Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Management of the American cockroach’s oothecae: The potential of entomopathogenic fungi control Mariah Valente Baggio-Deiblera,⁎, Marcelo da Costa Ferreiraa, Antonio Carlos Monteirob, Andressa de Souza-Polloc, Manoel Victor Franco Lemosd a Department of Plant Protection, UNESP, Jaboticabal, SP, Brazil bDepartment of Plant Production, UNESP, Jaboticabal, SP, Brazil c Department of Preventive Veterinary Medicine and Animal Reproduction, UNESP, Jaboticabal, SP, Brazil d Department of Applied Biology, UNESP, Jaboticabal, SP, Brazil A R T I C L E I N F O Keywords: Biocontrol Periplaneta americana Ovicide Ootheca Extrusion A B S T R A C T The Periplaneta americana species is an annoyance to man, causing allergies and damage to clothes and docu- ments. It has the ability to spread pathogens and requires control measures. Control with natural enemies is less aggressive and can currently be applied with less risk than other techniques, such as chemical control, which is the main method used worldwide to control its post-embryonic stages. The potential microbial control of nymphs and adults of this pest has been shown, but little is known about its oothecae. There are isolates of fungal species that can be used to achieve this aim, but they may have innate differences in their virulence and ability to spread. This study aimed to identify fungal isolates JAB 68 and IBCB 35 through genetic sequencing of the ITS1- 5.8S-ITS2 region, analyze their ability to synthesize chitinase, and investigate and compare their aggressiveness against P. americana oothecae and their influence on nymph eclosion. Fungal suspensions were inoculated into minimal medium containing glucose (control) as the sole carbon source and 1% colloidal chitin to determine the chitinolytic activity on the 4th, 7th and 10th days and sporulation on the 10th day. To obtain mortality, ex- trusion and the compiled number of hatched nymphs, oothecae were sprayed with suspensions of the isolates as follows: T1 – no application; T2 – aqueous solution of Tween 80® 0.1% (vehicle suspension for treatments T3 to T8); T3 – 2×109 conidia/mL of the JAB 68 isolate; T4 – 2×108 con./mL of the JAB 68 isolate; T5 – 2×107 con./mL of the JAB 68 isolate; T6 – 2×109 con./mL of the IBCB 35 isolate; T7 – 2×108 con./mL of the IBCB 35 isolate; T8 – 2×107 con./mL of the IBCB 35 isolate. The JAB 68 and IBCB 35 isolates were identified as belonging to the species Metarhizium anisopliae and Beauveria bassiana, respectively. Chitinolytic activity and extrusion were good parameters for evaluating the fungi’s action on oothecal control. The most aggressive en- tomopathogen was M. anisopliae isolate JAB 68, with shorter time for fungus extrusion at a concentration of 2×107 con./mL. B. bassiana reduced the number of hatched nymphs at a concentration of 2×108 con./mL. Both fungi are capable of infecting and killing P. americana’s oothecae and reducing the number of nymphs hatched. 1. Introduction Periplaneta americana (Linneaus, 1785) (Blattodea: Blattidae) are cosmopolitan cockroaches that occur frequently in urban sewage gal- leries and transit in anthropic environments, spreading pathogens; they are considered hazardous organisms to humans causing serious health problems such as allergies, asthma, and others (Thyssen et al., 2004; Pinto et al., 2007; Kassiri and Kazemi, 2012). These insects have ovi- parous reproduction with sets of 7–16 eggs inside of ovate capsules named oothecae. Females can lay approximately 27 oothecae per year, and 32 days is the average incubation period for eggs at 30 °C (Vianna et al., 2001), which clearly shows the great multiplication ability of this insect. P. americana control has been primarily accomplished by the ap- plication of chemical insecticides, either in liquid or powder formula- tions, targeting its post-embryonic stages because of the difficulty of these products have penetrating through ootheca to reach the eggs. P. americana oothecae are composed of proteins (87%), oxalate (8%), di- phenol (4%), lipids (1%) and calcium (6.5%) (Hackman and Goldberg, 1960; Kramer et al., 1991). The oxalate and calcium are responsible for https://doi.org/10.1016/j.jip.2018.02.008 