From the analysis of the secretions of adult males of Eurycantha calcarata, 52 compounds were identified: 19 carboxylic acids, 11 esters, 10 alcohols, 5 hydrocarbons, glycerol, monopalmitate, monooleate, monostearin, cholesterol, manoyl oxide and 7,9,-di-teter-butyl-1-oxaspiro(4, 5)deca-6,9-diene-2,8-dione. Differences in the occurrence of compounds in the analyses differ due to the way the secretions were collected and samples were extracted. Table 1. shows the sample number (used in tables below) along with a description of the collection, extraction method and number of identified compounds. Table 2 shows the number of compounds which were identified from each group in each analysis.
Table 1
Samples of the defensive secretion of the adult male of E. calcarata – sample number, used absorbent, method of extraction and number of identified compounds.
Sample number | Absorbent | Amount | Extraction method | Number of identified compounds |
1 | Silica gel (terrarium) | 1g | evaporation with nitrogen + silylating agent | 28 |
2 | Silica gel | 1g | evaporation in the air | 6 |
3 | Silica gel | 1g | evaporation with nitrogen | 5 |
4 | Silica gel | 2g | evaporation in the air | 7 |
5 | Silica gel | 2g | evaporation with nitrogen + silylating agent | 30 |
6 | Activated carbon | 1g | evaporation with nitrogen + silylating agent | 16 |
7 | Activated carbon | 1g | evaporation with nitrogen + silylating agent | 12 |
8 | Activated carbon | 2g | evaporation with nitrogen + silylating agent | 22 |
9 | Activated carbon | 2g | evaporation with nitrogen + silylating agent | 18 |
Table 2
Number of identified hydrocarbons, carboxylic acids, alcohols, esters and other groups of compounds (sample numbers from Table 1.).
Sample number | Absorbent | Amount | Extraction method | Hydrocarbons | Carboxylic acids | Alcohols | Esters | Other compounds |
1 | Silica gel (terrarium) | 1g | evaporation with nitrogen + silylating agent | 3 | 13 | 8 | 1 | 3 |
2 | Silica gel | 1g | evaporation in the air | 2 | 1 | 1 | 2 | 0 |
3 | Silica gel | 1g | evaporation with nitrogen | 1 | 4 | 0 | 0 | 0 |
4 | Silica gel | 2g | evaporation in the air | 3 | 1 | 0 | 2 | 1 |
5 | Silica gel | 2g | evaporation with nitrogen + silylating agent | 5 | 15 | 9 | 0 | 1 |
6 | Activated carbon | 1g | evaporation with nitrogen + silylating agent | 0 | 4 | 2 | 6 | 4 |
7 | Activated carbon | 1g | evaporation with nitrogen + silylating agent | 0 | 11 | 0 | 0 | 1 |
8 | Activated carbon | 2g | evaporation with nitrogen + silylating agent | 0 | 14 | 4 | 2 | 2 |
9 | Activated carbon | 2g | evaporation with nitrogen + silylating agent | 0 | 13 | 4 | 0 | 1 |
From Table 1 and 2 it can be concluded that the addition of a silylating agent increased the detectability of the compounds contained in the defense secretion of this insect. Analysing the presence of hydrocarbons in the defensive secretion of E. calcarata, we observe differences in the number of detected compounds depending on the absorbent that was used. Silica gel turned out to be a better absorbent because it captured more compounds than activated carbon. The amount of the absorbent used did not affect the quality of the analyses. When analysing the compounds detected in the samples, there are not significant differences in the occurrence of carboxylic acids from samples collected with the different absorbents. In samples 2–4 there are less identified acids. There is also no correlation in the amount of detected acids in relation to the amount of absorbent used. This suggests that the amount of absorbents does not affect the quality of the analyses. It is not possible to determine clear dependencies in the presence of alcohols in the defensive secretion of E. calcarata in relation to the used absorbents and their amounts. There are also no clear dependencies in the presence of esters in relation to the amount and type of the absorbent used.
