Is Isaria fumosorosea selective to Trichogramma pretiosum (Hymenoptera: Trichogrammatidae)?

Entomopathogenic fungi and the egg parasitoid Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) might be used together in biological control. However, the effects of these fungi on T. pretiosum are not known. Thus, this study aimed to determine the effect of the entomopathogenic fungus Isaria fumosorosea, on the biological parameters of T. pretiosum. Two isolates of I. fumosorosea (IBCB 367 and IBCB 394) were used for this purpose. (1) In a free choice test: cards (1.0 × 5.0 cm) with non-parasitized eggs of Anagasta kuehniella Zeller (Lepidoptera: Pyralidae) were either sprayed with 0.2 mL of the fungus suspension (1.0 × 109 conidia.mL-1) or with sterile distilled water containing Tween® 80 (0.01%), which were then offered to females of T. pretiosum. (2) No choice test: the isolates were sprayed at a concentration of 1.0 × 109 conidia.mL-1 on cards (1.0 × 5.0 cm) with A. kuehniella eggs. The control consisted of spraying sterile distilled water containing Tween® 80 (0.01%). Individual females of T. pretiosum were confi ned for 24 h with the cards. The number of eggs parasitized, percentage of emergence, longevity, duration of the egg-adult period and sex ratio were evaluated, as well as the longevity of the females that parasitized the eggs and the mortality of the emerging adults evaluated. IBCB 367 isolate repelled T. pretiosum. The pre-parasitism and post-parasitism sprays did not affect the number of eggs parasitized or the sex ratio, however, the pre-parasitism IBCB 394 treatment the females and males survived for longer, whereas the survival of females in post-parasitism treatment with the same isolate was reduced. The presence of conidia on and mycelium of the fungus in T. pretiosum was confi rmed using Scanning Electron Microscopy and a histological analysis. Isolates IBCB 367 and IBCB 394 from I. fumosorosea are selective to T. pretiosum in the laboratory.

When the cards were dry, one card spayed with the fungal isolate and another sprayed with water, the control, were identifi ed and fi xed with honey syrup inside a sterilized fl at bottom glass tube (8.0 cm × 2.5 cm Ø), each tube is a repetition, and 20 glass tubes were used for each treatment. Into each tube, a 24-h-old female (fed and mated) was placed and kept there for one day in the same climatic chamber (26 ± 2°C, 14 h photoperiod and R.H. 70 ± 10%). According to Nogueira de Sá & Parra (1994), Inoue & Parra (1998) and Pratissoli et al. (2005Pratissoli et al. ( , 2006, temperatures between 20 and 30 are the best for the parasitism of T. pretiosum, including on A. kuehniella eggs. All experiments were carried out under the above conditions. For each isolate and respective control there were 20 repetitions. The percentage parasitism by T. pretiosum was evaluated by comparing each isolate with the respective control. The results were analysed using Wilcoxon non-parametric test in the statistical program BioEstat 5.0 ® (Ayres et al., 2003).

Parasitism by T. pretiosum in no choice test
Pre-parasitism spraying: The suspensions of the isolates were sprayed on cards (1.0 × 5.0 cm), containing approximately 200 sterile and non-parasitized eggs of A. kuehniella, with 20 cards per isolate and 20 cards used for the control. After spraying and drying, the cards were offered to females of T. pretiosum (fed and mated), for 24 h.
The biological parameters evaluated in both bioassays were the number of blackened eggs of A. kuehniella eggs (parasitism signal) (Cônsoli et al., 1999), percentage of eggs from which adult parasitoids emerged, longevity of emerged adults, duration of the egg-adult period and sex ratio, as recorded by Hassan (1997) and using the equations proposed by Potrich et al. (2015). The longevities of the females of T. pretiosum that parasitized the eggs were recorded and, after death they were placed in a humid chamber to confi rm they were killed by the fungus.
The data were subjected to an analysis of variance and the means were compared using the Kruskal-Wallis non-parametric test. The relationship between the treatments (pre-and post-parasitism) was compared using the non-parametric Mann-Whitney test (two independent samples ) in the statistical program Bio-Estat 5.0 ® (Ayres et al., 2003).