Received 15 April 2017; Received in revised form 22 November 2017; Accepted 9 February 2018 ⁎ Corresponding author. E-mail address: mariahbdeibler@gmail.com (M.V. Baggio-Deibler). Journal of Invertebrate Pathology 153 (2018) 30–34 Available online 10 February 2018 0022-2011/ © 2018 Elsevier Inc. All rights reserved. T http://www.sciencedirect.com/science/journal/00222011 https://www.elsevier.com/locate/jip https://doi.org/10.1016/j.jip.2018.02.008 https://doi.org/10.1016/j.jip.2018.02.008 mailto:mariahbdeibler@gmail.com https://doi.org/10.1016/j.jip.2018.02.008 http://crossmark.crossref.org/dialog/?doi=10.1016/j.jip.2018.02.008&domain=pdf hardening the structure, making the passage of chemicals more diffi- cult. Nonetheless, the high protein content can facilitate penetration of entomopathogenic fungi, considering the proteolytic activity of species such as Metarhizium anisopliae (Metschnikoff, 1879) and Beauveria bassiana (Balsamo) Vuillemin (1912) (Petlamul and Prasertsan, 2012) that are used in biological control programs. Therefore, the discovery of a new strategy to control the eggs of this pest, such as microbial control with fungus, could reduce the number of nymphs and adults of this organism in the environment, preventing risks of disease dissemination or of chemical contamination by the pesticides used to control them in the internal and external environments. Intra- and inter-specific differences between fungi in pathogenicity and virulence to insects may occur depending on the species and the isolate used (Hubner-Campos et al., 2013; Islam et al., 2014). That is the reason why species identification is needed. One acceptable tool used for this purpose is genetic sequencing of the ITS1-5.8S-ITS2 region (Souza et al., 2013). The choice of entomopathogens as an insect control agent is per- formed using mortality and lethal time parameters. However, fungi may have other important differences, such as sporulation on the host's body (Alves and Lecuona, 1998) and secretion of enzymes such as chitinases related to deposition and degradation of chitin, hatching of eggs and insect exoskeleton degradation (Xiuli et al., 1988), which can be cor- related with the isolates’ virulence and pathogenicity (Pelizza et al., 2012). Despite the lack of chitin in the composition of oothecae, fungal chitinolytic activity study can be an interesting tool to determine dif- ferences in fungal aggressiveness toward the host prior to conducting experiments aiming at controlling the pest in field conditions. Only a few studies in the literature have reported fungal patho- genicity to cockroach oothecae. Isolates of Metarhizium spp. and B. bassiana have been considered the most promising for the control of P. americana oothecae (Mohan et al., 1999; Hubner-Campos et al., 2013). Penetration of Aspergillus westerdijkiae (Frisvad and Samson, 2004) through the integument of P. americana’s oothecae was shown by Baggio et al. (2016). The aim of this study was to identify fungal isolates JAB 68 and IBCB 35, analyze their ability to synthesize chitinase, and investigate and compare their aggressiveness against P. americana oothecae and their influence on nymph eclosion. 2. Material and methods The oothecae used were obtained from the mass rearing of the Laboratory of Nucleus Studies and Application Technology Development (NEDTA) Department of Plant Protection, Faculdade de Ciências Agrárias e Veterinárias (FCAV) from the Universidade Estadual Paulista (UNESP), Jaboticabal – São Paulo State, Brazil. The isolates JAB 68 and IBCB 35 were provided by the Applied Microbiology Laboratory of the Department of Plant Production, FCAV, UNESP, Jaboticabal. 2.1. Identification of fungal isolates by genetic sequencing of the ITS1-5.8S- ITS2 region Fungi were cultured for 7 days in 40mL of potato dextrose medium at 27 °C in the dark. The mycelium of each isolate was filtered and placed to dry at 60 °C for 12 h. They were macerated with liquid ni- trogen and DNA extraction was performed according to the Kuramae- Izioka Protocol (1997) with DNA precipitation in absolute ethanol. Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCC TCCGCTTATTGATAT GC-3′) (White et al., 1990) were used to amplify the fragment of ITS1-5.8S-ITS2. For PCR reactions, a 1× buffer, 2 mM MgCl2, 0.2 mM dNTPs, 1.0 U of Taq DNA polymerase (Invitrogen), 4 pmol primer, 60 ng of genomic DNA and ultrapure water were used to obtain a final volume of 20 µL. The amplification reactions were per- formed in a Nexus thermocycler (Eppendorf) using 1 cycle at 95 °C for 3min, 36 cycles at 94 °C for 40 s, 60 °C for 40 s and 72 °C for one minute and 1 cycle at 72 °C for 10min. The amplified DNA fragment was subjected to sequencing PCR using BigDye Terminator v3.1 (Applied Biosystems) according to the manu- facturer’s instructions. The primers used were the same ones used for the amplification step. DNA sequencing was performed on the ABI 3100 sequencer (Applied Biosystems). The sequences obtained were eval- uated using PHRED/PHRAP/CONSED (Ewing and Green, 1998; Green, 1996; Gordon et al., 1998). Sections of contiguous sequences of bases with quality equal or superior to 20 were accepted and used for ana- lysis. The obtained nucleotide sequences were compared to GenBank database sequences accessed through the National Center for Biotechnology Information (NCBI). For phylogenetic relationships analysis, ITS sequences from this study and others from GenBank were aligned by MUSCLE (Edgar, 2004) with the software MEGA 6.06 (Tamura et al., 2013). The same program was used to determine the most suitable evolutionary model to be ap- plied to sequences in Bayesian analysis using the Akaike Information Criterion (AIC) (Posada and Buckley, 2004). The phylogenetic tree was generated by MrBayes 3.2.3 software (Ronquist and Huelsenbeck, 2003) set for the Kimura-2 substitution model (Kimura, 1980) and Gamma distribution. The Markov Chain Monte Carlo (MCMC) algo- rithm performed analysis with four chains simultaneously, three hot and one cold. Four separate runs were performed with 1,000,000 generations, and the chains were sampled every 200 generations. At the analysis end, 25% of the trees were discarded as burn-in, considering a desirable standard deviation corresponding to less than 0.01. The tree generated by MrBayes was edited graphically by TreeGraph 2.3.0 software (Stöver and Müller, 2010). 2.2. Chitinolytic activity of fungal isolates Fungi were inoculated into minimal medium (Pontecorvo et al., 1953) with the only carbon sources being glucose (control) and 1% colloidal chitin. Commercial chitin (Sigma-Aldrich) was hydrolyzed with fuming hydrochloric acid (Merck) with sequential washes using deionized water to obtain colloidal chitin (Hankin and Anagnostakis, 1975). The Petri dishes were stored at 27 ± 0.5 °C in the dark. The diameter of fungal colonies and halos indicative of chitinolytic activity were measured with a millimetric ruler at 4, 7 and 10 days of incubation. With these data, the enzymatic index of chitin degradation was calculated, which corresponds to the ratio between the diameter of the colony and the degradation halo diameter (Rosato et al., 1981), and the lower the value obtained, the greater the chitinolytic activity. On the tenth day of cultivation, the conidia produced by colonies of fungi were collected and counted using a Neubauer chamber. The experimental design was completely randomized with four treatments and five replications. Analysis of variance was performed by F test and comparison of means by Tukey test (p≤ 0.05). 2.3. Infection of Periplaneta americana’s oothecae Oothecae 1–3 days of age were randomly selected, cleaned with damp paper and submitted to the following treatments: T1 – no appli- cation (control); T2 – aqueous solution of Tween 80® 0.1% (vehicle suspensions); T3 – suspension containing 2× 109 conidia/mL of the JAB 68 isolate; T4 – suspension with 2×108 con./mL of the JAB 68 isolate; T5 – suspension with 2×107 con./mL of the JAB 68 isolate; T6 – suspension with 2×109 con./mL of the IBCB 35 isolate; T7 – sus- pension with 2× 108 con./mL of the IBCB 35 isolate; T8 – suspension with 2× 107 con./mL of the IBCB 35 isolate. Groups of 10 oothecae were sprayed with 1mL of conidial suspen- sion with the aid of a manual sprayer. They were placed in Petri dishes containing moistened cotton at a temperature of 27 ± 0.5 °C, RH > 80% and the absence of light. Mortality was