Tables 3–7 shows the identified compounds in each analyses. The most numerous groups of compounds from all the analyses were carboxylic acids (Table 2), 19 different compounds were identified and the most abundant were hexadecanoic acid (Fig. 2) and octadecanoic acid (Table 3). Carboxylic acids have been found in the defensive secretions of insects. In Carabus lefebvrei pupa, carboxylic acids occur in the glandular secretion. Authors claim that the compounds including carboxylic acids found in the pygidial gland secretion have been regarded as the deterrent against predators19. Hexadecanoic acid is an organic chemical compound from the group of fatty acids, a solid substance. Excess carbohydrates are converted into hexadecanoic acid, which is a precursor to the production of longer-chain fatty acids20. It is assumed that the high amount of hexadecanoic acid is related to its function as a precursor for the production of longer chain fatty acids and that it may be involved in metabolic processes leading to the formation of other compounds. Octadecanoic acid, also known as stearic acid, is a white waxy solid with a mild tallow-like odour. Its esters are found in animal fats20. It probably performs the function of storing active substances because it is a solid and active compounds can be suspended in it. Among other insects, hexadecanoic and octadecanoic acids were also present in the highest amount in the defensive secretion of the bug Graphosoma lineatum (Hemiptera; Pentatomidae)21.
Table 3
Percentage content of identified carboxylic acids (sample numbers from Table 1.).
Compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
2-hydroxypropanoic acid | - | - | - | - | - | - | 2,50 | - | - |
Hexanoic acid | 0,35 | - | - | - | - | - | 1,91 | 3,83 | 3,38 |
Heptanoic acid | 0,10 | - | - | - | 0,06 | - | - | 0,76 | 1,01 |
Nonanoic acid | - | 3,29 | 0,83 | 1,91 | 2,38 | - | 7,61 | 8,29 | 9,40 |
Octanoic acid | 0,68 | - | - | - | 0,50 | - | - | 3,06 | - |
Decanoic acid | - | - | - | - | 0,21 | - | 0,24 | 0,55 | 0,53 |
Adipic acid | - | - | - | - | - | - | 1,70 | - | - |
Dodecanoic acid | 0,21 | - | - | - | 0,04 | - | - | 0,20 | 0,31 |
Azelaic acid | - | - | - | - | 0,22 | - | 2,03 | 0,29 | 0,53 |
Tetradecanoic acid | 0,37 | - | 4,91 | - | 0,22 | - | 0,77 | 0,69 | 0,94 |
Pentadecanoic acid | 0,33 | - | - | - | 0,36 | - | 0,53 | 0,82 | 0,79 |
Hexadecanoic acid | 11,82 | - | 24,92 | - | 10,47 | 8,45 | 35,90 | 28,86 | 34,70 |
Heptadecanoic acid | 0,51 | - | - | - | 0,60 | 0,34 | 0,59 | 1,16 | 0,71 |
Octadecanoic acid | 8,55 | - | 10,79 | - | 5,74 | 9,83 | 31,98 | 19,12 | 26,67 |
Eicosanoic acid | 0,55 | - | - | - | 0,62 | 4,95 | - | 0,92 | 0,81 |
Heneicosanoic acid | 0,15 | - | - | - | - | - | - | - | - |
Docosanoic acid | 1,27 | - | - | - | 1,76 | - | - | 1,96 | 2,14 |
Tetracosanoic acid | - | - | - | - | 0,44 | - | - | - | - |
Hexacosanoic acid | 0,42 | - | - | - | 0,20 | - | - | - | - |
Esters were the second most abundant group of compounds in the analyzes (Table 2). 9-hexadecanoic acid octadecyl ester (Fig. 3) and hexadecanoic acid tetradecyl ester had the highest percentage content (Table 4). Little is known about the octadecyl ester of 9-hexadecanoic acid and, so far, it has not been found in the defense secretions of insects. The only mention of its occurrence among these organisms is the record of its presence in Ceratitis capitata both before and during the breeding period22. Hexadecanoic acid tetradecyl ester, or myristyl palmitate, is referred to as a xenobiotic metabolite of bacterial and fungal origin. It comes from tetradecan-1-ol20. The presence of this compound in the secretion of Eurycantha calacarata may indicate the presence and use of endosymbionts (bacteria or fungi) in this insect. The second possibility is its origin as a xenometabolite from host plants and only its accumulation by phasmids. Determining the origin and possible functions of this compound in E. calcarata requires further research. 9-octadecanoic acid 9-octadecyl ester, 9-hexadecanoic acid eicosyl ester, 2,3-hydroxy tetradecanoic acid propyl ester, and hexadecanoic acid octadecyl ester are compounds that are not well understood. There is no information on their occurrence in plants and animals, they have not been identified as compounds found in insects, nor is there information on their occurrence in insect defensive secretions. The presence of these esters was found in only one analysis, but after verification and confirmation of their presence, this would be the first finding of the presence of these substances in insects. The main problem here is to determine whether these are esters that may come from background impurities. Some of the esters identified in the analyses have previously been shown in insects. This includes isopropyl myristate, found in three analyses. It is a polar compound that is used as a moisturizer in topical cosmetics and medical preparations to improve skin absorption. Isopropyl myristate has been extensively researched and used as a skin penetration enhancer20. Among insects, it has been detected in analyses of extracts from the head and the body of the ant Iridomyrmex humilis23,24. Its potential function in the phasmid may be to increase the absorption of irritating substances, thus strengthening their effect on the attacker.
