The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis  and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus

Agata Kaczmarek; Mieczysława Irena Boguś; Emilia Włóka; Anna Katarzyna Wrońska; Anna Krawiel; Michalina Kazek; Katarzyna Zalewska; Katarzyna Kłocińska-Biały; Martyna Sobocińska; Aleksandra Gliniewicz; Ewa Mikulak; Marta Matławska

doi:10.1371/journal.pone.0235785

. 2020 Jul 9;15(7):e0235785. doi: 10.1371/journal.pone.0235785

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus

Agata Kaczmarek ^1,^*, Mieczysława Irena Boguś ^1,², Emilia Włóka ¹, Anna Katarzyna Wrońska ¹, Anna Krawiel ², Michalina Kazek ¹, Katarzyna Zalewska ², Katarzyna Kłocińska-Biały ², Martyna Sobocińska ², Aleksandra Gliniewicz ³, Ewa Mikulak ³, Marta Matławska ³

Editor: Ahmed Ibrahim Hasaballah⁴

PMCID: PMC7347226 PMID: 32645074

Abstract

The interactions between entomopathogenic fungi and insects serve a classic example of a co-evolutionary arms race between pathogens and their target host. The cuticle, site of the first contact between insects and entomopathogenic fungus, is an important defensive barrier against pathogens. It is covered by a layer of lipids that appears to play a key role in these processes and cuticular free fatty acid (FFA) profiles are consider as a determinant of susceptibility, or resistance, to fungal infections. These profiles are species-specific. The cockroaches Blattella germanica (Blattodea: Blattidae) and Blatta orientalis (Blattodea: Ectobiidae) are unsusceptible to the soil fungus Conidiobolus coronatus (Entomophthorales: Ancylistaceae) infection, therefore we studied the profiles of FFAs in order to understand the defensive capabilities of the cockroaches. The fungus was cultivated for three weeks in minimal medium. Cell-free filtrate was obtained, assayed for elastase, N-acetylglucosaminidase, chitobiosidase and lipase activity, and then used for in vitro hydrolysis of the cuticle from wings and thoraces of adults and oothecae. The amounts of amino acids, N-glucosamine and FFAs released from the hydrolysed cuticle samples were measured after eight hours of incubation. The FFA profiles of the cuticle of adults, and the wings, thoraces and oothecae of both species were established using GC-MS and the results were correlated with the effectiveness of fungal proteases, chitinases and lipases in the hydrolyzation of cuticle samples. Positive correlations would suggest the existence of compounds used by the fungus as nutrients, whereas negative correlations may indicate that these compounds could be engaged in insect defence.

Introduction

Insect populations are regulated in part by the activity of entomopathogens. Entomopathogenic fungi are proposed as an eco-friendly alternative to chemical insecticides and as model organisms to study insect infection [1–4]. Unlike bacteria or viruses, fungi infect insects by direct penetration of the cuticle, followed by multiplication in the hemocoel [5].

Infection by entomopathogenic fungi is a multi-stage process comprising adhesion of fungal spores to the insect cuticle, germination and the penetration of invasive hyphae into the host body, hyphae propagation inside the hemocoel and colonization of the host internal organs, followed by the release of toxic secondary metabolites, which might result in host death [6]. The fungus penetrates the insect cuticle by a combination of mechanical pressure from growing hyphae and the enzymatic degradation of the proteins, chitin and lipids comprising the cuticle: proteases are produced first, followed by chitinases and lipases [7,8].

Two key factors influencing the infection process are the structure and composition of the host exoskeleton, and the efficiency of the immune system. Since the cuticle is the first point of contact between the insect and fungus, it is the first and most decisive defence mechanism in insects, and its composition varies greatly according to the species and the developmental stage [9–13]. This complex structure is covered by a waxy layer rich in lipids which play a key role in resistance to entomopathogenic fungi [8,14]. However, although many cuticular lipids have antimicrobial properties, other stimulate the germination process, growth and virulence of fungi; and variations in lipid profiles between species are reflected in differential susceptibility to infection [15–21]

The fungal proteases, chitinases and lipases used to degrade cuticle components play crucial roles in the infection process and are known to act in a coordinated fashion [5,22–24]. Some cuticular proteins display protease inhibition, and protect the insect by suppressing conidial germination and penetration [25,26]. Although no lipase and chitinase inhibitors have been identified in the cuticle so far, several natural chitinase and lipase inhibitors, mostly of microbial origin, have been described [9,27]. Further studies might bring more information on the presence of substances tempering the activity of fungal chitinases and lipases in the insect cuticle.

Previous studies on four medically-important fly species (Lucilia sericata, Calliphora vicina, Calliphora vomitoria and Musca domestica) identified correlations between the efficiency of cuticle digestion by fungal enzymes and the content of cuticular free fatty acids (FFAs), free fatty acid methyl esters (FAME), fatty alcohols, n-alkanes, sterols and several non-typical compounds [28].

The German cockroach (B. germanica), and the oriental cockroach (B. orientalis) are two of the most common species of cockroaches worldwide. They usually reside in human habitats, where they act as hosts for parasites, viruses, bacteria and pathogenic fungi and can cause severe allergic reactions in humans [29–32]. As these insects are difficult to eradicate, due to their high rates of reproduction and resistance to commonly-used pesticides, biological control strategies based on the use of entomopathogenic fungi are becoming an increasingly desirable option [33–35].

The aim of the present work was to identify any relationships between the cuticular FFA profiles of two cockroach species, B. orientalis and B. germanica, and the efficiency of fungal enzymes in hydrolysing the insect cuticle.

Results

Susceptibility of cockroaches to fungal infection

Exposure of B. orientalis and B. germanica imagines and oothecae to sporulating C. coronatus colonies showed high resistance of both cockroach species to fungal infection. No infection or mortality was observed in either the control or fungus-threated groups of B. orientalis. Mortality of fungus treated B. germanica was very low and comparable to the control groups (Table 1 and S1 Table).

Table 1. The susceptibility of B. orientalis and B. germanica to fungal infection.

Insect treatment		Tested object	N	Mortality [%±SD]
B. orientalis	control	imago	20	0 ± 0
	control	ootheca	10^*	0 ± 0^**
	fungal infection	imago	30	0 ± 0
	fungal infection	ootheca	30^*	0 ± 0^**
B. germanica	control	imago	25	4 ± 8
	control	ootheca	25^*	4 ± 4^**
	fungal infection	imago	30	3 ± 8
	fungal infection	ootheca	25^*	8 ± 5^**

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The insects were exposed to sporulating C. coronatus colonies as described in Materials and methods. The susceptibility to fungal infection is expressed as percentage of mortality in tested populations.

* the number of tested oothecae each containing on average 16 eggs (B. orientalis) and 40 eggs (B. germanica), respectively.

** the total percentage of larvae which died during the three days after hatching from oothecae. Percentage of larvae hatching from control and fungus exposed oothecae was 100% in both species (for raw data see supplementary S1 Table).

Enzyme activity in cell-free C. coronatus filtrate

The proteolytic, chitinolytic and lipolytic activities of the cell-free C. coronatus filtrate of were measured as described in the Materials and methods section. The highest activity was demonstrated by elastase (55.31±21.83 mM/min/mg protein): 24-times higher than NAGase (2.32±1.55 mM/min/mg protein; P = 0.0028, F_(3,6) = 196.60), 553-times higher than chitobiosidase (0.10±0.04 mM/min/mg protein; P = 0.0023, F_(3,6) = 258083.00), and 1844-times higher than lipase (0.03±0.001 mM/min/mg protein; P = 0.0023, F_(3,6) = 3411192.00).

Hydrolysis of cuticular proteins by C. coronatus enzymes

The effectiveness of fungal proteolytic enzymes in the culture medium during the 3 weeks of the C. coronatus in vitro cultivation was measured as the amounts of amino acids released from insect cuticle preparations. The greatest amounts of amino acids were produced during enzymatic digestion of B. germanica oothecae (978.29±45.49 μM/mg cuticle), and the least (127.86±52.69 μM/mg cuticle F_{(5, 12)} = 14.37, p = 0.0001) for B. orientalis oothecae. Higher concentrations of amino acids were released from the thoraces, wings and imago of B. germanica than B. orientalis. Also 7.7-times more amino acids were released from B. germanica oothecae than B. orientalis. Results are given in Fig 1 and supplementary S2 Table.

Fig 1 — Amino acid released during eight hours of incubation is presented as mean ± standard deviation μm/mg of cuticle from wings, thoraces and oothecae of the two cockroach species. Statistically significant differences are marked with the same letter (ANOVA, Tukey's HSD test, p<0.05, for raw data see S2 Table: protein).

Hydrolysis of cuticular chitin by C. coronatus enzymes

The effectiveness of hydrolysis by the C. coronatus chitinolytic enzymes was found to be similar in all samples, measured as the concentration of N-glucosamine (Fig 2 and S2 Table). The highest levels of N-glucosamine were observed for B. orientalis wings (66.70±0.80 μM/mg cuticle), and the lowest (43.49±3.80 μM/mg cuticle; F_(5,12) = 4.98, p = 0.0106) for B. orientalis thoraces.

Fig 2 — N-glucosamine released during eight hours of incubation is presented as mean ± standard deviation μm/mg of cuticle from wings, thoraces and oothecae of the two cockroach species. Statistically significant differences are marked with the same letter (ANOVA, Tukey's HSD test, p<0.05, for raw data see S2 Table: chitin).

Hydrolysis of cuticular lipids by C. coronatus enzymes

The release of free fatty acids, indicating lipolytic activity, was observed only after digestion of B. germanica oothecae (0.09±0.13 μM/mg cuticle), B. orientalis oothecae (0.02±0.03 μM/mg cuticle) and B. orientalis thoraces (0.15±0.16 μM/mg cuticle) (Fig 3 and S3 Table). These differences were not statistically significant (F_{(5, 12) =} 1.14, p = 0.3918).

Fig 3 — Free fatty acids released during eight hours of incubation are presented as mean ± standard deviation μm/mg of cuticle from wings, thoraces and oothecae of the two cockroach species (for raw data see S2 Table: lipid).

GC-MS analyses of cuticular FFAs

In almost all cases, significantly higher cuticular FFAs were extracted from B. germanica than B. orientalis: whole body extracts: 2.6 vs. 1.1 mg/g body; wings extracts: 52.8 vs. 11.4 mg/g wing; thoraces extracts: 30.1 vs. 1.8 mg/g thorax (Table 2). However, for the oothecae, FFA concentrations were 6.3 mg/g for B. germanica vs. 8.1 mg/g for B. orientalis. In both species, the highest levels of substances were found in the wings and thoraces.

Table 2. Numbers of B. orientalis and B. germanica used here and mass of the extracts.

Extracts made from:	N	Insect/body part mass (g)	Extract mass (mg)
Extracts made from:	N	Insect/body part mass (g)	I	II	III
B. orientalis
whole insects	12	5.98	5.61	0.95	5,40
oothecae	80	1.24	5.49	4.60	4.77
wings	539	0.88	6.84	3.20	3.21
thoraces	274	1.26	1.21	1.11	2.26
B. germanica
whole insects	100	6.32	14.22	2.54	36.25
oothecae	50	1.69	5.38	5.27	6.60
wings	377	0.27	9.25	5.00	9.67
thoraces	186	0.07	1.86	0.25	0.75

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N–total number of individuals; I–petroleum ether extract; II–dichloromethane extract; III–dichloromethane extract after sonification

Both species yielded low FFA content in whole body extracts: 5.46±0.70 μg/g b.w. for B. orientalis and 5.42±0.65μg/g b.w. for B. germanica. These differences increased for individual body parts (Table 3 and S3 Table).

Table 3. Fatty acid contents in the cuticular lipids (sum of I and II extracts) extracted from the whole adults, oothecae, wings and thoraces of B. orientalis and B. germanica (μg/g of insect body or structure) and their antifungal activity.