assessed daily for M.V. Baggio-Deibler et al. Journal of Invertebrate Pathology 153 (2018) 30–34 31 62 days. Oothecae without fungus extrusion and without hatching nymphs were disinfected with sodium hypochlorite solution (3%), washed with deionized water, dissected and pieces of their internal content de- posited on potato dextrose agar (PDA) medium to determine en- tomopathogen growth and confirm the causal agent of insect death of this stage. Oothecae with no hatching nymphs were considered for total oo- theca mortality analysis. The average time (in days) of fungus extrusion and the total number of hatched nymphs from all ten oothecae of each treatment were also evaluated. Data were compiled and the results submitted to statistical analysis by Sisvar program – V. 5.3. The experimental design was completely randomized with 8 treatments, 5 repetitions per treatment and 10 oo- thecae per repetition. The analysis of variance was conducted by F test in a factorial design, with eight treatments× 2 fungi for ootheca mortality and number of hatched nymphs per treatment, and 6 treat- ments× 2 fungi for the average time of fungal extrusion, with the comparison of means by Tukey test (p≤ 0.05). 3. Results and discussion 3.1. Identification of fungal isolates by genetic sequencing of the ITS1-5.8S- ITS2 region The technique of sequencing the ITS1-5.8S-ITS2 region is known as a precise and laborious method for cataloging molecular differences among fungi (Xu, 2006), and it proved to be an efficient tool for identifying the fungal species of this work. The nucleotide sequences of isolates JAB 68 and IBCB 35 aligned with 100% similarity to the GenBank sequences of M. anisopliae and B. bassiana, respectively. The phylogram presented three main groups, one group composed of sequences ofMetarhizium spp., the second composed of Beauveria spp. and the third was the external grouping (Fig. 1). The sequences were deposited in GenBank with the following encodings: KF958306 Metarhizium anisopliae JAB 68 and KF958305 Beauveria bassiana IBCB 35. In the phylogram, M. anisopliae JAB 68 grouped with other Metarhizium species from GenBank but presented genetic distance from them (Fig. 1). The same was observed for isolate IBCB 35, which grouped with other isolates of Beauveria from GenBank, also showing genetic distance from them. This result indicates that the JAB 68 and IBCB 35 isolates from Brazil could be identified by genetic sequencing of the ITS1-5.8S-ITS2 region (Fig. 1). Other studies also used the sequencing of the ITS1-5.8S-ITS2 region to identify isolates of B. bassiana and M. anisopliae with potential use in controlling insects such as Ostrinia nubilalis (Lepidoptera: Pyralidae) (Demir et al., 2012), Galleria mellonella larvae (Lepidoptera: Pyralidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) (Mora et al., 2016). 3.2. Chitinolytic activity of fungal isolates The analyzed fungal isolates showed differences in enzyme pro- duction and sporulation. The chitinolytic activity of B. bassiana isolate IBCB 35 was significantly higher (0.64) on the fourth day when com- pared with the JAB 68 isolate ofM. anisopliae (0.84), but on the seventh and tenth days of culture, no significant difference of enzyme synthesis capacity was shown between fungi. The amount of conidia produced by isolate IBCB 35 in medium containing chitin was smaller than that produced by the isolate JAB 68 grown in the same medium, suggesting that JAB 68 has a greater capacity to channel the nutritional benefits of chitin lysis for conidiogenesis. Chitinase secretion differs depending on the fungal isolate, and it decreases after 96 h of incubation (Petlamul and Prasertsan, 2012) as shown in Table 1. This occurs because the structure of the cell wall of these microorganisms is mainly composed of chitin (Seidl, 2008) and when in excess in their cells, chitinases can be harmful to secreting fungi. 