Histological analyses of T. pretiosum
The females of T. pretiosum used were those that were confi ned with cards either with eggs of A. kuehniella sprayed with one of the two isolates of I. fumosorosea or their respective controls. The parasitized eggs that were used, were those from which adults had not emerged. Both came from the no choice parasitism test.
The samples were fi xed in Bouin and dehydrated in an alcohol series and were later cleared by immersing them in xylol. After complete dehydration, paraffi nization and embedding in blocks of histological paraffi n wax (Histological Paraffi n / Beeswax 4 : 1), a microtome was used to cut 2 μm thick sections from the blocks with material, which were stained using the H/E method (Hematoxylin / Eosin).
The microscope slides with the sections were examined and photographed using an optical Stereo Microscope Zeiss ® in the However, the increasing use of entomopathogenic fungi has raised concerns about its effects on non-target organisms such as the natural enemies (Magalhães et al., 1998;Sosa-Gómez et al., 1998;Dalvi et al., 2007;Amaro et al., 2015;Oreste et al., 2016;Potrich et al., 2017Potrich et al., , 2018. Thus, it is important that studies on the selectivity of entomopathogenic fungi, used to control whitefl ies, take into consideration their effect on T. pretiosum in order to determine the safety of using both these biocontrol agents together to control pests of tomatoes and other crops. It is important to note that I. fumosorosea is a fungus that has been outstanding in controlling whitefl y and for which there are several commercial isolates.
Thus, it is important to know whether interactions between these control agents when applied simultaneous increase or decrease the effi ciency of biological control. Thus, this study aimed to determine the effect of the entomopathogenic fungus, I. fumosorosea, on the biological parameters of T. pretiosum.

Preparation of a monosporic culture of I. fumosorosea and the suspension used
Two isolates of I. fumosorosea were used, IBCB 367, which was isolated from soil from a coffee platation at Tabapuã -SP, and IBCB 394, isolated from soil from a sugar plantation at Espírito Santo do Pinhal -SP. These isolates were multiplied in sporulation medium (M.E.) in Petri dishes and incubated at 26 ± 2°C, 14 h photophase and 70 ± 10% RH, for eight days. Conidia were then collected using a sterilized spatula and stored in glass tubes in a freezer at -10°C .
To obtain monosporic cultures, conidia were suspended in 10 mL of sterile distilled water containing Tween ® 80 (0.01%). The suspension was stirred for one minute by vortexing and quantifi ed using a Neubauer's chamber (1.0 × 10 2 conidia.mL -1 ). Then, 0.1 ml was spread using a Drigalsky loop in Petri dish containing the culture medium M.E. and incubated for eight days under the same conditions as described above. Subsequently, in order, to obtain a monosporic culture a colony was transferred to another plate. Suspensions for use in the bioassays were prepared using sterile distilled water containing Tween ® 80 (0.01%), shaken and quantifi ed in Neubauer's chamber (1.0 × 10 9 conidia.mL -1 ).

Parasitism by T. pretiosum in free choice test
The treatments were sprayed on two cards of 1.0 × 5.0 cm, with approximately 200 non-parasitized eggs of A. kuehniella, using a Pneumatic Sagyma ® airbrush coupled to a Fanem ® constant pressure pump, 1.2 kgf.cm -1 (procedure also used in other experiments) at a fi xed distance of 30 cm with lateral protection. Of the fungus suspensions (1.0 × 10 9 conidia.mL -1 ) 0.2 mL was sprayed on one of the cards and sterile distilled water containing Tween ® 80 (0.01%) was sprayed on the other card, this volume was suffi cient to cover the eggs on each card. The procedures followed were those used by Potrich et al. (2015).
Laboratory of Photomicroscopy of Unioeste -Câmpus Cascavel. The tissues of the parasitoids sprayed with the isolates were compared with the tissues of the uninfected parasitoids (fom control), in addition to the spot (tissue in adults or tissue and vitelum in eggs) that showed growth of the fungus.

Scanning Electron Microscopy (SEM) study
The suspensions of entomopathogenic fungi (1.0 × 10 9 conidia/ mL) were sprayed on cards as described above. Females (24-hold) of T. pretiosum (fed and mated) were confi ned with these cards for 1 day. The same procedure was followed for the controls. After this period, the parasitoids were prepared for studying using a SEM.
The samples (20 T. pretiosum females per treatment) were fi xed for 4 h in a solution consisting of 2% Paraformaldehyde, 2% Glutaraldehyde and Phosphate Buffer (PO 4 0.1 M). Then they were washed in phosphate buffer and fi xed in 1% Osmium Tetroxide solution (OsO 4 ) for 2 h and then washed in Phosphate Buffer. After this procedure, the samples were dehydrated with a fi nal dehydration using CO 2 at the Critical Point. The samples were mounted on metallic supports (stubs) with silver glue under a Zeiss ® Stereo Microscope. The stubs with the samples were sputter coated with gold using a BAL-TEC metallizer SCD-050 and then viewed under a SEM at high vacuum and an electron beam intensity of 20 KV, with the images recorded as digital photomicrographs. Samples of the material sprayed with the two isolates of the entomopathogenic fungus were compared with the control material, in terms of the presence / absence of conidia on the body of the parasitoid and the places where these conidia were located.

