cedrela fissilis*, *Tapirira guianensis*, and *Simarouba versicolor* were evaluated and found to inhibit the activity of the pectinase enzyme present in the faecal liquid of *A. sexdens rubropilosa* [61,78]. These enzymes appear to be essential for the nutrition of the ants and the fungus in plant materials [71].

Plant extracts that inhibit enzymatic activity may be useful for the control of leaf-cutting ants and constitute a new approach with respect to methods for controlling these insects. This type of control should be evaluated further to determine the viability and effectiveness of its use in the field and confirm its suggested potential. Phytochemical analyses of active extracts are being conducted with the purpose of isolating the substances associated with enzymatic inhibition.