FFA	Antifungal activity^*	adults		oothecae		wings		thoraces
FFA	Antifungal activity^*	B. orientalis	B. germanica	B. orientalis	B. germanica	B. orientalis	B. germanica	B. orientalis	B. germanica
Butanoic acid C4:0	NDT	NDC ^A	0.04±0.02 ^A,B,C,DE,F,G	NDC ^B	NDC ^C	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Pentanoic acid C5:0	NDT	NDC ^A	0.04±0.00 ^{A,B,C,D,E,F,G}	NDC ^B	NDC ^C	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Hexanoic acid C6:0	YES	0.10±0.04 ^A,F	0.43±0.56 ^B,G	0.36±0.12 ^C,H	0.88±0.07 ^D,I	4.32±0.16 ^A,B,C,D,E	14.58±0.36 ^A,B,C,D,J	0.63±0.05 ^E,J	13.93±0.60 ^E,F,G,H,I
Heptanoic acid C7:0	NDT	NDC ^A,B,C	NDC ^D,E,F	0.20±0.07 ^A,E,O,G,H	0.14±0.01 ^B,F,I,J,K	NDC ^O,I,L	NDC ^G,J,M	NDC ^H,K,N	0.12±0.06 ^C,D,L,M,N
Octanoic acid C8:0	NDT	0.11±0.01 ^A,C	0.14±0.01 ^B,D	0.56±0.19 ^E,F	0.47±0.02 ^G,H	2.07±0.06 ^{A,B,C,D,E,F,G,H,I}	7.32±0.93 ^A,B,E,G,I	0.35±0.00 ^I,R	6.24±0.86 ^C,D,F,H,O,R
Nonanoic acid C9:0	NDT	0.29±0.01 ^A	0.23±0.02 ^B	1.01±0.37 ^C	0.82±0.05 ^D	3.84±0.24 ^A,B,C,D,E	15.12±0.77 ^A,B,C,D,E,	0.40±0.04 ^E	17.18±1.06 ^A,B,C,D,E
Decanoic acid C10:0	NDT	0.05±0.04 ^A	0.03±0.01 ^B	0.09±0.02 ^C	0.08±0.01 ^D	0.19±0.08 ^E	NDC ^F	0.03±0.01 ^G	3.30±2.90 ^{A,B,C,D,E,F,G}
Dodecanoic acid C12:0	NDT	0.19±0.00 ^A	0.10±0.01 ^B	0.74±0.02 ^C	0.29±0.03 ^D	1.65±0.23 ^A,B,C,D,E	12.66±0.63 ^A,B,C,D,E	0.29±0.02 ^E	13.89±0.37 ^A,B,C,D,E
Tridecanoic acid C13:0	YES	NDC ^A	NDC ^B	0.18±0.00 ^{A,B,C,D,E,F,G}	NDC ^C	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Tetradecenoic acid C14:1	YES	NDC ^A	NDC ^B	NDC ^C	0.02±0.00 ^{A,B,C,D,E,F,G}	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Tetradecanoic acid C14:0	NDT	0.15±0.03 ^A,B	0.09±0.01 ^C,D	3.74±0.34 ^A,C,E,F	0.28±0.01 ^E,G,H	3.65±0.18 ^B,D,G,I,	71.18±1.28 ^A,C,G,J	0.78±0.02 ^F,I,J	85.30±0.78 ^A,C,H,I,
Pentadecenoic acid C15:1	NDT	NDC ^A	NDC ^B	NDC ^C	0.04±0.00 ^{A,B,C,D,E,F,G}	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Pentadecanoic acid C15:0	SLIGHT	0.03±0.00 ^A,B	0.03±0.01 ^C,D	1.70±0.11 ^A,C,E,F	0.10±0.01 ^E,G,H	1.77±0.12 ^B,D,G,I	8.08±0.22 ^A,C,G,J	0.32±0.01 ^F,I,J	9.48±0.34 ^A,C,H,I
Hexadecenoic acid C16:1	NDT	NDC ^A	0.06±0.03 ^B	6.43±1.68 ^C	0.50±0.04 ^D	2.89±0.20 ^E	297.38±4.77 ^A,B,C,D,E,J	0.98±0.02 ^J	331.30±7.27 ^A,B,C,D,E,J
Hexadecanoic acid C16:0	NDT	2.17±0.22 ^A,B	1.30±0.73 ^C,D	106.74±12.43 ^A,C,E,F	7.41±0.51 ^E,G	96.01±20.83 ^B,D,G,H	2888.11±20.58 ^A,C,G,H	16.75±0.05 ^F,H	3229.99±43.68 ^A,C,G,H
Heptadecenoic acid C17:1	SLIGHT	NDC ^A	NDC ^B	1.27±0.15 ^C	0.04±0.01 ^D	0.95±0.11 ^E	14.28±1.22 ^A,B,C,D,E,J	0.45±0.02 ^J	20.93±0.99 ^A,B,C,D,E,J
Heptadecanoic acid C17:0	NO	0.03±0.01 ^A,B	0.47±0.74 ^D	1.57±0.13 ^C	0.12±0.00 ^F,E	2.46±0.07 ^A,F	29.16±1.46 ^B,D,C,E,G	0.56±0.02 ^G	36.74±1.18 ^B,D,C,E,G
Octadecatrienoic acid C18:3	SLIGHT	NDC ^A	NDC ^B	9.10±1.66 ^A,B,C,D,E,F	2.12±0.25 ^A,B,C,D,E,F	NDC ^C	NDC ^D	NDC ^E	NDC ^F
Octadecadienoic acid C18:2	NDT	0.38±0.11 ^A	0.50±0.01 ^B	78.74±20.91 ^C	1.07±0.02 ^A,D	120.56±1.44 ^A,B,E	1700.88±62.15 ^A,B,C,D,E	30.43±0.53 ^E	2437.16±51.51 ^A,B,C,D,E
Octadecenoic acid C18:1	NDT	0.56±0.03 ^A	0.95±0.04 ^B	211.05±52.85 ^A,B,C,D	3.89±0.20 ^C,E	195.24±2.34 ^A,B,E,F	4542.11±52.74 ^A,B,D,E	38.29±1.71 ^D,F	5300.31±45.7 ^A,B,C,F
Octadecanoic acid C18:0	NDT	0.80±0.13 ^A	0.90±0.07 ^B	44.18±11.20 ^A,B,C,D	NDC ^C	79.08±14.47 ^A,B,C,D	808.21±12.00 ^A,B,C,D	12.32±0.12 ^D	863.89±13.33 ^A,B,C,D
Nonadecenoic acid C19:1	SLIGHT	NDC ^A	NDC ^B	NDC ^C	NDC ^D	NDC ^E	NDC ^F	0.56±0.07 ^{A,B,C,D,E,F,G}	NDC ^G
Nonadecanoic acid C19:0	NDT	NDC ^A	NDC ^B	0.46±0.09 ^C	NDC ^D	NDC ^E	NDC ^F	NDC ^G	4.79±1.36 ^{A,B,C,D,E,F,G}
Eicosatetraenoic acid C20:4	YES	NDC ^A	NDC ^B	NDC ^C	NDC ^D	4.80±0.19 ^E	116.51±4.00 ^A,B,C,D,E,F	2.38±0.06 ^F	208.87±5.99 ^A,B,C,D,E,F
Eicosatrienoic acid C20:3	SLIGHT	NDC ^A	NDC ^B	0.51±0.86 ^C	NDC ^D	5.14±0.12 ^A,B,C,D,E	10.02±1.52 ^A,B,C,D,E	0.35±0.13 ^E	28.01±1.67 ^A,B,C,D,E
Eicosadienoic acid C20:2	SLIGHT	NDC ^A	NDC ^B	NDC ^C	NDC ^D	NDC ^E	8.73±5.73 ^A,B,C,D,E,F	NDC ^F	7.53±6.55
Eicosenoic acid C20:1	SLIGHT	NDC ^A	NDC ^B	NDC ^C	NDC ^D	NDC ^E	36.99±4.61 ^A,B,C,D,E,F	NDC ^F	18.83±2.17 ^A,B,C,D,E,F
Eicosanoic acid C20:0	NO	NDC ^A	0.03±0.00 ^B	1.78±0.24 ^A,B,C,D	0.25±0.01 ^C	4.35±0.41 ^A,B,C,D	14.11±0.37 ^A,B,C,D	0.50±0.03 ^D	15.23±0.44 ^A,B,C,D
Docosanoic acid C22:0	NDT	0.13±0.06 ^A	0.04±0.00 ^B	2.43±1.42 ^A,B,C,E,F	0.46±0.01 ^C	1.44±0.20 ^D	NDC ^E	0.33±0.11 ^F	11.39±0.50 ^{A,B,C,D, E,F}
Tetracosanoic acid C24:0	NO	0.23±0.10 ^A,G	0.05±0.00 ^B,H	5.08±1.31 ^A,B,C,D,E,F	0.43±0.02 ^C,I	NDC ^D,J	NDC ^E,K	NDC ^F,L	6.26±0.58 ^G,H,I,J,K,L
Hexacosanoic acid C26:0	NO	0.24±0.06 ^A	NDC ^B	2.82±1.62 ^A,B,C,D,E,F	0.38±0.02 ^C	NDC ^D	NDC ^E	NDC ^F	5.15±0.75 ^A,B,C,D,E,F
Sum of FFA		5.46±0.70 ^A,B,	5.42±0.65 ^C,D	481.80±134.56 ^A,C,E,F	19.89±1.02 ^E,G	530.40±34.94 ^B,D,G,H	10595.44±7.97 ^A,B,C,D,G,H	106.71±1.28 ^F,H	12675.83±45.53 ^A,B,C,D,G,H

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FFA- free fatty acids. SD—standard deviation; Extract I- petroleum ether extract; Extract II- dichloromethane extracts; NDT–not determined; NDC–not detected; statistically significant differences are marked with the same letters (ANOVA, Test HSD Tukey, p<0.05), for raw data see supplementary S3 Table

*data from [18,36]. Tested fungal species: Aspergillus niger, Beauveria bassiana, Candida albicans, Candida lipolytica, Candida tropicalis, Lecanicillium lecanii, Metarhizium anisopliae, Paecilomyces fumosoroseus, Peacilomyces lilacinus

The highest total FFA level was observed in B. germanica wings (10595.44±7.97 μg/g) and thoraces (12675.83±45.53 μg/g). In contrast, 20 and 120-times lower FFA content was found in B. orientalis wings (530.40±34.94 μg/g) and thoraces (106.71±1.28 μg/g) (F_(7,16) = 31770.00, p<0.0001). Regarding the oothecae, the total FFA content of B. orientalis (481.80±134.56 μg/g) was 24 times higher than B. germanica (19.89±1.02 μg/g).

Individual FFAs present in each extract were identified and quantified. Example mass spectra of the trimethylsilyl (TMS) esters of hexadecenoic acid (C16:0) and hexadecenoic acid (C16:1) are given in Fig 4.

Fig 4 — Mass spectra of the trimethylsilyl (TMS) ester of hexadecenoic acid (A) and hexadecanoic acid (B).

In the cuticular extracts of adult B. orientalis, fifteen FFAs were detected: thirteen saturated (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C22:0, C24:0, C26:0), and two unsaturated (C18:2 and C18:1). In the oothecae, twenty three acids were found: seventeen saturated (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C19:0, C20:0, C22:0, C24:0. C26:0) and six unsaturated (C16:1, C17:1, C18:2, C18:1, C18:3, C20:3). Eighteen were demonstrated in wings cuticle: twelve saturated (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C22:0) and six unsaturated (C16:1, C17:1, C18:2, C18:1, C20:4, C20:3). Nineteen FFAs were found in the thoraces extract: 12 saturated (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C22:0) and seven unsaturated (C16:1, C17:1, C18:2, C18:1, C19:1, C20:4, C20:3). Of these C7:0, C13:0, C18:3, and C19:0 were detected only in the oothecae, while C16:1, C17:1, C20:3 and C20:0 were absent from the whole B. orientalis adults cuticular extract but were found in extracts from their oothecae, wings and thoraces.

Eighteen FFAs were found in cuticular extracts from B. germanica adults (whole body): fifteen saturated (C4:0, C5:0, C6:0, C8:0, C9:0 C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C22:0, C24:0) and three unsaturated (C16:1, C18:2, C18:1). C4:0, C5:0, C16:1, C20:0 were absent in analogical extracts from B. orientalis, while C26:0 present in B. orientalis was absent from B. germanica. Twenty-one FFAs were found in B. germanica oothecae: seventeen saturated (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C20:0, C22:0, C24:0, C26:0) and seven unsaturated (C14:1, C15:1, C16:1, C17:1, C18:2, C18:1, C18:3). C14:1, C15:1 and C18:3 were found only in the oothecae while C7:0 and C26:0 were found in both oothecae and thoraces. Comparing the oothecae of the two species, C14:1 and C15:1 were found only in B. germanica while C13:0, C18:0, C19:0 and C20:3 were only present in B. orientalis. Most oothecae FFAs common to both species were present in significantly different amounts (Table 3). Eighteen FFAs were found on the wings cuticle: ten saturated (C6:0, C8:0, C9:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0) and eight unsaturated (C16:1, C17:1, C18:2, C18:1, C20:4, C20:3, C20:2, C20:1). Interestingly, the C10:0 missing in B. germanica wings was detected in other B. germanica extracts, as well as in B. orientalis wings. C20:1, C20:2 and C22:0 were absent in B. orientalis but present in B. germanica. All shared FFAs were present in significantly higher amounts in B. germanica, ranging from 1.9 (C20:3) to 102.9 times (C16:1). Twenty-four FFAs were detected in the B. germanica thoraces: sixteen saturated (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C19:0, C20:0, C22:0, C24:0, C26:0) and eight unsaturated (C16:1, C17:1, C18:2, C18:1, C20:4, C20:3, C20:2, C20:1). C19:1 was present only in B. orientalis thoraces but no other extract. C7:0, C19:0, C20:1, C20:2, C24:0 and C26:0 were absent from B. orientalis but present in B. germanica thoraces. All common FFAs were significantly more abundant in B. germanica: 17.8 (C8:0) to 338 times (C16:1).

Four FFAs predominated in all cuticular extracts: C16:0, C18:2, C18:1 and C18:0 (Table 3). C16:0 predominated in whole insect extracts in both species (2.17±0.22 μg/g bw B. orientalis; 1.30±0.73 μg/g bw B. germanica). C16:0 was strong in B. germanica wings (2888.11±20.58 μg/g wings) and thoraces (3229.99±43.68 μg/g thoraces). C18:1 predominated in B. orientalis oothecae, (211.05±52.85 μg/g), wings (195.24±2.34 μg/g) and thoraces (38.29±1.71 μg/g), as well as in B. germanica wings (4542.11±52.74 μg/g) and thoraces (5300.31±45.7 μg/g thoraces). The wings and thoraces of B. germanica also demonstrated high concentrations of C18:0 (808.21±12.00 μg/g wings, 863.89±13.33 μg/g thoraces) and C18:2 (1700.88±62.15 μg/g wings, 2437.16±51.51 μg/g thoraces).

GC-MS analyses of internal FFAs

In almost all cases, significantly higher amounts were extracted from B. germanica than from B. orientalis. The yields were obtained from B. germanica whole body (61.51±3.53 μg/g) were17-times greater than for B. orientalis (3.62±0.17 μg/g) (Table 4 and S3 Table). A similar situation was observed for the wings (197.34±1.80 μg/g B. orientalis vs. 2291.14±31.31 μg/g B. germanica) and thoraces (266.96±1.44 μg/g B. orientalis vs. 1570.31±11.94 μg/g B. germanica); the opposite was found for oothecae (163.24±23.69 μg/g B. orientalis vs. 41.21±5.31 μg/g B. germanica) (Table 4). Fifteen FFAs were detected in B. orientalis adult whole body extracts: thirteen saturated (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C16:0, C17:0, C18:0, C20:0, C22:0, C24:0, C26:0) and two unsaturated (C18:2, C18:1). Similarly, fifteen FFAs were found in B. germanica adults, but twelve were saturated (C5:0, C6:0, C7:0, C8:0, C9:0 C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0) and three were unsaturated (C16:1, C18:2, C18:1). C5:0 was found only in the body of B. germanica, not in any other analysed internal lipid fraction. Most shared FFAs were more abundant in adult B. germanica than B. orientalis.

Table 4. Fatty acid contents in the internal lipids (extract III) of the adults, oothecae, wings and thoraces of B. orientalis and B. germanica (μg/g of insect body or structure) and their antifungal activity.