P arasitism by T. pretiosum in choice test
When T. pretiosum females had a choice, they preferred to parasitize eggs of A. kuehniella that were not treated with IBCB 367. However, the same behaviour was not recorded for isolate IBCB 394, which was not repellent to T. pretiosum (Table 1).

Parasitism by T. pretiosum in no choice test
There was no difference in the number of parasitized A. kuehniella eggs when they were treated with both I. fumosorosea isolates, prior or after parasitism, in relation to the control. There was also no difference in the number of parasitized eggs when compared to prior spraying times or after parasitism (Table 2).
When the isolates IBCB 367 and IBCB 394 were sprayed on A. kuehniella eggs prior to parasitism, they did not affect the percentage from which adults of T. pretiosum emerged, their sex ratio or the egg-adult developmental time of male and female T. pretiosum. Moreover, the longevities of the females and males that emerged from eggs treated with isolate IBCB 394 was longer than those of the control (Table 3).
Both isolates of I. fumosorosea sprayed on A. kuehniella eggs after parasitism by T. pretiosum did not affect the biological parameters of the adult parasites that emerged. However, isolate IBCB 394 caused a signifi cative reduction in the longevity of females (Table 3).
Comparing the biological parameters of T. pretiosum that emerged from A. kuehniella eggs sprayed before and after parasitism, confi rmed that the longevity of T. pretiosum females and males was reduced when the eggs were sprayed after they were parasitized ( Table 3). The percentage emergence was also lower but only in the IBCB 394 treatment.
The longevity of the females that came into contact with eggs sprayed with isolates of I. fumosorosea was not affected (Table 4). Of the females that parasitized the eggs and came into contact with the isolates of I. fumosorosea, 20% of those that came into contact with isolate IBCB 394 and 55% with IBCB 367 were killed by the fungus.

Histological analyses and SEM study of T. pretiosum
The histology study of T. pretiosum revealed the presence of hyphae, conidia and phialides ( Fig. 1A and B). The tissues with the highest number of hyphae were integument, adipose tissue and nerve tissue (head region), while no hyphae were observed in the muscles. The SEM study revealed conidia in folds in the wings and on the surface of T. pretiosum ( Fig. 2A and B). 19.2 ± 5.14 b p 0.0001 The total number of eggs parasitized by the confi ned female was considered 100% and the percentage on each card (Treatment × Control) was calculated. Averages followed by the same lowercase letter in the column do not differ from each other based on the Wilcoxon test (p < 0.05).