3.3. Infection of Periplaneta americana oothecae Both fungi were able to infect the oothecae of P. americana and showed similar results for total mortality (F= 23.6055, df= 7, p < 0.0001) (Fig. 2). The entomopathogens sprayed on P. americana oothecae caused great mortality, differing from the control with no fungus application and the negative control (Tween 80® 0.1%). There was no significant difference in insect mortality considering the evaluated fungal con- centrations and species of fungi. The results were similar to a study of the horizontal transmission of M. anisopliae in Blattella germanica (Linnaeus, 1767) in which ootheca viability was reduced 48–85% (Quesada-Moraga et al., 2004). Hubner-Campos et al. (2013) found a lower percentage of P. americana oothecae killed by fungi than that presented in this work, with approximately 31% mortality for isolate IP Fig. 1. Bayesian analysis of ITS sequences of isolates JAB 68 and IBCB 35 and species-type sequences from the GenBank database showing clustering of isolates according to their genetic similarity. Numbers between branches refer to Bayesian support values. M.V. Baggio-Deibler et al. Journal of Invertebrate Pathology 153 (2018) 30–34 32 46 of M. anisopliae and 23% mortality for IP 34 of M. robertsii at the concentration 5×105 con./mL. Promising results have been verified for a fungal isolate of Aspergillus westerdijkiae species, confirming its infection, colonization and extrusion on oothecae of P. americana with an ootheca mortality rate of 60% caused by the fungus at a concentration of 3×108 con./mL (Baggio et al., 2016). There was no relationship between the concentration of fungal conidia and extrusion time (Treatment F=3.821, df= 2, p=0.0363, Fungus: F= 68.762, df= 1, p < 0.0001. Treatment vs Fungi: F= 0.878; df= 2; p= 0.4287), but it was observed that M. anisopliae was more effective, taking from 15 to 21 days for its extrusion on the ootheca surface, while for B. bassiana it occurred 29–32 days after in- oculation (Fig. 3). The same interspecific difference in performance of fungi was found in infections of three species of white grubs (Dhoj-GC et al., 2008), ticks of the species Boophilus annulatus (Say, 1821) (Pirali- Kheirabadi et al., 2007), and the large traces of wax from Galleria mellonella (Linnaeus, 1758) and Tenebrio molitor (Linnaeus, 1758) (Oreste et al., 2012). The total number of hatched nymphs was significantly affected by the fungal species applied (F=15.8670, df= 7, p < 0.0001), with little difference between the conidia concentrations used. The suspen- sion of 2×108 con./mL can be considered the most effective because it provided approximately 75% and 95% reduction in the number of hatched nymphs from the application of M. anisopliae and B. bassiana, respectively, when compared to control (Fig. 4). This event shows the potential for reduction of offspring by the microbial agents used. A Table 1 Enzymatic activity index and sporulation of isolates IBCB 35 of Beauveria bassiana and JAB 68 of Metarhizium anisopliae in minimal medium containing chitin as a specific substrate for detecting chitinolytic activity. Treatments Enzymatic activity index Sporulation1 (× 108 conidia/mm2 colony) 4 days 7 days 10 days No chitin+M. anisopliae – – – 2.08 ± 0.35b Chitin+M. anisopliae 0.84 ± 0.02b 0.90 ± 0.00a 0.90 ± 0.01a 1.25 ± 0.15b No chitin+ B. bassiana – – – 1.71 ± 0.24b Chitin+ B. bassiana 0.64 ± 0.08a 0.94 ± 0.01a 0.90 ± 0.01a 0.22 ± 0.05a F test 13.86 38.71 93.81 14.74 Original mean (± standard error of the mean), but statistical analysis was performed by the F test with 1data transformed into +x 1 . Means followed by the same letter in the column do not differ by Tukey test (p≤ 0.05). For all variables p < 0.0001 and df=3. Fig. 2. Ootheca mortality of Periplaneta americana sprayed with suspensions of different concentrations of isolates JAB 68 of Metarhizium anisopliae and IBCB 35 of Beauveria bassiana under laboratory conditions. Original mean (± standard error of the mean); those followed by the same letter do not differ significantly by Tukey test (p≤ 0.05). The statistical analysis was conducted by F test with data transformed into arcsine x/100 . Fig. 3. Average time for extrusion of isolates JAB 68 of Metarhizium anisopliae and IBCB 35 of Beauveria bassiana on oothecae of Periplaneta americana treated with different concentrations of fungal suspensions in laboratory conditions. Original mean (± stan- dard error of the mean), but statistical analysis was performed by the F test with data transformed into +x( 1) . Similar capital letters between treatments (fungi) and similar lowercase letters between concentrations of conidia tested do not differ statistically by Tukey test (p≤ 0.05). Fig. 4. Total number of hatched nymphs from Periplaneta americana oothecae sprayed with fungal suspensions of different concentrations in laboratory conditions. Original mean (± standard error of the mean), but statistical analysis was performed by the F test with data transformed into +x( 1) . Treatments with the same lowercase letter do not differ by Tukey test (p≤ 0.05). M.V. Baggio-Deibler et al. Journal of Invertebrate Pathology 153 (2018) 30–34 33 similar result was observed for hatched larvae of Choristoneura rosa- ceana (Harris, 1841), decreasing by 90% following the treatment of eggs with suspensions of B. bassiana at a concentration of 1×108 con./ mL (Cossentine, 2014). Cockroach control is a worldwide problem, especially in the em- bryonic phase, due to a lack of chemical products available to kill this specific stage. Therefore, it requires new combat strategies involving new research. The promising results of this study showed that M. ani- sopliae and B. bassiana caused high mortality to P. americana oothecae in all evaluated conidia concentrations, suggesting that they can be successfully used in the control of this insect’s embryonic stage as an alternative to chemical control. However, control of this stage of the insect's life cycle is still a challenge, especially in field conditions, be- cause the environments these insects inhabit probably exert a great influence on the pathogenic action of fungi, suggesting the need for further specific studies for field application. 4. Conclusion The fungal isolates JAB 68 and IBCB 35, identified as Metarhizium anisopliae and Beauveria bassiana species, respectively, are pathogenic to the eggs of Periplaneta americana. Their chitinolytic activity and ex- trusion were good parameters in evaluating the action of the fungi on ootheca control. Both fungi are capable of infecting and killing oo- thecae and reducing the number of nymphs hatched. There is high potential for using conidial suspensions of these isolates to control the insect, leading to a possible reduction of chemical applications to control its post-embryonic stages. The concentration of the conidia suspensions used exerted little influence on the fungal action. The most aggressive isolate was JAB 68 of M. anisopliae at a concentration of 2× 107 con./mL, presenting shorter extrusion time on oothecae. Isolate IBCB 35 of B. bassiana significantly reduced the number of hatched nymphs at a concentration of 2× 108 con./mL. Acknowledgements To Ana Carolina Peterossi, Dinalva Alves Mochi and all the students from NEDTA-FCAV/UNESP for helping conduct the experiments. To Jake Deibler from Rent-a-Hero Studios LLC for providing the graphic design of the graphical abstract. 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http://refhub.elsevier.com/S0022-2011(17)30154-4/h9010 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0165 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0165 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0165 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0170 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0170 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0170 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0170 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0175 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0175 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0175 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0180 http://refhub.elsevier.com/S0022-2011(17)30154-4/h0180 Management of the American cockroach’s oothecae: The potential of entomopathogenic fungi control Introduction Material and methods Identification of fungal isolates by genetic sequencing of the ITS1-5.8S-ITS2 region Chitinolytic activity of fungal isolates Infection of Periplaneta americana’s oothecae Results and discussion Identification of fungal isolates by genetic sequencing of the ITS1-5.8S-ITS2 region Chitinolytic activity of fungal isolates Infection of Periplaneta americana oothecae Conclusion Acknowledgements References