FFA	Antifungal activity^*	adults		oothecae		wings		thoraces
FFA	Antifungal activity^*	B. orientalis	B. germanica	B. orientalis	B. germanica	B. orientalis	B. germanica	B. orientalis	B. germanica
Butanoic acid C4:0	NDT	NDC	NDC	NDC	NDC	NDC	NDC	NDC	NDC
Pentanoic acid C5:0	NDT	NDC ^A	0.18±0.06 ^A,B,C,D,E,F	NDC ^B	NDC ^C	NDC ^D	NDC ^E	NDC ^E	NDC ^F
Hexanoic acid C6:0	YES	0.07±0.02 ^A,B	0.94±0.38 ^A,C,	0.33±0.13 ^D	0.49±0.04 ^E	1.41±0.09 ^B,D,E,F	10.82±0.49 ^B,C,D,E,F	0.66±0.04 ^F	6.81±0.29 ^B,C,D,E,F
Heptanoic acid C7:0	NDT	NDC ^A	0.18±0.04 ^B	0.06±0.01 ^C	0.08±0.03 ^D	NDC ^E	NDC ^F	NDC ^G	0.81±0.39 ^{A,B,C,D,E,F,G}
Octanoic acid C8:0	NDT	0.05±0.03 ^A	0.31±0.05 ^B	0.20±0.06 ^C	0.18±0.05 ^D	0.61±0.02 ^E	3.32±0.2 ^A,B,C,D,E,F	0.38±0.01 ^F	2.26±0.31 ^A,B,C,D,E,F
Nonanoic acid C9:0	NDT	0.08±0.02 ^A,B,C	1.06±0.08 ^A	0.42±0.06 ^D	0.75±0.09 ^B	0.87±0.06 ^C	8.20±0.50 ^A,B,C,D,E	0.50±0.03 ^E	4.15±0.32 ^{A,B, C,D,E}
Decanoic acid C10:0	NDT	0.02±0.00	0.08±0.07	0.09±0.03	0.05±0.01	NDC	NDC	0.09±0.04	NDC
Dodecanoic acid C12:0	NDT	0.12±0.02 ^A	0.30±0.01 ^B,C	0.35±0.03 ^D,E	0.23±0.03 ^F,G	0.54±0.08 ^A	2.09±0.34 ^A,B,D,F,H,L	0.34±0.00 ^H,I	2.27±0.13 ^A,C,E,G,I,M
Tridecanoic acid C13:0	YES	NDC	NDC	NDC	NDC	NDC	NDC	NDC	NDC
Tetradecenoic acid C14:1	YES	NDC	NDC	NDC	NDC	NDC	NDC	NDC	NDC
Tetradecanoic acid C14:0	NDT	0.04±0.01 ^A,B,C,D	0.80±0.02 ^A	1.17±0.19 ^B	0.40±0.08 ^B,E,F	1.41±0.11 ^C,E	12.11±0.22 ^A,B,C,D	1.40±0.06 ^D,F	6.31±0.43 ^{A, B,C,D}
Pentadecenoic acid C15:1	NDT	NDC	NDC	NDC	NDC	NDC	NDC	NDC	NDC
Pentadecanoic acid C15:0	SLIGHT	NDC ^A,B	0.09±0.01 ^C,D	0.45±0.06 ^E,F	0.11±0.01 ^G,K	0.77±0.06 ^A,C,G,L	2.16±0.39 ^A,C,E,G,M	0.62±0.04 ^B,M	1.88±0.32 ^B,D,F,K,L
Hexadecenoic acid C16:1	NDT	NDC ^A,B,C,D	1.35±0.10 ^A	1.88±0.28 ^B	1.48±0.17 ^C	1.27±0.04 ^D	42.52±0.34 ^A,B,C,D	2.16±0.04 ^A,D	24.33±0.48 ^A,B,C,D
Hexadecanoic acid C16:0	NDT	1.12±0.11 ^A,B,C	25.61±1.32 ^A,D	41.54±5.23 ^A,E	16.96±1.98 ^A,E,F	30.14±0.21 ^B,E	467.16±2.21 ^A,E,F	37.47±0.41 ^C,D,F	273.13±1.15 ^A,E,F
Heptadecenoic acid C17:1	SLIGHT	NDC ^A,B	NDC ^C,D	0.32±0.04 ^E,F	NDC ^G,H	0.79±0.08 ^A,C,G	2.55±0.29 ^A,B,C,E,G	0.10±0.05 ^B,D,F,H	1.49±0.41 ^A,C,E,G
Heptadecanoic acid C17:0	NO	0.03±0.01 ^A,B	0.22±0.06 ^C,F	0.56±0.06 ^D	0.15±0.04 ^E,G	1.00±0.02 ^A	5.95±0.67 ^A,C,D,E,H	1.10±0.02 ^B,F,G,H	3.28±0.46 ^A,C,D,E,H
Octadecatrienoic acid C18:3	SLIGHT	NDC ^A,B	NDC ^C,D	2.42±0.32 ^A,C,E,F,G,H	2.68±0.36 ^B,D,I,J,K,L	NDC ^E,I	NDC ^F,J	NDC ^G,K	NDC ^H,L
Octadecadienoic acid C18:2	NDT	0.60±0.06 ^A	8.36±0.39 ^B	31.32±7.73 ^A,B,C	4.12±0.65 ^C	53.85±1.41 ^A,B,C	503.35±7.27 ^A,B,C	78.62±0.68 ^A,B,C	430.98±3.13 ^A,B,C
Octadecenoic acid C18:1	NDT	0.46±0.03 ^A,B	14.69±1.11 ^C,D	61.99±6.85 ^A,C,E	12.71±1.66 ^E,F	74.11±2.31 ^B,D	981.41±27.67 ^A,C,E,F	103.23±2.27 ^A,C,E,	651.29±6.23 ^A,C,E,F
Octadecanoic acid C18:0	NDT	0.36±0.04 ^A,B	7.33±0.60 ^A,C	11.86±1.32 ^A,D,E,	NDC ^C,D,F	24.78±0.70 ^A,F	174.09±3.12 ^A,F,G	26.99±0.,34 ^B,C,E,G,	75.62±1.95 ^A,F,G
Nonadecanoic acid C19:0	NDT	NDC ^A	NDC ^B	0.17±0.03 ^A,B,C,D,E,F	NDC ^C	NDC ^D	NDC ^E	0.44±0.05 ^A,B,C,D,E,F	NDC ^F
Nonadecenoic acid C19:1	SLIGHT	NDC ^A	NDC ^B	NDC ^C	NDC ^D	NDC ^E	NDC ^F	1.31±0.07 ^{A,B,C,D,E,F,G}	NDC ^G
Eicosatetraenoic acid C20:4	YES	NDC ^A	NDC ^B	0.77±0.23 ^C	NDC ^D	NDC ^E	48.75±2.11 ^A,B,C,D,E	7.03±0.22 ^A,B,C,D,E	59.13±1.43 ^A,B,C,D,E
Eicosatrienoic acid C20:3	SLIGHT	NDC ^A,B	NDC ^C,D	0.70±0.13 ^E,F	0.11±0.02 ^G,H	4.12±0.20 ^A,C,E,G,I	26.66±2.32 ^A,C,E,G,I,J	0.98±0.01 ^I,J	3.95±0.54 ^B,D,F,H,J
Eicosadienoic acid C20:2	SLIGHT	NDC ^A,B	NDC ^C,D	0.59±0.12 ^A,C,E,F,G,H	NDC ^E,I	NDC ^F,J	NDC ^G,K	0.78±0.24 ^B,D,I,J,K,L	NDC ^H,L
Eicosenoic acid C20:1	SLIGHT	NDC	NDC	NDC	NDC	NDC	NDC	NDC	NDC
Eicosanoic acid C20:0	NO	0.07±0.02 ^A,B	NDC ^C,D	0.80±0.10 ^A,C,E,F	0.16±0.02 ^E,N	1.67±0.09 ^A,C,E,F,G	NDC ^F,H	1.20±0.06 ^B,D,N,G,H,I	2.76±0.34 ^A,C,E,F,I
Docosanoic acid C22:0	NDT	0.14±0.01 ^A	NDC ^B	1.53±0.20 ^A,B,C,D,E,F	0.26±0.06 ^C	NDC ^D	NDC ^E	0.64±0.15 ^F	7.23±0.49 ^A,B,C,D,E,F
Tetracosanoic acid C24:0	NO	0.28±0.01 ^A	NDC ^B	2.02±0.24 ^C	0.25±0.08 ^D	NDC ^E	NDC ^F	NDC ^G	12.61±1.64 ^{A,B,C,D,E,F,G}
Hexacosanoic acid C26:0	NO	0.19±0.04 ^A	NDC ^B	1.69±0.24 ^{A,B,C,D,E,F,G}	NDC ^C	NDC ^D	NDC ^E	NDC ^F	NDC ^G
Sum of FFA		3.62±0.17 ^A,B	61.51±3.53 ^A,B	163.24±23.69 ^B,C	41.21±5.31 ^C,D	197.34±1.8 ^A,D	2291.14±31.31 ^A,BC,	266.96±1.44 ^A,B,C	1570.31±11.94 ^A,BC

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FFA- free fatty acids; SD—standard deviation; Extract III- dichloromethane extracts after sonification; NDT–not determined; NDC–not detected; statistically significant differences are marked with the same letters (ANOVA, Test HSD Tukey, p<0.05), for raw data see supplementary S3 Table

* data from [18,36] Tested fungal species: Aspergillus niger, Beauveria bassiana, Candida albicans, Candida lipolytica, Candida tropicalis, Lecanicillium lecanii, Metarhizium anisopliae, Paecilomyces fumosoroseus, Peacilomyces lilacinus

A greater variety of FFA types were found in the oothecae of both species. Twenty-four in B. orientalis, including sixteen saturated (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C19:0, C20:0, C22:0, C24:0, C26:0) and eight unsaturated (C16:1, C17:1, C18:2, C18:1, C18:3, C20:4, C20:3, C20:2) and eighteen in B. germanica: thirteen saturated (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C20:0, C22:0, C24:0) and five unsaturated (C16:1, C18:2, C18:1, C18:3, C20:3). C18:3 was found exclusively in the oothecae of both species. Most shared FFAs were more abundant in B. orientalis (Table 4).

Fifteen FFAs were found in B. orientalis wings extracts: ten saturated (C6:0, C8:0, C9:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0) and five unsaturated (C16:1, C17:1, C18:2, C18:1, C20:3). Fifteen were found in B. germanica wings, but nine were saturated (C6:0, C8:0, C9:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0) and six unsaturated (C16:1, C17:1, C18:2, C18:1, C20:4, C20:3). C20:4 was present in B. germanica wings but not in B. orientalis wings, and vice versa for C20:0. All shared FFAs were significantly more abundant in B. germanica: 2.8 (C15:0) to 15.5 times (C16:0).

Twenty-one FFAs were found in B. orientalis thoraces, thirteen saturated (C6:0, C8:0, C9:0, C10:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C19:0, C20:0, C22:0) and eight unsaturated (C16:1, C17:1, C18:2, C18:1, C19:1, C20:4, C20:3, C20:2), while 19 FFAs were detected in B. germanica thoraces: thirteen saturated (C6:0, C7:0, C8:0, C9:0, C12:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0, C22:0, C24:0) and six unsaturated (C16:1, C17:1, C18:2, C18:1, C20:4, C20:3). C19:1 was present only in B. germanica thoraces, and in no other extracts, while C10:0, C19:0, C20:2 and C24:0 were absent in B. germanica thoraces but present in B. orientalis thoraces. All shared FFAs were significantly more abundant in B. germanica: 2.9 (C17:0) to 14.9 times (C17:1).

Four FFAs predominated in all analysed internal extracts: C16:0, C18:2, C18:1 and C18:0 (Table 4). C16:0 was the most abundant FFA in all whole insect extracts (1.12±0.11 μg/g b.w. B. orientalis; 1325.61±1.32 μg/g b.w. B. germanica). C16:0 was abundant in B. germanica wings (467.16±2.21μg/g) and thoraces (273.13±1.15 μg/g), but less so in the oothecae (41.54±1.15 μg/g B. orientalis; 16.96±1.98 μg/g B. germanica). The highest concentrations of C18:1 were measured in B. germanica wings (981.41±27.67 μg/g) and thoraces (651.29±6.23 μg/g), as well as high concentrations of C18:2 (503.35±7.27 μg/g wings, 430.98±3.13 μg/g thoraces) and C18:0 (174.09±3.12 μg/g wings, 75.62±1.95 μg/g thoraces). C16:0, C18:2, C18:1 and C18:0 predominated in B. orientalis extracts.

B. orientalis demonstrated higher concentrations of FFAs from the cuticle (except C16:0, C17:0, C18:0, C18:1, C18:2), while in B. germanica higher FFA levels were detected inside the body. For the oothecae, B. germanica had a similar profile, while for B. orientalis, all FFAs, except C14:0 and C20:4, were more abundant in extracts I and II than extract III.

Regarding the wings, higher concentrations of all FFAs were found in the cuticle for both species, apart from C20:3 for B. germanica. Regarding the thoraces, higher concentrations of FFAs were present in the combined extracts I and II for B. germanica (except for C7:0 and C24:0), while all FFAs were more abundant in extract III for B. orientalis (Tables 3 and 4).

Correlations between rates of cuticle hydrolysis and FFAs profiles

The correlation coefficients obtained between cuticle hydrolysis and FFA profile varied from |0.1| to |1.0| (Tables 5 and 6). Scatterplots indicate both positive and negative linear correlations with various strengths. A correlation between cuticle FFAs concentration and the effectiveness of fungal enzymes was recognized in cases where a strong correlation (r ≥0.6 or r ≤−0.6) was observed in both cockroach species. Proteolytic degradation of the cuticle was negatively correlated with cuticular C10:0 in oothecae, and C6:0, C9:0, C16:0 and C20:0 in thoraces, and positively correlated with C17:0 in oothecae, C6:0 in wings, C12:0 and C20:3 in thoraces (Table 5). Fungal protease efficiency negatively correlated with internal C15:0, C16:0, C17:0 and C20:3 levels for thoraces, and positively correlated with internal C6:0 and C14 for wings (Table 6). Fungal chitinase efficiency positively correlated with cuticular C12:0, C14:0, C17:1 and C20:3 content in oothecae, C16:1 in wings, and C6:0 and C10:0 in thoraces, and negatively correlated with C18:0 in wings and thoraces, and with C18:2 in wings (Table 5). In thoraces, chitin degradation positively correlated with internal C6:0 and C20:3, and negatively correlated with internal C17:1, C18:2 and C20:4.

Table 5. Correlation between the concentration of compounds identified in the cuticle of two cockroach species and the efficiency of C. coronatus proteases, chitinases and lipases in degrading the cockroach cuticle.