Table 4
Percentage content of identified esters (sample numbers from Table 1.).
Compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Isopropyl laurate | - | 0,15 | - | - | - | - | - | - | - |
Dodecanoic acid 1-methylethylene ester | - | - | - | 0,12 | - | - | - | - | - |
2,2,4-Trimethyl-3-carboxy isopropyl pentanoic acid isobutyl ester | 0,47 | - | - | - | - | - | - | 1,91 | - |
Isopropyl myristate | - | 0,57 | - | 0,64 | - | - | - | 0,21 | - |
2,3-Hydroxy tetradecanoic acid propyl ester | - | - | - | - | - | 2,02 | - | - | - |
Hexadecanoic acid tetradecyl ester | - | - | - | - | - | 5,17 | - | - | - |
9-hexadecanoic acid octadecyl ester | - | - | - | - | - | 7,55 | - | - | - |
Hexadecanoic acid octadecyl ester | - | - | - | - | - | 2,33 | - | - | - |
9-octadecanoic acid, 9-octadecyl ester | - | - | - | - | - | 4,69 | - | - | - |
9-hexadecanoic acid eicosyl ester | - | - | - | - | - | 4,90 | - | - | - |
From the analyses the two most abundant alcohols are octacosanol (Fig. 4) and triacontanol (Table 5). Researchers describe the alcohols found in insect defense secretions as compounds found in smaller amounts and they do not assign them a greater role in it; however, some insects use alcohols for defense, though not directly. The larvae of many beetles Chrysomelidae sequester phenolglucosides such as salicin from their food plants like Salix and Populus spp. Salicin is hydrolysed in the glandular reservoir of the defense glands. The resulting salicylic alcohol (saligenin) is oxidized by extracellular oxidase. The product, salicylaldehyde, accumulates as the main defense compound of these beetles25. It seems that alcohols may be used in a similar way by Eurycantha calcarata, but this requires further research and determining whether they are only accumulated, or whether they are subject to further metabolic transformations. Octacosanol is a straight-chain aliphatic fatty alcohol that is used as a nutritional supplement. This organic compound is the main component of a natural product wax extracted from plants. Octacosanol is reported to possess cholesterol-lowering effects, antiaggregatory properties, cytoprotective use, and ergogenic properties. It has been studied as a potential therapeutic agent for the treatment of Parkinson's disease20. Triacontanol is an ultra-long-chain primary fatty alcohol20 that has shown significant inhibition of feeding activity against the insects, Spilosoma obliqua and Spodoptera litura26. It is possible that insects use it as a deterrent.
Table 5
Percentage content of identified alcohols (sample numbers from Table 1.).
Compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Dodecanol | 0,21 | 0,88 | - | - | 0,22 | - | - | 0,33 | 0,22 |
Tetradecanol | 0,14 | - | - | - | 0,21 | - | - | - | - |
Hexadecanol | 0,33 | - | - | - | 0,37 | 0,16 | - | 0,63 | 0,42 |
Oktadecanol | - | - | - | - | 0,29 | 2,58 | - | - | - |
Eicosanol | 0,06 | - | - | - | - | - | - | - | - |
Docosanol | 0,47 | - | - | - | 0,59 | - | - | - | - |
Hexacosanol | 0,78 | - | - | - | 1,37 | - | - | - | - |
Octacosanol | 8,97 | - | - | - | 12,77 | - | - | 10,64 | 8,21 |
Triacontanol | 5,98 | - | - | - | 8,38 | - | - | 5,99 | 3,62 |
Dotriacontanol | - | - | - | - | 2,24 | - | - | - | - |
The hydrocarbons occurring in the largest amounts turned out to be tridecane (Fig. 5) and tetradecane (Table 6); although these two compounds were present in the control sample, they were more than twice as high in the defensive secretion samples (Table 6). It is assumed that in a closed terrarium where insects are living, volatile compounds emitted by them will also be present in the atmosphere of the insectarium. Tridecane is a straight-chain alkane with 13 carbon atoms and it is a component of essential oils isolated from plants. In its pure form it is an oily, yellow, transparent liquid with a characteristic smell. Repeated or prolonged contact of tridecane with the skin may irritate it and cause redness, leading to inflammation. Exposure to high vapour concentrations can cause headache and stupor20. In insects tridecane occurs in the volatile secretion of the bug Cosmopepla lintneriana (Hemiptera; Pentatomidae)27. The Pentatomidae family are known for using volatile odorants as a predator deterrent, which they release when threatened28. The secretion of this bug, consisting mainly of tridecane, acts as a deterrent to birds and lizards27. Tridecane was also found in Graphosoma lineatum secretions. It has been identified as a toxic, irritant or repellent compound, that is released by these bugs in response to irritation, and it is believed that the secretion of this compound by the insect also acts as a predator deterrent21. This alkane was also found in the secretion from the metatoracal glands of another Pentatomide species – Piezodorus guildinii, where it performed the same functions. Tridecane is known as a toxin, irritant or repellent released by Pentatomidae in response to irritation. This suggests that this compound acts as a chemical defense for these species29. Tridecane was present in both sexes in the defensive secretion of Parastizopus transgariepinus30. Chemioreaction experiments shows that tridecan was attractive to females but not males, which indicates that tridecane could also be a sex pheromone. Tetradecane is a straight chain alkane consisting of 14 carbon atoms. It is a natural compound found in plants in essential oils, known to be s secondary plant metabolite20. Interestingly, it has recently been shown31 that the amount of volatile tetradecane in maize roots increases after the invasion of the larvae of the beetle Holotrichia parallela (Melolonthidae). These studies show that tetradecane can act as a signal to induce defenses and prepare plants for the following attacks, including metabolic changes31. It was also shown that the growth of H. parallela larvae was inhibited when fed with maize roots after exposure to tetradecane. This compound can be extracted from plants and used by E. calcarata as a compound to deter potential predators. There is also the possibility that tetradecane is being used as a signal of impending danger to other individuals. Both tridecane and tetradecane were also present in the protective secretion of ground beetles (Carabidae) of the following genera: Brachinus, Stenaptinus, Metrius, Goniotropis, Pachyteles, Ozaena and Homopterus32.
Table 6
Percentage content of identified hydrocarbons (sample numbers from Table 1.).
Compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Dodecane | 0,68 | - | - | 1,76 | 0,83 | - | - | - | - |
Tridecane | 0,93 | 2,62 | - | 2,95 | 1,33 | - | - | - | - |
Tetradecane | 21,71 | 59,86 | 23,39 | 66,05 | 34,14 | - | - | - | - |
Heptacosane | - | - | - | - | 0,47 | - | - | - | - |
Hentriaceontane | - | - | - | - | 0,97 | - | - | - | - |
Table 7
Percentage content of identified compounds from other groups of compounds (sample numbers from Table 1.)y.
Compound | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
Glycerol | 6,11 | - | - | - | 3,63 | 0,48 | 14,24 | 3,34 | 4,41 |
7,9,-di-teter-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione | - | - | - | - | - | - | - | 1,12 | - |
Manoyl oxide | - | - | - | 0,54 | - | - | - | - | - |
Monopalmitate | 1,36 | - | - | - | - | 9,81 | - | - | - |
Monooleate | - | - | - | - | - | 2,82 | - | - | - |
Monostearin | 0,16 | - | - | - | - | - | - | - | - |
Cholesterol | - | - | - | - | - | 21,44 | - | - | - |