FFA	Effect on C. coronatus^*	Proteases			Chitinases			Lipases
FFA	Effect on C. coronatus^*	oothecae	wings	thoraces	oothecae	wings	thoraces	oothecae	wings	thoraces
C4:0	Negative	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C4:0	Negative	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
C5:0	Negative	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C5:0	Negative	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
C6:0	Negative	BO (0.92)	BO (0.79)	BO (-0.90)	BO (0.84)	BO (-0.55)	BO (0.64)	BO (-0.94)	BO (NDC)	BO (0.57)
C6:0	Negative	BG (-0.98)	BG (0.95)	BG (-0.66)	BG (0.25)	BG (0.60)	BG (0.92)	BG (0.81)	BG (NDC)	BG (NDC)
C7:0	Negative	BO (0.96)	BO (NDC)	BO (NDC)	BO (0.77)	BO (NDC)	BO (NDC)	BO (-0.98)	BO (NDC)	BO (NDC)
C7:0	Negative	BG (-0.99)	BG (NDC)	BG (-0.28)	BG (-0.07)	BG (NDC)	BG (-0.83)	BG (0.59)	BG (NDC)	BG (NDC)
C8:0	Negative	BO (0.88)	BO (0.96)	BO (1.00)	BO (0.88)	BO (-0.83)	BO (-0.45)	BO (-0.91)	BO (NDC)	BO (-0.37)
C8:0	Negative	BG (-0.97)	BG (-0.18)	BG (0.17)	BG (-0.18)	BG (0.50)	BG (0.88)	BG (0.49)	BG (NDC)	BG (NDC)
C9:0	Negative	BO (0.98)	BO (0.81)	BO (-0.80)	BO (0.71)	BO (-0.58)	BO (-0.45)	BO (-0.99)	BO (NDC)	BO (-0.53)
C9:0	Negative	BG (-0.97)	BG (-0.76)	BG (-0.97)	BG (-0.18)	BG (-0.20)	BG (0.54)	BG (0.49)	BG (NDC)	BG (NDC)
C10:0	Negative	BO (-0.88)	BO (0.13)	BO (0.92)	BO (-0.11)	BO (-0.44)	BO (0.99)	BO (0.85)	BO (NDC)	BO (0.98)
C10:0	Negative	BG (-0.75)	BG (NDC)	BG (-0.33)	BG (-0.61)	BG (NDC)	BG (1.00)	BG (0.05)	BG (NDC)	BG (NDC)
C12:0	Negative	BO (0.93)	BO (-0.51)	BO (0.80)	BO (0.83)	BO (0.20)	BO (-0.81)	BO (-0.95)	BO (NDC)	BO (-0.75)
C12:0	Negative	BG (0.32)	BG (-0.85)	BG (0.75)	BG (0.92)	BG (-1.00)	BG (-0.86)	BG (0.46)	BG (NDC)	BG (NDC)
C13:0	Negative	BO (0.11)	BO (NDC)	BO (NDC)	BO (-0.76)	BO (NDC)	BO (NDC)	BO (-0.04)	BO (NDC)	BO (NDC)
C13:0	Negative	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
C14:1	Negative	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C14:1	Negative	BG (0.28)	BG (NDC)	BG (NDC)	BG (0.94)	BG (NDC)	BG (NDC)	BG (0.50)	BG (NDC)	BG (NDC)
C14:0	Negative	BO (0.79)	BO (0.08)	BO (0.90)	BO (0.95)	BO (-0.39)	BO (1.00)	BO (-0.83)	BO (NDC)	BO (0.19)
C14:0	Negative	BG (-0.73)	BG (-0.65)	BG (-0.96)	BG (0.73)	BG (-1.00)	BG (0.56)	BG (1.00)	BG (NDC)	BG (NDC)
C15:1	Negative	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C15:1	Negative	BG (-0.05)	BG (NDC)	BG (NDC)	BG (1.00)	BG (NDC)	BG (NDC)	BG (0.76)	BG (NDC)	BG (NDC)
C15:0	Positive	BO (-0.46)	BO (0.10)	BO (0.40)	BO (0.47)	BO (-0.42)	BO (-0.98)	BO (0.40)	BO (NDC)	BO (-0.96)
C15:0	Positive	BG (-0.93)	BG (-1.00)	BG (0.06)	BG (-0.42)	BG (-0.80)	BG (0.93)	BG (-0.90)	BG (NDC)	BG (NDC)
C16:1	Negative	BO (0.92)	BO (-0.92)	BO (-0.10)	BO (0.84)	BO (1.00)	BO (0.99)	BO (-0.95)	BO (NDC)	BO (1.00)
C16:1	Negative	BG (-0.93)	BG (0.73)	BG (-0.98)	BG (0.43)	BG (1.00)	BG (0.51)	BG (0.91)	BG (NDC)	BG (NDC)
C16:0	Negative	BO (0.91)	BO (0.28)	BO (-0.90)	BO (0.86)	BO (-0.57)	BO (-0.15)	BO (-0.94)	BO (NDC)	BO (-0.24)
C16:0	Negative	BG (-0.70)	BG (0.98)	BG (-0.99)	BG (-0.66)	BG (0.90)	BG (0.46)	BG (-0.03)	BG (NDC)	BG (NDC)
C17:1	Negative	BO (0.97)	BO (0.19)	BO (-0.70)	BO (0.75)	BO (-0.49)	BO (0.82)	BO (-0.98)	BO (NDC)	BO (0.76)
C17:1	Negative	BG (0.28)	BG (0.35)	BG (0.89)	BG (0.94)	BG (0.90)	BG (-0.71)	BG (0.50)	BG (NDC)	BG (NDC)
C17:0	Negative	BO (0.99)	BO (0.99)	BO (-1.00)	BO (0.44)	BO (-0.99)	BO (0.24)	BO (-0.98)	BO (NDC)	BO (0.15)
C17:0	Negative	BG (0.89)	BG (0.12)	BG (0.80)	BG (0.39)	BG (0.70)	BG (0.33)	BG (-0.30)	BG (NDC)	BG (NDC)
C18:3	Negative	BO (0.99)	BO (NDC)	BO (NDC)	BO (0.66)	BO (NDC)	BO (NDC)	BO (-1.00)	BO (NDC)	BO (NDC)
C18:3	Negative	BG (-0.52)	BG (NDC)	BG (NDC)	BG (-0.82)	BG (NDC)	BG (NDC)	BG (-0.26)	BG (NDC)	BG (NDC)
C18:2	Negative	BO (0.90)	BO (0.71)	BO (0.50)	BO (0.87)	BO (-0.90)	BO (0.69)	BO (-0.93)	BO (NDC)	BO (0.76)
C18:2	Negative	BG (-0.78)	BG (-0.53)	BG (0.93)	BG (0.67)	BG (-0.90)	BG (-0.65)	BG (0.99)	BG (NDC)	BG (NDC)
C18:1	Negative	BO (0.98)	BO (0.30)	BO (-0.70)	BO (0.71)	BO (0.02)	BO (-0.47)	BO (-0.99)	BO (NDC)	BO (-0.55)
C18:1	Negative	BG (-0.99)	BG (0.39)	BG (0.29)	BG (0.22)	BG (0.90)	BG (0.82)	BG (0.80)	BG (NDC)	BG (NDC)
C18:0	Negative	BO (-0.98)	BO (0.33)	BO (-0.30)	BO (-0.73)	BO (-0.62)	BO (-0.87)	BO (0.99)	BO (NDC)	BO (-0.91)
C18:0	Negative	BG (NDC)	BG (-0.77)	BG (0.90)	BG (NDC)	BG (-1.00)	BG (-0.69)	BG (NDC)	BG (NDC)	BG (NDC)
C19:1	NDT	BO (NDC)	BO (NDC)	BO (-0.80)	BO (NDC)	BO (NDC)	BO (0.77)	BO (NDC)	BO (NDC)	BO (0.71)
C19:1	NDT	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
C19:0	NDT	BO (0.99)	BO (NDC)	BO (NDC)	BO (0.69)	BO (NDC)	BO (NDC)	BO (-1.00)	BO (NDC)	BO (NDC)
C19:0	NDT	BG (NDC)	BG (NDC)	BG (0.27)	BG (NDC)	BG (NDC)	BG (0.83)	BG (NDC)	BG (NDC)	BG (NDC)
C20:4	NDT	BO (NDC)	BO (-0.26)	BO (0.40)	BO (NDC)	BO (0.55)	BO (-0.98)	BO (NDC)	BO (NDC)	BO (-0.96)
C20:4	NDT	BG (NDC)	BG (0.86)	BG (0.08)	BG (NDC)	BG (1.00)	BG (0.92)	BG (NDC)	BG (NDC)	BG (NDC)
C20:3	NDT	BO (-0.35)	BO (-0.88)	BO (1.00)	BO (0.58)	BO (0.99)	BO (-0.24)	BO (0.28)	BO (NDC)	BO (-0.14)
C20:3	NDT	BG (NDC)	BG (0.25)	BG (0.86)	BG (NDC)	BG (-0.40)	BG (-0.75)	BG (NDC)	BG (NDC)	BG (NDC)
C20:2	NDT	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C20:2	NDT	BG (NDC)	BG (-0.67)	BG (0.83)	BG (NDC)	BG (-1.00)	BG (-0.79)	BG (NDC)	BG (NDC)	BG (NDC)
C20:1	Negative	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)	BO (NDC)
C20:1	Negative	BG (NDC)	BG (1.00)	BG (1.00)	BG (NDC)	BG (0.80)	BG (-0.32)	BG (NDC)	BG (NDC)	BG (NDC)
C20:0	Negative	BO (0.99)	BO (-0.63)	BO (-0.80)	BO (0.46)	BO (0.35)	BO (0.81)	BO (-0.98)	BO (NDC)	BO (0.76)
C20:0	Negative	BG (-0.40)	BG (-0.41)	BG (-0.97)	BG (-0.89)	BG (-0.90)	BG (0.08)	BG (-0.38)	BG (NDC)	BG (NDC)
C22:0	NDT	BO (0.89)	BO (-0.95)	BO (0.10)	BO (0.87)	BO (1.00)	BO (0.95)	BO (-0.92)	BO (NDC)	BO (0.97)
C22:0	NDT	BG (-0.58)	BG (NDC)	BG (0.94)	BG (-0.78)	BG (NDC)	BG (-0.62)	BG (-0.19)	BG (NDC)	BG (NDC)
C24:0	NDT	BO (0.66)	BO (NDC)	BO (NDC)	BO (0.99)	BO (NDC)	BO (NDC)	BO (-0.71)	BO (NDC)	BO (NDC)
C24:0	NDT	BG (0.06)	BG (NDC)	BG (-0.86)	BG (0.99)	BG (NDC)	BG (-0.21)	BG (0.68)	BG (NDC)	BG (NDC)
C26:0	NDT	BO (0.99)	BO (NDC)	BO (NDC)	BO (0.69)	BO (NDC)	BO (NDC)	BO (-0.99)	BO (NDC)	BO (NDC)
C26:0	NDT	BG (-0.84)	BG (NDC)	BG (-0.90)	BG (-0.48)	BG (NDC)	BG (-0.13)	BG (0.20)	BG (NDC)	BG (NDC)
SUM		BO (-0.91)	BO (0.34)	BO (-0.80)	BO (-0.85)	BO (-0.62)	BO (-0.35)	BO (0.94)	BO (NDC)	BO (-0.44)
SUM		BG (-0.87)	BG (0.64)	BG (0.68)	BG (-0.43)	BG (0.00)	BG (0.49)	BG (0.25)	BG (NDC)	BG (NDC)

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Correlation coefficients (r) are presented in brackets. BO–B. orientalis; BG–B. germanica, NDC–not detected; NDT–not determined

* data concerning compounds’ effects on the in vitro growth, sporulation and virulence of C. coronatus are from [15].

Table 6. Correlation between the concentration of internal FFAs of two cockroach species and the efficiency of C. coronatus proteases, chitinases and lipases in degrading the cockroach cuticle.

FFA	Effect on C. coronatus^*	Proteases			Chitinases			Lipase
FFA	Effect on C. coronatus^*	oothecae	wings	thoraces	oothecae	wings	thoraces	oothecae	wings	thoraces
C6:0	Negative	BO (0.66)	BO (0.67)	BO (0.06)	BO (-0.25)	BO (-0.40)	BO (0.96)	BO (-0.60)	BO (NDC)	BO (0.98)
C6:0	Negative	BG (-0.88)	BG (0.96)	BG (-0.80)	BG (-0.41)	BG (0.57)	BG (0.85)	BG (0.27)	BG (NDC)	BG (NDC)
C7:0	Negative	BO (0.89)	BO (NDC)	BO (NDC)	BO (0.12)	BO (NDC)	BO (NDC)	BO (-0.90)	BO (NDC)	BO (NDC)
C7:0	Negative	BG (-0.79)	BG (NDC)	BG (-0.30)	BG (-0.56)	BG (NDC)	BG (1.00)	BG (0.11)	BG (NDC)	BG (NDC)
C8:0	Negative	BO (0.91)	BO (-0.76)	BO (-0.40)	BO (0.16)	BO (0.93)	BO (-0.80)	BO (-0.90)	BO (NDC)	BO (-0.85)
C8:0	Negative	BG (-0.90)	BG (-0.20)	BG (1.00)	BG (-0.38)	BG (-0.76)	BG (-0.32)	BG (0.30)	BG (NDC)	BG (NDC)
C9:0	Negative	BO (0.94)	BO (-0.75)	BO (1.00)	BO (0.26)	BO (0.92)	BO (-0.25)	BO (-0.90)	BO (NDC)	BO (-0.16)
C9:0	Negative	BG (-0.86)	BG (-0.43)	BG (-1.00)	BG (-0.45)	BG (-0.89)	BG (0.39)	BG (0.23)	BG (NDC)	BG (NDC)
C10:0	Negative	BO (1.00)	BO (NDC)	BO (-0.97)	BO (0.59)	BO (NDC)	BO (0.44)	BO (1.00)	BO (NDC)	BO (0.36) BG (NDC)
C10:0	Negative	BG (-0.99)	BG (NDC)	BG (NDC)	BG (-0.10)	BG (NDC)	BG (NDC)	BG (0.56)	BG (NDC)	BO (0.36) BG (NDC)
C12:0	Negative	BO (0.38)	BO (0.58)	BO (0.95)	BO (-0.55)	BO (-0.28)	BO (0.08)	BO (-0.30)	BO (NDC)	BO (0.17)
C12:0	Negative	BG (-0.81)	BG (0.28)	BG (-1.00)	BG (-0.53)	BG (-0.37)	BG (0.44)	BG (0.14)	BG (NDC)	BG (NDC)
C14:0	Negative	BO (0.86)	BO (0.66)	BO (0.19)	BO (0.05)	BO (-0.87)	BO (-1.00)	BO (-0.80)	BO (NDC)	BO (-1.00)
C14:0	Negative	BG (-0.87)	BG (0.88)	BG (-1.00)	BG (-0.44)	BG (0.39)	BG (0.01)	BG (0.24)	BG (NDC)	BG (NDC)
C15:0	Positive	BO (0.76)	BO (0.54)	BO (-0.67)	BO (-0.11)	BO (-0.78)	BO (-0.57)	BO (-0.70)	BO (NDC)	BO (-0.65)
C15:0	Positive	BG (-0.26)	BG (-0.98)	BG (-0.90)	BG (-0.95)	BG (-0.66)	BG (-0.16)	BG (-0.52)	BG (NDC)	BG (NDC)
C16:1	Negative	BO (0.79)	BO (-1.00)	BO (-0.98)	BO (-0.07)	BO (0.92)	BO (0.44)	BO (-0.70)	BO (NDC)	BO (0.35)
C16:1	Negative	BG (-0.84)	BG (-0.04)	BG (-0.30)	BG (-0.49)	BG (-0.65)	BG (1.00)	BG (0.18)	BG (NDC)	BG (NDC)
C16:0	Negative	BO (0.75)	BO (-0.75)	BO (-0.92)	BO (-0.13)	BO (0.50)	BO (-0.16)	BO (-0.70)	BO (NDC)	BO (-0.25)
C16:0	Negative	BG (-0.80)	BG (0.42)	BG (-1.00)	BG (-0.55)	BG (-0.23)	BG (0.04)	BG (0.11)	BG (NDC)	BG (NDC)
C17:1	Negative	BO (0.72)	BO (-0.62)	BO (-0.08)	BO (-0.17)	BO (0.84)	BO (-0.95)	BO (-0.70) BG (NDC)	BO (NDC)	BO (-0.98)
C17:1	Negative	BG (NDC)	BG (-0.89)	BG (0.00)	BG (NDC)	BG (-0.42)	BG (-0.95)	BO (-0.70) BG (NDC)	BG (NDC)	BG (NDC)
C17:0	Negative	BO (0.84)	BO (0.06)	BO (-0.63)	BO (0.03)	BO (-0.38)	BO (0.90)	BO (-0.80)	BO (NDC)	BO (0.86)
C17:0	Negative	BG (-0.64)	BG (-0.62)	BG (-1.00)	BG (-0.73)	BG (0.00)	BG (0.13)	BG (-0.11)	BG (NDC)	BG (NDC)
C18:3	Negative	BO (0.31)	BO (NDC)	BO (NDC)	BO (-0.62)	BO (NDC)	BO (NDC)	BO (-0.20)	BO (NDC)	BO (NDC)
C18:3	Negative	BG (-0.82)	BG (NDC)	BG (NDC)	BG (-0.52)	BG (NDC)	BG (NDC)	BG (0.15)	BG (NDC)	BG (NDC)
C18:2	Negative	BO (0.61)	BO (0.00)	BO (-0.48)	BO (-0.31)	BO (0.32)	BO (-0.74)	BO (-0.60)	BO (NDC)	BO (-0.80)
C18:2	Negative	BG (-0.84)	BG (-0.81)	BG (0.90)	BG (-0.49)	BG (-0.27)	BG (-0.76)	BG (0.19)	BG (NDC)	BG (NDC)
C18:1	Negative	BO (0.72)	BO (1.00)	BO (0.21)	BO (-0.16)	BO (-0.96)	BO (0.90)	BO (-0.70)	BO (NDC)	BO (0.94)
C18:1	Negative	BG (-0.78)	BG (-0.90)	BG (-1.00)	BG (-0.58)	BG (-0.44)	BG (0.31)	BG (0.08)	BG (NDC)	BG (NDC)
C18:0	Negative	BO (0.53) BG (NDC)	BO (-0.71)	BO (0.88)	BO (-0.40) BG (NDC)	BO (0.44)	BO (-0.26)	BO (-0.50) BG (NDC)	BO (NDC)	BO (-0.35)
C18:0	Negative	BO (0.53) BG (NDC)	BG (0.91)	BG (-1.00)	BO (-0.40) BG (NDC)	BG (0.46)	BG (0.23)	BO (-0.50) BG (NDC)	BG (NDC)	BG (NDC)
C19:1	NDT	BO (NDC)	BO (NDC)	BO (0.07) BG (NDC)	BO (NDC)	BO (NDC)	BO (0.96) BG (NDC)	BO (NDC)	BO (NDC)	BO (0.98)
C19:1	NDT	BG (NDC)	BG (NDC)	BO (0.07) BG (NDC)	BG (NDC)	BG (NDC)	BO (0.96) BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
C19:0	NDT	BO (0.92) BG (NDC)	BO (NDC)	BO (0.93) BG (NDC)	BO (0.20) BG (NDC)	BO (NDC)	BO (0.15) BG (NDC)	BO (-0.90) BG (NDC)	BO (NDC)	BO (0.24 BG (NDC)
C19:0	NDT	BO (0.92) BG (NDC)	BG (NDC)	BO (0.93) BG (NDC)	BO (0.20) BG (NDC)	BG (NDC)	BO (0.15) BG (NDC)	BO (-0.90) BG (NDC)	BG (NDC)	BO (0.24 BG (NDC)
C20:4	NDT	BO (0.47) BG (NDC)	BO (NDC)	BO (0.69)	BO (-0.46) BG (NDC)	BO (NDC)	BO (-0.86)	BO (-0.40) BG (NDC)	BO (NDC)	BO (-0.81)
C20:4	NDT	BO (0.47) BG (NDC)	BG (0.53)	BG (-0.40)	BO (-0.46) BG (NDC)	BG (0.94)	BG (-0.76)	BO (-0.40) BG (NDC)	BG (NDC)	BG (NDC)
C20:3	NDT	BO (0.63)	BO (-0.30)	BO (-0.76)	BO (-0.29)	BO (-0.02)	BO (0.81)	BO (-0.60)	BO (NDC)	BO (0.75)
C20:3	NDT	BG (0.19)	BG (-0.89)	BG (-0.60)	BG (-0.99)	BG (-0.98)	BG (0.93)	BG (-0.84)	BG (NDC)	BG (NDC)
C20:2	NDT	BO (0.85)	BO (NDC)	BO (-0.89) BG (NDC)	BO (0.03) BG (NDC)	BO (NDC)	BO (0.64) BG (NDC)	BO (-0.80) BG (NDC)	BO (NDC)	BO (0.57)
C20:2	NDT	BG (NDC)	BG (NDC)	BO (-0.89) BG (NDC)	BO (0.03) BG (NDC)	BG (NDC)	BO (0.64) BG (NDC)	BO (-0.80) BG (NDC)	BG (NDC)	BG (NDC)
C20:0	Negative	BO (0.98)	BO (0.22)	BO (-0.86)	BO (0.38)	BO (0.11) BG (NDC)	BO (0.70)	BO (-1.00)	BO (NDC)	BO (0.63)
C20:0	Negative	BG (-0.29)	BG (NDC)	BG (-0.30)	BG (-0.93)	BO (0.11) BG (NDC)	BG (1.00)	BG (-0.48)	BG (NDC)	BG (NDC)
C22:0	NDT	BO (0.49)	BO (NDC)	BO (1.00)	BO (-0.44)	BO (NDC)	BO (-0.13)	BO (-0.40)	BO (NDC)	BO (-0.04)
C22:0	NDT	BG (-0.42)	BG (NDC)	BG (-1.00)	BG (-0.88)	BG (NDC)	BG (0.05)	BG (-0.36)	BG (NDC)	BG (NDC)
C24:0	NDT	BO (0.76)	BO (NDC)	BO (NDC)	BO (-0.11)	BO (NDC)	BG (0.52) BG (NDC)	BO (-0.70)	BO (NDC)	BO (NDC)
C24:0	NDT	BG (-0.99)	BG (NDC)	BG (-1.00)	BG (-0.09)	BG (NDC)	BG (0.52) BG (NDC)	BG (0.57)	BG (NDC)	BG (NDC)
C26:0	NDT	BO (0.95)	BO (NDC)	BO (NDC)	BO (0.27)	BO (NDC)	BO (NDC)	BO (-0.90)	BO (NDC)	BO (NDC)
C26:0	NDT	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)	BG (NDC)
SUM		BO (0.68)	BO (0.93)	BO (-0.38)	BO (-0.22)	BO (-0.77)	BO (0.99)	BO (-0.60)	BO (NDC)	BO (0.97)
SUM		BG (-0.80)	BG (-0.91)	BG (-1.00)	BG (-0.55)	BG (-0.47)	BG (0.13)	BG (0.12)	BG (NDC)	BG (NDC)

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Correlation coefficients (r) are presented in brackets. BO–B. orientalis; BG–B. germanica, NDC–not detected, NDT–not determined

*data concerning compounds’ effects on the in vitro growth, sporulation and virulence of C. coronatus are from [15]

Chitin degradation negatively correlated to a lesser extent with C18:3 in oothecae (B. orientalis r = -0.62, B. germanica r = -0.52) (Table 6). In both cockroach species, C18:3 was detected only in the oothecae. No positive or negative correlations were found between cuticular FFA content and efficiency of fungal lipases. Internal C20:3 was negatively correlated with lipid degradation in oothecae. A slightly less distinct negative correlation was also observed for C15:0 (B. orientalis r = -0.70, B. germanica r = -0.52) (Tables 5 and 6).

Discussion

Although chemical pesticides are among the most popular methods of controlling cockroach infestations, their disadvantages have spurred the search for new strategies, including the use of entomopathogenic fungi [34,37,38]. C. coronatus is a cosmopolitan soil fungus that selectively attacks various insect species [39]. Our findings indicate that B. orientalis and B. germanica are not susceptible to infection by C. coronatus, but not to infection by other entomopathogenic fungi, such as Metarhizium anisopliae, Beauveria bassiana and Purpureocillium lilacinum [35,40–42]

The mycelia of C. coronatus cultivated in vitro secrete a plethora of enzymes, however, the activities of fungal enzymes measured in vitro are not necessarily correlated with their importance in the infection process occurring in nature [15,43–46]. The enzymatic cocktail released by C. coronatus mycelia degrades cuticle samples from susceptible insects far more effectively than those from resistant species and/or developmental stages [11,28,47]. Similar differences were observed in the present study for B. orientalis and B. germanica, particularly regarding the digestion of oothecae proteins by fungal proteases; this might indicate higher levels of total protein in B. germanica oothecae than in B. orientalis, or of proteins susceptible to digestion by C. coronatus proteases.

¹³C –NMR spectroscopy revealed higher levels of proteins in the cuticle of B. germanica oothecae than for B. orientalis [48]; this may be due to the higher protein requirement of developing nymphs [49] and/or differences in their physiology: B. orientalis females deposit oothecae as soon as they are formed while B. germanica females retain the oothecae until nymphs are ready to hatch.

In contrast to the oothecae, the two cockroach species released similar, low amounts of amino acids, suggesting their protein content was low in wings and had similar protein compositions. C. coronatus proteases have been found to be highly effective against the wings proteins of four fly species (L. sericata, C. vicina, C. vomitoria, M. domestica) and those of G. mellonella [28,47]. It could suggest a lower content of degradable proteins in the cockroach wings compared to other insects we have studied in terms of efficiency of cuticular protein digestion by C. coronatus proteases. Similar high concentrations of amino acids were released from the digested thoraces of B. orientalis and B. germanica, suggesting a high abundance of similar proteins. Taken together, our findings suggest that the protein composition of the cuticle varies considerably across the body of the insect.

In contrast, no significant differences were found in the effectiveness of chitin hydrolysis in all samples of both cockroach species, indicating no species-specific variation and similar spatial distribution of chitin in the bodies. However, the C. coronatus chitinolytic enzymes demonstrated greater efficiency against both cockroach species compared to four fly species [28] and the wax moth [47] suggesting higher levels of chitin in cockroach cuticles. N-glucosamine was released from oothecae incubated with C. coronatus enzymatic cocktail containing chitinases, thus confirming the presence of chitin. It has long been assumed that chitin was absent from oothecae [50], however, this belief has been challenged by recent studies [48,51–53].

The lipases present in the C. coronatus enzyme cocktail demonstrated less hydrolytic activity against the cuticle samples than the proteases and chitinases; FFAs were only released from the oothecae of both species and thoraces of B. orientalis. Similar results have been noted against the previously described four fly species and G. mellonella [28,47]. It appears that in C. coronatus, lipases play a lesser role in the development of an infection to that of proteases and chitinases, in contrast with the pivotal role of lipolytic activity during M. anisopliae infection [27].

GC-MS identified several FFAs in B. orientalis and B. germanica cuticle samples, these being odd-numbered FFAs: seven saturated (C5:0, C7:0, C9:0, C13:0, C15:0, C17:0, C19:0) and three unsaturated (C15:1, C17:1, C19:1). The presence of odd-numbered FFAs on the surface of insects is rare. Traces of pentadecenoic acid (C15:1) were identified in the cuticular lipids of Acyrthosiphon pisum [54] and chlorpyrifos-treated B. germanica males [55]. This FFA is also characteristic for the cuticle of C. vomitoria males [36] and Nezara viridula infected by the plant pathogenic fungus Paecilomyces spp [56]. In present studies C15:1 was detected only in cuticular extracts from B. germanica oothecae.

The cuticular and internal FFAs identified in this work are similar to those previously identified for B. orientalis and B. germanica [38,55,57]. Slight discrepancies in the presence and quantity of individual FFAs result from variation in the use of GC-MS instruments, extraction and derivatization procedures, and from the different starting materials: we used both males and females pooled together, while Paszkiewicz et al. examined B. orientalis females and B. germanica males only. Most previously examined insect species indicate higher abundance of FFAs in the internal lipids than in cuticular lipids [16,18,36]; however, Chorthippus brunneus appears to be an exception, as are the present results [58]. Higher amounts of FFAs were found in wings and thoraces (g^-1 of tissue) than in the whole body of adults (g^-1 insect body); this could be due to the high number of these light body parts (B. orientalis: 539 wings and 274 thoraces; B. germanica 377 wings and 186 thoraces) required to extract sufficient amounts of lipids for GC-MS analyses. B. orientalis display a clear wing dimorphism; the present study used equal amounts of reduced and leathery female wings and longer, membranous male wings.

Species specific differences in cuticular FFA profiles were found between cockroach species: C4:0 and C5:0 was present only in B. germanica, while C19:1 was found only in the thoraces of B. orientalis indicating an uneven spatial distribution. The physiological functions of these FFAs in cockroaches remain unknown. While C18:3 was found solely in the oothecae of both species, its exact role is unknown; however, it is likely to protect against fungal attack as C18:3 inhibits C. coronatus growth and the growth and germination of B. bassiana and Paecilomyces fumosoroseus [21,59]. The origin of C13:0, detected only in B. orientalis oothecae remains obscure. The same applies to C14:1 and C15:1, found only in B. germanica oothecae. C13:0, C14:1 and C15:1 have demonstrated antifungal activity against C. coronatus and several pathogenic fungi [15,60]. Eleven FFAs (C6:0, C7:0, C8:0, C9:0, C10:0, C12:0, C16:0, C18:1, C18:2, C18:3 and C20:0) known to inhibit key factors determining the ability of C. coronatus to infect insects, i.e. hyphal growth, sporulation and virulence [15], were found in the oothecae of both species, indicating multiple investments in protecting cockroach eggs and developing offspring. The cockroach ootheca is formed from the secretions of two colleterial glands containing proteins, enzymes and catechol derivatives [53]. The method of delivery of lipids to the ootheca is poorly understood [61], and the presence and amount of each cuticular FFA is the result of a number of poorly-understood processes of synthesis, degradation and distribution in the insect body and transportation to the target sites [28]

The efficiency in degrading cockroach cuticle samples by C. coronatus proteases was found to be negatively correlated with concentrations of C6:0, C9:0, C10:0, C16:0 and C20:0. This suggests that these FFAs may play a protective role against fungal assault. However, this inference is weakened by the positive correlations found between fungal protease efficiency and concentrations of C6:0 in the wings, C12:0 and C20:3 in the thoraces, and C17:0 in the oothecae.

In the case of C. coronatus, the role of chitinases is even more complex, as both negative and positive correlations were found regarding the same FFAs, but these differed according to body part. Obviously, more experiments are necessary to demonstrate the impact of each FFA detected in cockroach cuticle on the activity of fungal enzymes engaged in the initial stage of fungal attack.

The present study partly elucidates the mechanisms underlying the non-susceptibility of two species of cockroaches, B. germanica and B. orientalis to fungal infection and highlights the role of FFAs in that process. Further studies on the role played by cuticular lipids in the interaction between the invading fungus and the insect host will shed greater light on the complexity of the infection process.

Materials and methods

Insects

B. orientalis and B. germanica were cultured in the laboratory at 25°C, 70% relative humidity (RH), and a 12:12-hour photoperiod. The insects were cultured on standard rodent food (Agropol, Poland). For cuticle preparations, both adults and oothecae were used.

Entomopathogenic fungus

The entomopathogenic fungus was C. coronatus (isolate no. 3491), originally isolated from Dendrolaelaps spp. (Mesostigmata: Digamasellidae), obtained from the collection of Professor Bałazy (Polish Academy of Sciences, Research Centre for Agricultural and Forest Environment, Poznań, Poland). The fungus was maintained in 90 mm Petri dishes at 20°C in a 12:12-hour light/dark cycle to stimulate sporulation [62] on Sabouraud agar medium (SAM). The medium was supplemented by homogenized G. mellonella larvae to a final concentration of 10% wet weight. This addition enhances sporulation and virulence of the SAM cultures of C. coronatus. At seven days, conidia were harvested by flooding the plates with sterile water; 100μL portions of suspension, each containing approximately 50 conidia, were taken for inoculations.

To obtain the mixture of fungal enzymes to hydrolyze the insect cuticle, C. coronatus was cultivated at 20°C in 500-ml Erlenmeyer flasks containing 250ml of minimal medium as described by Bania and co-workers but without shaking [43]. After three weeks, the mycelia were removed by filtration through Whatman no. 1 filter paper. The cell-free filtrates were assayed for their protein concentrations and protease, chitinase and lipase activities, and taken for in vitro hydrolysis of cockroach cuticle preparations.

The same C. coronatus cell-free filtrate was used in studies of cuticle hydrolysis in four medically-important fly species and Galleria mellonella [28,47].

Infection of insects with C. coronatus

B. orientalis and B. germanica adults were exposed for 24 hours at 20˚C to fully-grown and sporulating C. coronatus colonies, around 10 per Petri dish. Controls were exposed for 24 hours to sterile Sabouraud agar medium. After exposure, the insects were transferred to new, clean Petri dishes with appropriate food, and observed for seven days.

Oothecae were exposed in the same way within 24 hours of being laid by the females. The effectiveness of fungus penetration into the oothecae and their impact on developing insects was measured as the percentage of larvae that were dead within three days of hatching.

Cuticle preparation

Frozen adults of B. orientalis and B. germanica were briefly (5–10 min) rinsed in tap water and then thoroughly dried with a paper towel. The wings were dissected, and the remnants of the muscles were removed. The cuticles were dissected from thoraces in 10 mM ice-cold Tris-HCl buffer (pH 7.0) and carefully cleaned of remnants of fat body, muscles and other tissues. Empty oothecae were cleaned inside to remove the remnants left by eggs and hatching larvae. All prepared cuticle pieces were washed three times in 10mM ice-cold Tris-HCl buffer (pH 7.0), allowed to dry on ice-cold towels and stored at −20°C until use.

Enzymatic assays

Elastase, N-acetylglucosaminidase (NAGase), chitinase and lipase activity were measured in C. coronatus cell-free filtrates according to Boguś and co-workers [28]. Measurements were taken spectrophotometrically and spectrofluorimetrically (BioTek Synergy HT, USA) in 96-well polystyrene plates using suitable synthetic substrates (Merck, Germany). Elastolytic activity was measured using N-succinyl-alanine-alanine-proline-leucine-p-nitroanilide in 100mM Tris-HCl buffer containing 20mM CaCl₂ (pH 8.0). The reactions were performed in plate wells containing 2 μl of cell-free filtrate comprising fungal enzymes, 0.5mM final substrate concentration, and reaction buffer to a final volume of 200 μl. The reaction was started by the addition of the substrate, and readings were taken at A₄₁₀ to create a progress curve. Chitobiosidase activity was measured using a 0.003mM final concentration of 4-methylumbelliferyl 𝛽-D-N-N′-diacetylchitobioside in 50mM Tris-HCl buffer (pH 7.0). Fluorescence was read at Ex = 340 nm and Em = 450 nm. NAGase activity was measured using a 0.3mM final concentration of 4-nitrophenyl-N-acetyl-𝛽-D-glucosaminide in 10mM Tris-HCl buffer (pH 7.0). Absorbance was read at 405 nm. Lipase activity was measured using a 0.01mM final concentration of 4-methylumbelliferyl oleate in 50mM Tris-HCl buffer (pH 10.0). Fluorescence was read at Ex = 360 nm and Em = 450 nm.

Determination of protein concentration

The protein concentration of the cell-free filtrate of C. coronatus was determined with the Bio-Rad Protein Assay (USA), according to Bradford. Briefly, an acidic dye (Coomassie Brilliant Blue) was added to the protein solution, and the absorbance was measured at 595 nm with a microplate reader. Absorbances were measured using BioTek Synergy HT. Bovine serum albumin (BSA) was used as the standard.

Hydrolysis of insect cuticle incubated with cell-free filtrate of C. coronatus

The insect cuticle samples were divided into 50 mg portions, ground in liquid nitrogen and then washed four times in 10 mM Tris-HCl buffer (pH 7.0); 10 mg of ground cuticle was suspended in 1ml of the 10mM Tris-HCl buffer (pH 7.0), 800 μl of which was mixed with 228 μl of the C. coronatus cell-free filtrate containing elastase, NAGase, chitobiosidase and lipase. The reaction mixture was incubated for eight hours at 30°C. The reaction cocktail was divided into 20 μl portions and immediately frozen to stop further hydrolysis. Two negative controls were added, one consisting of reaction buffer with 1 mg of cuticle but without the cell-free C. coronatus filtrate (C1), and the other consisting of buffer with C. coronatus filtrate but without the insect cuticle (C2). The free amino acids produced by hydrolysis of the cuticle by proteases were measured according to Adler-Nissen, with some modifications [63]. The samples and the controls were mixed with 0.1% picrylsulfonic acid (Merck, Germany) and read at A₃₄₀. The absorbance of the negative controls was subtracted from the samples. The amounts of N-glucosamine released by chitinase hydrolysis were measured using the D-glucosamine Assay Kit (Megazyme, Ireland) according to the producer’s manual. The concentrations of free fatty acids (FFAs) released by lipases were determined with the use of the EnzymChrom TM Free Fatty Acid Assay Kit (BioAssay Systems, USA). Three independent replications of all procedures were performed. The hydrolytic efficiency of the fungal enzymes was calculated per mg of cuticle. No determination of cuticle protein, chitin and lipid content was not performed due to due to the amounts of insect-derived material being insufficient.

Extraction of free fatty acids (FFAs)

Cuticular and internal lipid components of insects were extracted, separated and analysed by GC-MS. Whole adults, oothecae, wings and thoraces isolated from adults (mass in Table 2) were extracted first in 20 ml of petroleum ether (Merck, Germany) for 5 min (extract I) and then again in 20 ml of dichloromethane (Merck, Germany) for 5 min (extract II) to yield cuticular lipids. The insects and cuticle preparations were sonicated with dichloromethane to produce Extract III containing internal lipids. The extracts were placed in glass flasks and evaporated under nitrogen.

Derivatization method

Trimethylsilyl esters (TMS) of FFAs were obtained by adding 100 μl of a BSTFA: TMCS mixture (99:1) (Merck, Germany) to 1 mg of sample and heating for 1h at 100°C. The TMS of fatty acids were then analysed by GC-MS.

GC-MS analyses

The analyses were carried out on a GCMS-QP2010 with mass detector (Shimadzu, Japan). Helium was used as the carrier gas at a column head pressure of 65.2 kPa. A DB-5 MS (Zebron, Phenomenex, USA) column was used (thickness 0.25 μm, length 30 m, diameter 0.25 μm). The column oven temperature cycle was 80^ºC for 3 min then 80°C to 310ºC at 4°C/min; the final temperature was then held for 10 min. The ion source temperature was 200°C and the interface temperature was 310^ºC. Split mode was used with a split ratio of 10. All compounds were identified based on fragmentation patterns and mass-to-charge ions of the TMS derivatives and the NIST 11 library. The mass spectrum of the fatty acid trimethylsilyl esters comprised M+ (molecular ion), [M-15]+, and fragment ions at m/z 117, 129, 132, and 145. GC analysis used the 19-methylarachidic acid (1 mg/ml; Merck, Germany) as an internal standard (IS). The contents were calculated from the relative peak areas that were compared to the IS peak area and expressed as a percentage (%, w/w) of total extracts. Response factors of one were assumed for all constituents.

Statistics

The findings were tested by the parametric t-test and one-way analysis of variance (ANOVA), where appropriate. Tukey’s test was used for post hoc analysis. Each test was performed separately. All analyses were performed using Statistica 6 software (StatSoft Polska, Poland). Differences were significant at p<0.05.

Supporting information

S1 Table. The resistance of B. orientalis and B. germanica to fungal infection–raw data.

(XLSX)

Click here for additional data file.^{(14.7KB, xlsx)}

S2 Table. Hydrolysis of cuticular protein, chitin and lipid by C. coronatus enzymes–raw data.

(XLSX)

Click here for additional data file.^{(16KB, xlsx)}

S3 Table. GC-MS analysis of cuticular lipids by C. coronatus enzymes–raw data.

(XLSX)

Click here for additional data file.^{(63.2KB, xlsx)}

Acknowledgments

We appreciate Lena Siecińska and Ewelina Mokijewska for their technical support.

Data Availability

Data supporting of this article are included within the article and in its supplementary information files.

Funding Statement

This work was partly supported by National Centre for Research and Development grant POIG.01.04.00-14-019/12 and by the Marshal’s Office of the Mazowieckie Voivodeship grant RPMA.01.02.00-14-5626/16 to the Biomibo company. There was no additional external funding received for this study. Biomibo provided support in the form of salary to A. Krawiel, KZ, KKB and MS, the purchase of chemicals, and made laboratory equipment available for A. Kaczmarek, AKW and MK. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Qu S, Wang S. Interaction of entomopathogenic fungi with the host immune system. Dev Comp Immunol. 2018;83: 96–103. 10.1016/j.dci.2018.01.010 [DOI] [PubMed] [Google Scholar]
2.Araújo JPM, Hughes DP. Diversity of Entomopathogenic Fungi. Which Groups Conquered the Insect Body? In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 1–39. [DOI] [PubMed] [Google Scholar]
3.Butt TM, Coates CJ, Dubovskiy IM, Ratcliffe NA. Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions. In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 307–364. [DOI] [PubMed] [Google Scholar]
4.Lu HL, St. Leger RJ. Insect Immunity to Entomopathogenic Fungi. In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 251–285. [DOI] [PubMed] [Google Scholar]
5.Ortiz-Urquiza A, Keyhani NO. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects. 2013;4: 357–374. 10.3390/insects4030357 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Vega FE, Meyling N V., Luangsa-Ard JJ, Blackwell M. Fungal entomopathogens. 2nd ed In: Vega FE, Kaya HK, editors. Insect Pathology. 2nd ed Academic Press,; 2012. pp. 171–220. [Google Scholar]
7.St. Leger RJ, Charnley AK, Cooper RM. Cuticle-degrading enzymes of entomopathogenic fungi: Synthesis in culture on cuticle. J Invertebr Pathol. 1986;48: 85–95. 10.1016/0022-2011(86)90146-1 [DOI] [Google Scholar]
8.Gillespie JP, Bailey AM, Cobb B, Vilcinskas A. Fungi as elicitors of insect immune responses. Arch Insect Biochem Physiol. 2000;44: 49–68. [DOI] [PubMed] [Google Scholar]
9.Andersen SO. Cuticular Sclerotization and Tanning. In: Gilbert LI, editor. Insect Molecular Biology and Biochemistry. Academic Press; 2012. pp. 167–192. [Google Scholar]
10.Vilcinskas A, Gotz P. Parasitic fungi and their interactions with the insect immune system. Adv Parasitol. 1999;43: 267–313. 10.1016/s0065-308x(08)60244-4 [DOI] [Google Scholar]
11.Boguś MI, Kedra E, Bania J, Szczepanik M, Czygier M, Jabłoński P, et al. Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection. J Insect Physiol. 2007;53: 909–922. 10.1016/j.jinsphys.2007.02.016 [DOI] [PubMed] [Google Scholar]
12.Locke M. The wigglesworth lecture: Insects for studying fundamental problems in biology. J Insect Physiol. 2001;47: 495–507. 10.1016/s0022-1910(00)00123-2 [DOI] [PubMed] [Google Scholar]
13.Moussian B. Recent advances in understanding mechanisms of insect cuticle differentiation. Insect Biochem Mol Biol. 2010;40: 363–375. 10.1016/j.ibmb.2010.03.003 [DOI] [PubMed] [Google Scholar]
14.Pedrini N, Ortiz-Urquiza A, Huarte-Bonnet C, Zhang S, Keyhani NO. Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: Hydrocarbon oxidation within the context of a host-pathogen interaction. Front Microbiol. 2013;4 10.3389/fmicb.2013.00024 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Boguś MI, Czygier M, Gołębiowski M, Kędra E, Kucińska J, Mazgajska J, et al. Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus. Exp Parasitol. 2010;125: 400–408. 10.1016/j.exppara.2010.04.001 [DOI] [PubMed] [Google Scholar]
16.Gołębiowski M, Maliński E, Boguś MI, Kumirska J, Stepnowski P. The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection. Insect Biochem Mol Biol. 2008;38: 619–627. 10.1016/j.ibmb.2008.03.005 [DOI] [PubMed] [Google Scholar]
17.Gołębiowski M, Boguś MI, Paszkiewicz M, Stepnowski P. Cuticular lipids of insects as potential biofungicides: Methods of lipid composition analysis. Anal Bioanal Chem. 2011;399: 3177–3191. 10.1007/s00216-010-4439-4 [DOI] [PubMed] [Google Scholar]
18.Gołębiowski M, Urbanek A, Oleszczak A, Dawgul M, Kamysz W, Boguś MI, et al. The antifungal activity of fatty acids of all stages of Sarcophaga carnaria L. (Diptera: Sarcophagidae). Microbiol Res. 2014;169: 279–286. 10.1016/j.micres.2013.07.011 [DOI] [PubMed] [Google Scholar]
19.James RR, Buckner JS, Freeman TP. Cuticular lipids and silverleaf whitefly stage affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J Invertebr Pathol. 2003;84: 67–74. 10.1016/j.jip.2003.08.006 [DOI] [PubMed] [Google Scholar]
20.Kerwin JL. Fatty acid regulation of the germination of Erynia variabilis conidia on adults and puparia of the lesser housefly, Fannia canicularis. Can J Microbiol. 1984;30: 158–161. 10.1139/m84-025 [DOI] [Google Scholar]
21.Saito T, Aoki J. Toxicity of Free Fatty Acids on the Larval Surfaces of Two Lepidopterous Insects towards Beauveria bassiana (Bals.) Vuill. and Paecilomyces fumoso-roseus (Wize) Brown et Smith (Deuteromycetes: Moniliales). Appl Entomol Zool. 1983;18: 225–233. 10.1303/aez.18.225 [DOI] [Google Scholar]
22.Fan Y, Fang W, Guo S, Pei X, Zhang Y, Xiao Y, et al. Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase. Appl Environ Microbiol. 2007;73: 295–302. 10.1128/AEM.01974-06 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Zhang YJ, Feng MG, Fan YH, Luo ZB, Yang XY, Wu D, et al. A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontrol Sci Technol. 2008;18: 551–563. 10.1080/09583150802082239 [DOI] [Google Scholar]
24.Fang W, Feng J, Fan Y, Zhang Y, Bidochka MJ, Leger RJS, et al. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana. J Invertebr Pathol. 2009;102: 155–159. 10.1016/j.jip.2009.07.013 [DOI] [PubMed] [Google Scholar]
25.Strachecka A, Paleolog J, Olszewski K, Borsuk G. Influence of amitraz and oxalic acid on the cuticle proteolytic system of apis mellifera L. workers. Insects. 2012;3: 821–832. 10.3390/insects3030821 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Li Y, Zhao P, Liu H, Guo X, He H, Zhu R, et al. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi. Insect Biochem Mol Biol. 2015;57: 11–19. 10.1016/j.ibmb.2014.11.006 [DOI] [PubMed] [Google Scholar]
27.Beys da Silva WO, Santi L, Schrank A, Vainstein MH. Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection. Fungal Biol. 2010;114: 10–15. 10.1016/j.mycres.2009.08.003 [DOI] [PubMed] [Google Scholar]
28.Boguś MI, Włóka E, Wrońska A, Kaczmarek A, Kazek M, Zalewska K, et al. Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus. Med Vet Entomol. 2017;31: 23–35. 10.1111/mve.12202 [DOI] [PubMed] [Google Scholar]
29.Arruda LK, Vailes LD, Ferriani VPL, Santos ABR, Pomés A, Chapman MD. Cockroach allergens and asthma. J Allergy Clin Immunol. 2001;107: 419–428. 10.1067/mai.2001.112854 [DOI] [PubMed] [Google Scholar]
30.Arruda LK, Chapman MD. The role of cockroach allergens in asthma. Curr Opin Pulm Med. 2001;7: 14–19. 10.1097/00063198-200101000-00003 [DOI] [PubMed] [Google Scholar]
31.Sookrung N, Chaicumpa W. A revisit to cockroach allergens. Asian Pacific J Allergy Immunol. 2010;28: 95–106. [PubMed] [Google Scholar]
32.Zhang F, Sun XX, Zhang XC, Zhang S, Lu J, Xia YM, et al. The interactions between gut microbiota and entomopathogenic fungi: a potential approach for biological control of Blattella germanica (L.). Pest Manag Sci. 2018;74: 438–447. 10.1002/ps.4726 [DOI] [PubMed] [Google Scholar]
33.Gabrys B, Capinera JL, Legaspi JC, Legaspi BC, Long LS, Capinera JL, et al. Cockroaches (Blattodea). Encyclopedia of Entomology. Springer, Dordrecht; 2008. pp. 937–948. [Google Scholar]
34.Gutierrez A, García J, Alzogaray R, Urrutia M, López Lastra C. Susceptibility of different life stages of Blattella germanica (Blattodea: Blattellidae) and Periplaneta fuliginosa (Blattodea: Blattidae) to entomopathogenic fungi. Int J Curr Microbiol Appl Sci. 2014;3: 614–621. [Google Scholar]
35.Quesada-Moraga E, Santos-Quirós R, Valverde-García P, Santiago-Álvarez C. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J Invertebr Pathol. 2004;87: 51–58. 10.1016/j.jip.2004.07.002 [DOI] [PubMed] [Google Scholar]
36.Gołębiowski M, Cerkowniak M, BoguŚ MI, Włóka E, Dawgul M, Kamysz W, et al. Free fatty acids in the cuticular and internal lipids of Calliphora vomitoria and their antimicrobial activity. J Insect Physiol. 2013;59: 416–429. 10.1016/j.jinsphys.2013.02.001 [DOI] [PubMed] [Google Scholar]
37.Davari B, Limoee M, Khodavaisy S, Zamini G, Izadi S. Toxicity of entomopathogenic fungi, Beauveria bassiana and Lecanicillium muscarium against a field-collected strain of the german cockroach Blattella germanica (L.) (Dictyoptera: Blattellidae). Trop Biomed. 2015;32: 463–470. [PubMed] [Google Scholar]
38.Paszkiewicz M, Gołebiowski M, Sychowska J, Boguś MI, Włóka E, Stepnowski P. The effect of the entomopathogenic fungus Conidiobolus coronatus on the composition of cuticular and internal lipids of Blatta orientalis females. Physiol Entomol. 2016;41: 111–120. 10.1111/phen.12133 [DOI] [Google Scholar]
39.Boguś MI, Scheller K. Extraction of an insecticidal protein fraction from the parasitic fungus Conidiobolus coronatus (Entomophthorales). Acta Parasitol. 2002;47: 66–72. [Google Scholar]
40.González M, López S, Rosell G, Goldarazena A, Guerrero A. Cuticular and internal chemical composition of biting midges Culicoides spp. (diptera: Ceratopogonidae), potential vectors of viral diseases. Nat Prod Commun. 2014;9: 1103–1108. 10.1177/1934578x1400900811 [DOI] [PubMed] [Google Scholar]
41.Lopes RB, Alves SB. Differential susceptibility of adults and nymphs of Blattella germanica (L.) (Blattodea: Blattellidae) to infection by metarhizium anisopliae and assessment of delivery strategies. Neotrop Entomol. 2011;40: 368–374. 10.1590/S1519-566X2011000300010 [DOI] [PubMed] [Google Scholar]
42.Yiğit Ş, Oksal E, Atay T, Akça İ, Kepenekci İ. Effect of Entomopathogenic Fungus, Purpureocillium lilacinum (syn: Paecilomyces lilacinus) on Blatta orientalis and Shelfordella tartara under Laboratory Conditions. 6th Entomopathogens & Mocrobial Control Congres. 2017. [Google Scholar]
43.Bania J, Samborski J, Bogus M, Polanowski A. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. Arch Insect Biochem Physiol. 2006;62: 186–196. 10.1002/arch.20134 [DOI] [PubMed] [Google Scholar]
44.Freimoser FM, Screen S, Hu G, St. Leger R. EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle. Microbiology. 2003;149: 1893–1900. 10.1099/mic.0.26252-0 [DOI] [PubMed] [Google Scholar]
45.Wieloch W, Boguś MI, Ligeza M, Koszela-Piotrowska I, Szewczyk A. Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane. Toxicon. 2011;58: 369–379. 10.1016/j.toxicon.2011.07.007 [DOI] [PubMed] [Google Scholar]
46.Włóka E. Characterization of proteo-, chitino- and lipolytic enzymes of parasitic fungus Conidiobolus coronatus. Wiadomości Parazytol. 2010;56: 83–85. [PubMed] [Google Scholar]
47.Wrońska AK, Boguś MI, Włóka E, Kazek M, Kaczmarek A, Zalewska K. Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus. PLoS One. 2018;13 10.1371/journal.pone.0192715 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Kramer KJ, Christensen AM, Morgan TD, Schaefer J, Czapla TH, Hopkins TL. Analysis of cockroach oothecae and exuviae by solid-state 13C-NMR spectroscopy. Insect Biochem. 1991;21: 149–156. 10.1016/0020-1790(91)90045-G [DOI] [Google Scholar]
49.Mosson HJ, Short JE, Schenkerb R, Edwardsa JP. The effects of the insect growth regulator lufenuron on oriental cockroach, Blatta orientalis, and german cockroach, Blattella germanica, populations in simulated domestic environments. Pestic Sci. 1995;45: 237–246. 10.1002/ps.2780450307 [DOI] [Google Scholar]
50.Pryor MGM. On the hardening of the ootheca of Blatta orientalis. Proc R Soc London Ser B—Biol Sci. 1940;128: 378–393. 10.1098/rspb.1940.0017 [DOI] [Google Scholar]
51.Kaya M, Muhammad M, Akyuz B, Bulut E, Sofi K, Zelencova L, et al. Extraction and characterization of chitins from coachroach Ootheca. 9 th International Scientific Conference THE VITAL NATURE SIGN. Kaunas, Lithuania; 2015. 10.13140/RG.2.1.4307.4405 [DOI]
52.Nation Sr. JL. Insect Physiology and Biochemistry. 3rd ed CRC Press; 2015. [Google Scholar]
53.Sugumaran M. Chemistry of Cuticular Sclerotization. In: Simpson SJ, editor. Advances in Insect Physiology. Academic Press; 2010. pp. 151–209. [Google Scholar]
54.Stránský K, Ubik K, Holman J, Streibl M. Chemical composition of compounds produced by the pea aphid Acyrthosiphon pisum (HARRIS): Pentane extract of surface lipids. Collect Czechoslov Chem Commun. 1973;38: 770–780. 10.1135/cccc19730770 [DOI] [Google Scholar]
55.Paszkiewicz M, Sikora A, Boguś MI, Włóka E, Stepnowski P, Gołębiowski M. Effect of exposure to chlorpyrifos on the cuticular and internal lipid composition of Blattella germanica males. Insect Sci. 2016;23: 94–104. 10.1111/1744-7917.12200 [DOI] [PubMed] [Google Scholar]
56.Raafat I, Meshrif WS, Husseiny EM El, El-Hariry M, Seif AI. Nezara viridula (Hemiptera: Pentatomidae) Cuticle as a Barrier for Beauveria bassiana and Paecilomyces sp. Infection. African Entomol. 2015;23: 75–87. 10.4001/003.023.0128 [DOI] [Google Scholar]
57.Gutierrez AC, Gołębiowski M, Pennisi M, Peterson G, García JJ, Manfrino RG, et al. Cuticle Fatty Acid Composition and Differential Susceptibility of Three Species of Cockroaches to the Entomopathogenic Fungi Metarhizium anisopliae (Ascomycota, Hypocreales). J Econ Entomol. 2015;108: 752–760. 10.1093/jee/tou096 [DOI] [PubMed] [Google Scholar]
58.Gołębiowski M, Cerkowniak M, Ostachowska A, Boguś MI, Stepnowski P. Determination of cuticular and internal fatty acids of Chorthippus brunneus males and females using HPLC-LLSD and GC–MS. Biomed Chromatogr. 2016;30: 1318–1323. 10.1002/bmc.3688 [DOI] [PubMed] [Google Scholar]
59.Babiarz E, Boguś MI, Czygier M, Kucińska J, Samborski J, Szafranek J. Influence of free fatty acids on growth, sporulation and virulence of the parasitic fungus Conidiobolus coronatus. Wiadomości Parazytol. 2001;47: 763–768. [PubMed] [Google Scholar]
60.Gołębiowski M, Cerkowniak M, Urbanek A, Dawgul M, Kamysz W, Boguś MI, et al. Antimicrobial activity of untypical lipid compounds in the cuticular and internal lipids of four fly species. J Appl Microbiol. 2014;116: 269–287. 10.1111/jam.12370 [DOI] [PubMed] [Google Scholar]
61.Young HP, Larabee JK, Gibbs AG, Schal C. Relationship between tissue-specific hydrocarbon profiles and lipid melting temperatures in the cockroach Blattella germanica. J Chem Ecol. 2000;26: 1245–1263. 10.1023/A:1005440212538 [DOI] [Google Scholar]
62.Callaghan AA. Light and spore discharge in Entomophthorales. Trans Br Mycol Soc. 1969;53: 87–97. 10.1016/s0007-1536(69)80010-0 [DOI] [Google Scholar]
63.Adler-Nissen J. Determination of the Degree of Hydrolysis of Food Protein Hydrolysates by Trinitrobenzenesulfonic Acid. J Agric Food Chem. 1979;27: 1256–1262. 10.1021/jf60226a042 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. The resistance of B. orientalis and B. germanica to fungal infection–raw data.

(XLSX)

Click here for additional data file.^{(14.7KB, xlsx)}

S2 Table. Hydrolysis of cuticular protein, chitin and lipid by C. coronatus enzymes–raw data.

(XLSX)

Click here for additional data file.^{(16KB, xlsx)}

S3 Table. GC-MS analysis of cuticular lipids by C. coronatus enzymes–raw data.

(XLSX)

Click here for additional data file.^{(63.2KB, xlsx)}

Data Availability Statement

Data supporting of this article are included within the article and in its supplementary information files.

[pone.0235785.ref001] 1.Qu S, Wang S. Interaction of entomopathogenic fungi with the host immune system. Dev Comp Immunol. 2018;83: 96–103. 10.1016/j.dci.2018.01.010 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref002] 2.Araújo JPM, Hughes DP. Diversity of Entomopathogenic Fungi. Which Groups Conquered the Insect Body? In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 1–39. [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref003] 3.Butt TM, Coates CJ, Dubovskiy IM, Ratcliffe NA. Entomopathogenic Fungi: New Insights into Host-Pathogen Interactions. In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 307–364. [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref004] 4.Lu HL, St. Leger RJ. Insect Immunity to Entomopathogenic Fungi. In: Lovett B, St. Leger RJ, editors. Advances in Genetics. Academic Press; 2016. pp. 251–285. [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref005] 5.Ortiz-Urquiza A, Keyhani NO. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects. 2013;4: 357–374. 10.3390/insects4030357 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0235785.ref006] 6.Vega FE, Meyling N V., Luangsa-Ard JJ, Blackwell M. Fungal entomopathogens. 2nd ed In: Vega FE, Kaya HK, editors. Insect Pathology. 2nd ed Academic Press,; 2012. pp. 171–220. [Google Scholar]

[pone.0235785.ref007] 7.St. Leger RJ, Charnley AK, Cooper RM. Cuticle-degrading enzymes of entomopathogenic fungi: Synthesis in culture on cuticle. J Invertebr Pathol. 1986;48: 85–95. 10.1016/0022-2011(86)90146-1 [DOI] [Google Scholar]

[pone.0235785.ref008] 8.Gillespie JP, Bailey AM, Cobb B, Vilcinskas A. Fungi as elicitors of insect immune responses. Arch Insect Biochem Physiol. 2000;44: 49–68. [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref009] 9.Andersen SO. Cuticular Sclerotization and Tanning. In: Gilbert LI, editor. Insect Molecular Biology and Biochemistry. Academic Press; 2012. pp. 167–192. [Google Scholar]

[pone.0235785.ref010] 10.Vilcinskas A, Gotz P. Parasitic fungi and their interactions with the insect immune system. Adv Parasitol. 1999;43: 267–313. 10.1016/s0065-308x(08)60244-4 [DOI] [Google Scholar]

[pone.0235785.ref011] 11.Boguś MI, Kedra E, Bania J, Szczepanik M, Czygier M, Jabłoński P, et al. Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection. J Insect Physiol. 2007;53: 909–922. 10.1016/j.jinsphys.2007.02.016 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref012] 12.Locke M. The wigglesworth lecture: Insects for studying fundamental problems in biology. J Insect Physiol. 2001;47: 495–507. 10.1016/s0022-1910(00)00123-2 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref013] 13.Moussian B. Recent advances in understanding mechanisms of insect cuticle differentiation. Insect Biochem Mol Biol. 2010;40: 363–375. 10.1016/j.ibmb.2010.03.003 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref014] 14.Pedrini N, Ortiz-Urquiza A, Huarte-Bonnet C, Zhang S, Keyhani NO. Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: Hydrocarbon oxidation within the context of a host-pathogen interaction. Front Microbiol. 2013;4 10.3389/fmicb.2013.00024 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0235785.ref015] 15.Boguś MI, Czygier M, Gołębiowski M, Kędra E, Kucińska J, Mazgajska J, et al. Effects of insect cuticular fatty acids on in vitro growth and pathogenicity of the entomopathogenic fungus Conidiobolus coronatus. Exp Parasitol. 2010;125: 400–408. 10.1016/j.exppara.2010.04.001 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref016] 16.Gołębiowski M, Maliński E, Boguś MI, Kumirska J, Stepnowski P. The cuticular fatty acids of Calliphora vicina, Dendrolimus pini and Galleria mellonella larvae and their role in resistance to fungal infection. Insect Biochem Mol Biol. 2008;38: 619–627. 10.1016/j.ibmb.2008.03.005 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref017] 17.Gołębiowski M, Boguś MI, Paszkiewicz M, Stepnowski P. Cuticular lipids of insects as potential biofungicides: Methods of lipid composition analysis. Anal Bioanal Chem. 2011;399: 3177–3191. 10.1007/s00216-010-4439-4 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref018] 18.Gołębiowski M, Urbanek A, Oleszczak A, Dawgul M, Kamysz W, Boguś MI, et al. The antifungal activity of fatty acids of all stages of Sarcophaga carnaria L. (Diptera: Sarcophagidae). Microbiol Res. 2014;169: 279–286. 10.1016/j.micres.2013.07.011 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref019] 19.James RR, Buckner JS, Freeman TP. Cuticular lipids and silverleaf whitefly stage affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J Invertebr Pathol. 2003;84: 67–74. 10.1016/j.jip.2003.08.006 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref020] 20.Kerwin JL. Fatty acid regulation of the germination of Erynia variabilis conidia on adults and puparia of the lesser housefly, Fannia canicularis. Can J Microbiol. 1984;30: 158–161. 10.1139/m84-025 [DOI] [Google Scholar]

[pone.0235785.ref021] 21.Saito T, Aoki J. Toxicity of Free Fatty Acids on the Larval Surfaces of Two Lepidopterous Insects towards Beauveria bassiana (Bals.) Vuill. and Paecilomyces fumoso-roseus (Wize) Brown et Smith (Deuteromycetes: Moniliales). Appl Entomol Zool. 1983;18: 225–233. 10.1303/aez.18.225 [DOI] [Google Scholar]

[pone.0235785.ref022] 22.Fan Y, Fang W, Guo S, Pei X, Zhang Y, Xiao Y, et al. Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase. Appl Environ Microbiol. 2007;73: 295–302. 10.1128/AEM.01974-06 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0235785.ref023] 23.Zhang YJ, Feng MG, Fan YH, Luo ZB, Yang XY, Wu D, et al. A cuticle-degrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontrol Sci Technol. 2008;18: 551–563. 10.1080/09583150802082239 [DOI] [Google Scholar]

[pone.0235785.ref024] 24.Fang W, Feng J, Fan Y, Zhang Y, Bidochka MJ, Leger RJS, et al. Expressing a fusion protein with protease and chitinase activities increases the virulence of the insect pathogen Beauveria bassiana. J Invertebr Pathol. 2009;102: 155–159. 10.1016/j.jip.2009.07.013 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref025] 25.Strachecka A, Paleolog J, Olszewski K, Borsuk G. Influence of amitraz and oxalic acid on the cuticle proteolytic system of apis mellifera L. workers. Insects. 2012;3: 821–832. 10.3390/insects3030821 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0235785.ref026] 26.Li Y, Zhao P, Liu H, Guo X, He H, Zhu R, et al. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi. Insect Biochem Mol Biol. 2015;57: 11–19. 10.1016/j.ibmb.2014.11.006 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref027] 27.Beys da Silva WO, Santi L, Schrank A, Vainstein MH. Metarhizium anisopliae lipolytic activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection. Fungal Biol. 2010;114: 10–15. 10.1016/j.mycres.2009.08.003 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref028] 28.Boguś MI, Włóka E, Wrońska A, Kaczmarek A, Kazek M, Zalewska K, et al. Cuticle hydrolysis in four medically important fly species by enzymes of the entomopathogenic fungus Conidiobolus coronatus. Med Vet Entomol. 2017;31: 23–35. 10.1111/mve.12202 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref029] 29.Arruda LK, Vailes LD, Ferriani VPL, Santos ABR, Pomés A, Chapman MD. Cockroach allergens and asthma. J Allergy Clin Immunol. 2001;107: 419–428. 10.1067/mai.2001.112854 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref030] 30.Arruda LK, Chapman MD. The role of cockroach allergens in asthma. Curr Opin Pulm Med. 2001;7: 14–19. 10.1097/00063198-200101000-00003 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref031] 31.Sookrung N, Chaicumpa W. A revisit to cockroach allergens. Asian Pacific J Allergy Immunol. 2010;28: 95–106. [PubMed] [Google Scholar]

[pone.0235785.ref032] 32.Zhang F, Sun XX, Zhang XC, Zhang S, Lu J, Xia YM, et al. The interactions between gut microbiota and entomopathogenic fungi: a potential approach for biological control of Blattella germanica (L.). Pest Manag Sci. 2018;74: 438–447. 10.1002/ps.4726 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref033] 33.Gabrys B, Capinera JL, Legaspi JC, Legaspi BC, Long LS, Capinera JL, et al. Cockroaches (Blattodea). Encyclopedia of Entomology. Springer, Dordrecht; 2008. pp. 937–948. [Google Scholar]

[pone.0235785.ref034] 34.Gutierrez A, García J, Alzogaray R, Urrutia M, López Lastra C. Susceptibility of different life stages of Blattella germanica (Blattodea: Blattellidae) and Periplaneta fuliginosa (Blattodea: Blattidae) to entomopathogenic fungi. Int J Curr Microbiol Appl Sci. 2014;3: 614–621. [Google Scholar]

[pone.0235785.ref035] 35.Quesada-Moraga E, Santos-Quirós R, Valverde-García P, Santiago-Álvarez C. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (Anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J Invertebr Pathol. 2004;87: 51–58. 10.1016/j.jip.2004.07.002 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref036] 36.Gołębiowski M, Cerkowniak M, BoguŚ MI, Włóka E, Dawgul M, Kamysz W, et al. Free fatty acids in the cuticular and internal lipids of Calliphora vomitoria and their antimicrobial activity. J Insect Physiol. 2013;59: 416–429. 10.1016/j.jinsphys.2013.02.001 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref037] 37.Davari B, Limoee M, Khodavaisy S, Zamini G, Izadi S. Toxicity of entomopathogenic fungi, Beauveria bassiana and Lecanicillium muscarium against a field-collected strain of the german cockroach Blattella germanica (L.) (Dictyoptera: Blattellidae). Trop Biomed. 2015;32: 463–470. [PubMed] [Google Scholar]

[pone.0235785.ref038] 38.Paszkiewicz M, Gołebiowski M, Sychowska J, Boguś MI, Włóka E, Stepnowski P. The effect of the entomopathogenic fungus Conidiobolus coronatus on the composition of cuticular and internal lipids of Blatta orientalis females. Physiol Entomol. 2016;41: 111–120. 10.1111/phen.12133 [DOI] [Google Scholar]

[pone.0235785.ref039] 39.Boguś MI, Scheller K. Extraction of an insecticidal protein fraction from the parasitic fungus Conidiobolus coronatus (Entomophthorales). Acta Parasitol. 2002;47: 66–72. [Google Scholar]

[pone.0235785.ref040] 40.González M, López S, Rosell G, Goldarazena A, Guerrero A. Cuticular and internal chemical composition of biting midges Culicoides spp. (diptera: Ceratopogonidae), potential vectors of viral diseases. Nat Prod Commun. 2014;9: 1103–1108. 10.1177/1934578x1400900811 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref041] 41.Lopes RB, Alves SB. Differential susceptibility of adults and nymphs of Blattella germanica (L.) (Blattodea: Blattellidae) to infection by metarhizium anisopliae and assessment of delivery strategies. Neotrop Entomol. 2011;40: 368–374. 10.1590/S1519-566X2011000300010 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref042] 42.Yiğit Ş, Oksal E, Atay T, Akça İ, Kepenekci İ. Effect of Entomopathogenic Fungus, Purpureocillium lilacinum (syn: Paecilomyces lilacinus) on Blatta orientalis and Shelfordella tartara under Laboratory Conditions. 6th Entomopathogens & Mocrobial Control Congres. 2017. [Google Scholar]

[pone.0235785.ref043] 43.Bania J, Samborski J, Bogus M, Polanowski A. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. Arch Insect Biochem Physiol. 2006;62: 186–196. 10.1002/arch.20134 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref044] 44.Freimoser FM, Screen S, Hu G, St. Leger R. EST analysis of genes expressed by the zygomycete pathogen Conidiobolus coronatus during growth on insect cuticle. Microbiology. 2003;149: 1893–1900. 10.1099/mic.0.26252-0 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref045] 45.Wieloch W, Boguś MI, Ligeza M, Koszela-Piotrowska I, Szewczyk A. Coronatin-1 isolated from entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella hemocytes in vitro and forms potassium channels in planar lipid membrane. Toxicon. 2011;58: 369–379. 10.1016/j.toxicon.2011.07.007 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref046] 46.Włóka E. Characterization of proteo-, chitino- and lipolytic enzymes of parasitic fungus Conidiobolus coronatus. Wiadomości Parazytol. 2010;56: 83–85. [PubMed] [Google Scholar]

[pone.0235785.ref047] 47.Wrońska AK, Boguś MI, Włóka E, Kazek M, Kaczmarek A, Zalewska K. Cuticular fatty acids of Galleria mellonella (Lepidoptera) inhibit fungal enzymatic activities of pathogenic Conidiobolus coronatus. PLoS One. 2018;13 10.1371/journal.pone.0192715 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0235785.ref048] 48.Kramer KJ, Christensen AM, Morgan TD, Schaefer J, Czapla TH, Hopkins TL. Analysis of cockroach oothecae and exuviae by solid-state 13C-NMR spectroscopy. Insect Biochem. 1991;21: 149–156. 10.1016/0020-1790(91)90045-G [DOI] [Google Scholar]

[pone.0235785.ref049] 49.Mosson HJ, Short JE, Schenkerb R, Edwardsa JP. The effects of the insect growth regulator lufenuron on oriental cockroach, Blatta orientalis, and german cockroach, Blattella germanica, populations in simulated domestic environments. Pestic Sci. 1995;45: 237–246. 10.1002/ps.2780450307 [DOI] [Google Scholar]

[pone.0235785.ref050] 50.Pryor MGM. On the hardening of the ootheca of Blatta orientalis. Proc R Soc London Ser B—Biol Sci. 1940;128: 378–393. 10.1098/rspb.1940.0017 [DOI] [Google Scholar]

[pone.0235785.ref051] 51.Kaya M, Muhammad M, Akyuz B, Bulut E, Sofi K, Zelencova L, et al. Extraction and characterization of chitins from coachroach Ootheca. 9 th International Scientific Conference THE VITAL NATURE SIGN. Kaunas, Lithuania; 2015. 10.13140/RG.2.1.4307.4405 [DOI]

[pone.0235785.ref052] 52.Nation Sr. JL. Insect Physiology and Biochemistry. 3rd ed CRC Press; 2015. [Google Scholar]

[pone.0235785.ref053] 53.Sugumaran M. Chemistry of Cuticular Sclerotization. In: Simpson SJ, editor. Advances in Insect Physiology. Academic Press; 2010. pp. 151–209. [Google Scholar]

[pone.0235785.ref054] 54.Stránský K, Ubik K, Holman J, Streibl M. Chemical composition of compounds produced by the pea aphid Acyrthosiphon pisum (HARRIS): Pentane extract of surface lipids. Collect Czechoslov Chem Commun. 1973;38: 770–780. 10.1135/cccc19730770 [DOI] [Google Scholar]

[pone.0235785.ref055] 55.Paszkiewicz M, Sikora A, Boguś MI, Włóka E, Stepnowski P, Gołębiowski M. Effect of exposure to chlorpyrifos on the cuticular and internal lipid composition of Blattella germanica males. Insect Sci. 2016;23: 94–104. 10.1111/1744-7917.12200 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref056] 56.Raafat I, Meshrif WS, Husseiny EM El, El-Hariry M, Seif AI. Nezara viridula (Hemiptera: Pentatomidae) Cuticle as a Barrier for Beauveria bassiana and Paecilomyces sp. Infection. African Entomol. 2015;23: 75–87. 10.4001/003.023.0128 [DOI] [Google Scholar]

[pone.0235785.ref057] 57.Gutierrez AC, Gołębiowski M, Pennisi M, Peterson G, García JJ, Manfrino RG, et al. Cuticle Fatty Acid Composition and Differential Susceptibility of Three Species of Cockroaches to the Entomopathogenic Fungi Metarhizium anisopliae (Ascomycota, Hypocreales). J Econ Entomol. 2015;108: 752–760. 10.1093/jee/tou096 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref058] 58.Gołębiowski M, Cerkowniak M, Ostachowska A, Boguś MI, Stepnowski P. Determination of cuticular and internal fatty acids of Chorthippus brunneus males and females using HPLC-LLSD and GC–MS. Biomed Chromatogr. 2016;30: 1318–1323. 10.1002/bmc.3688 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref059] 59.Babiarz E, Boguś MI, Czygier M, Kucińska J, Samborski J, Szafranek J. Influence of free fatty acids on growth, sporulation and virulence of the parasitic fungus Conidiobolus coronatus. Wiadomości Parazytol. 2001;47: 763–768. [PubMed] [Google Scholar]

[pone.0235785.ref060] 60.Gołębiowski M, Cerkowniak M, Urbanek A, Dawgul M, Kamysz W, Boguś MI, et al. Antimicrobial activity of untypical lipid compounds in the cuticular and internal lipids of four fly species. J Appl Microbiol. 2014;116: 269–287. 10.1111/jam.12370 [DOI] [PubMed] [Google Scholar]

[pone.0235785.ref061] 61.Young HP, Larabee JK, Gibbs AG, Schal C. Relationship between tissue-specific hydrocarbon profiles and lipid melting temperatures in the cockroach Blattella germanica. J Chem Ecol. 2000;26: 1245–1263. 10.1023/A:1005440212538 [DOI] [Google Scholar]

[pone.0235785.ref062] 62.Callaghan AA. Light and spore discharge in Entomophthorales. Trans Br Mycol Soc. 1969;53: 87–97. 10.1016/s0007-1536(69)80010-0 [DOI] [Google Scholar]

[pone.0235785.ref063] 63.Adler-Nissen J. Determination of the Degree of Hydrolysis of Food Protein Hydrolysates by Trinitrobenzenesulfonic Acid. J Agric Food Chem. 1979;27: 1256–1262. 10.1021/jf60226a042 [DOI] [PubMed] [Google Scholar]

PERMALINK

The interaction between cuticle free fatty acids (FFAs) of the cockroaches Blattella germanica and Blatta orientalis and hydrolases produced by the entomopathogenic fungus Conidiobolus coronatus

Agata Kaczmarek

Mieczysława Irena Boguś

Emilia Włóka

Anna Katarzyna Wrońska

Anna Krawiel

Michalina Kazek

Katarzyna Zalewska

Katarzyna Kłocińska-Biały

Martyna Sobocińska

Aleksandra Gliniewicz

Ewa Mikulak

Marta Matławska

Roles

Abstract

Introduction

Results

Susceptibility of cockroaches to fungal infection

Table 1. The susceptibility of B. orientalis and B. germanica to fungal infection.

Enzyme activity in cell-free C. coronatus filtrate

Hydrolysis of cuticular proteins by C. coronatus enzymes

Fig 1. Hydrolysis of cuticular protein by C. coronatus proteases.

Hydrolysis of cuticular chitin by C. coronatus enzymes

Fig 2. Hydrolysis of cuticular chitin by C. coronatus chitinases.

Hydrolysis of cuticular lipids by C. coronatus enzymes

Fig 3. Hydrolysis of cuticular lipids by C. coronatus lipases.

GC-MS analyses of cuticular FFAs

Table 2. Numbers of B. orientalis and B. germanica used here and mass of the extracts.

Table 3. Fatty acid contents in the cuticular lipids (sum of I and II extracts) extracted from the whole adults, oothecae, wings and thoraces of B. orientalis and B. germanica (μg/g of insect body or structure) and their antifungal activity.

Fig 4.

GC-MS analyses of internal FFAs

Table 4. Fatty acid contents in the internal lipids (extract III) of the adults, oothecae, wings and thoraces of B. orientalis and B. germanica (μg/g of insect body or structure) and their antifungal activity.

Correlations between rates of cuticle hydrolysis and FFAs profiles

Table 5. Correlation between the concentration of compounds identified in the cuticle of two cockroach species and the efficiency of C. coronatus proteases, chitinases and lipases in degrading the cockroach cuticle.

Table 6. Correlation between the concentration of internal FFAs of two cockroach species and the efficiency of C. coronatus proteases, chitinases and lipases in degrading the cockroach cuticle.

Discussion

Materials and methods

Insects

Entomopathogenic fungus

Infection of insects with C. coronatus

Cuticle preparation

Enzymatic assays

Determination of protein concentration

Hydrolysis of insect cuticle incubated with cell-free filtrate of C. coronatus

Extraction of free fatty acids (FFAs)

Derivatization method

GC-MS analyses

Statistics

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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