DISCUSSION
The T. pretiosum repellency is not related to the species I. fumosorosea, but to the isolate used, as observed by Isaria sp. IBCB 367 and Isaria sp. IBCB 394 causing differences in the parasitism of T. pretiosum, with IBCB 367 being repellent. Females of T. pretiosum are able to identify substances on host eggs that are attractive, toxic or repellent by walking backwards and forwards over the eggs and touching them with their antennae on which there are sense organs that can detect these substances (Vinson, 1997;Srivastava et al., 2017). Toxic substances produced by the entomopathogenic fungus were probably present in the suspensions, which were obtained by scraping the culture medium to obtain conidia that would have been collected along with pieces of the fungus and the toxins (exotoxins) they contain. Although females of T. pretiosum are able to recognize toxic substances on the surface of eggs (Klomp & Teerink, 1962;Vinson, 1998) they may reject a host egg or may determine its nutritional quality by inserting their ovipositors and if suitable parasitize them.
According to IOBC/WPRS, if a product causes less than a 30% reduction in the parasitism capacity of Trichogramma, it is classifi ed as innocuous to this parasitoid (Hassan, 1997;Hassan et al., 2000;Rampelotti-Ferreira et al., 2017), as the isolates evaluated here are (Table 2).
When the isolate IBCB 394 was applied after parasitism, the small difference in the percentage of adults that  Averages followed by the same letter in a column do not differ signifi cantly from each other based on the Kruskal Wallis test. emerged (the percentage emergence was higher when previously sprayed), although signifi cant, may be due to the eggs of the host being infected and not suitable for the development of the parasitoid. The infection of eggs by the fungus may have been due to the introduction of conidia adhering to the ovipositor of the parasitoid or by the conidia germinating on the surface of the egg and then entering via the hole made by the ovipositor or the direct action of the fungus. Thus, fungus may have penetrated and colonized host eggs internally, consuming the nutrients at the exact stage where they were necessary for the development of the immature phases of the parasitoid especially that the nutritional value of the host egg is directly related to the development of T. pretiosum, mainly of the females of this parasitoid. The sex ratio of T. pretiosum was not affected by the isolates used, which were between 0.61 and 0.74. The same is reported for the parasitoid Trichogramma atopovirilia Oatman & Platner, 1983 (Hymenoptera: Trichogrammatidae), emerging from eggs sprayed with a solution of L. lecanii, for which the sex ratio is 0.81 and does not differ from that recorded in the control (Dalvi et al., 2007). Potrich et al. (2009) also report that the sex ratio of T. pretiosum emerging from A. kuehniella eggs sprayed with M. anisopliae and B. bassiana both before and after being parasitized does no differ from that recorded for the control, which is similar to that recorded in the present study. Using B. bassiana, spraying before or after parasitism, also did not affect the sex ratio of the offspring (Potrich et al., 2015).
No change was recorded in the egg-adult period of males and females of T. pretiosum that emerged from eggs sprayed with the isolates of I. fumosorosea. A previous study by Potrich et al. (2009) also report no difference in the eggadult period of T. pretiosum emerging from eggs sprayed with B. bassiana and M. anisopliae, in comparison with previous or post-parasitism application. This is important since an increase in the egg-adult period would adversely affect the bio-control effectiveness of this parasitoid.
Comparison of the pre-and post-parasitism application of IBCB 367 and IBCB 394 revealed that spraying prior to parasitism resulted in an increase in the longevity of the females and males that emerged, and this higher longevity may be related to parasitoid development in the host egg and indicate that the fungal suspension is detrimental. When host eggs are a rich source of nutrients, large and more vigorous adults may emerge, which live for longer. However, parasitoids that develope in less nutritious eggs, or t hose infected with fungus, may emerge smaller and less vigorous or die, or as a result of coming into contact with the isolate become infected and have a short adult life. A reduction in adult longevity is also reported for the parasitoid Trichogramma galloi Zucchi, 1988 (Hymenoptera: Trichogrammatidae) that emerge from the eggs of the sugarcane boror Diatraea saccharalis (Fabr., 1794) (Lepidoptera: Pyralidae) treated with isolate IPA159E of M. anisopliae (Broglio-Micheletti et al., 2006). Potrich et al. (2017) report that application of isolates of the fungus M. anisopliae affect the longevity of males and females of T. pretiosum similar to the negative effects of the isolates' IBCB 367 and IBCB 394 on the longevity of females after post-parasitism treatments in this study.
However, the isolates of I. fumosorosea may not have resulted in the death of the females that came in contact with sprayed eggs as the fungus may have infected the body of T. pretiosum after death. Death due to infection with entomopathogenic fungi is reported for adults of the parasitoid, Aphelinus asychis Walker (Hymenoptera: Aphelinidae), treated with I. fumosorosea isolate ARSEF 4501 (Mesquita et al., 1999). Dalvi et al. (2007) also do not report a statistical difference in the longevity of T. atopovirilia females that come in contact with L. lecanii and those in the control. Simi- larly, Potrich et al. (2009) andPotrich et al. (2015), report that the longevity of T. pretiosum females that come into contact with isolates of the fungi B. bassiana and M. anisopliae when parasitizing eggs of A. kuehniella is not affected.
Despite the high mortality (55%) of females of T. pretiosum that came into contact with the isolate I. fumosorosea, IBCB 394, and the effects of the post-parasitism treatments' on female longevity, both isolates did not affect the other biological parameters of the adults. In addition, these isolates did not affect the number of eggs parasitized compared to the control.
These results are important since they indicate that the effectiveness of T. pretiosum in parasitizing the eggs of pest insects is not affected. This is also highlighted by Bueno et al. (2009), who emphasize that it is parasitism that determines the effi ciency of biological control in the fi eld. When isolates of M. anisopliae were applied before and after parasitism, they also did not affect the number of eggs parasitized by T. pretiosum or the sex ratio of the emerging adults (Potrich et al., 2017).
The histological study of the immature stages of T. pretiosum revealed indications of infection of different tissues with I. fumosorosea. This may indicate that this entomopathogen can infect eggs and adversely affect the development of T. pretiosum, especially by decreasing the availability nutrients for the development of female parasitoids.
According to Sosa-Gómez et al. (1998) it is logical to infer that the development of a parasitoid within an entomopathogen-infected host is likely to be adversely affected. However, these authors comment that in many cases the parasitoids continuing to develope normally in hosts infected with entomopathogens. If this is the case and it does not affect longevity this is important because short lived females will parasitize fewer eggs of the host and their control effi ciency will be lower.
The SEM study revealed conidia of I. fumosorosea adhering to the body of T. pretiosum that are diffi cult to clean, which renders it vulnerable to infection by entomopathogens. Although this parasitoid can clean itself by rubbing its wings and body with its legs, however, there are regions that are diffi cult to clean, where conidia can adhere and germinate. Joints, intersegmental membranes, folds in the wings, buccal parts and ventral regions, are the most diffi cult for T. pretiosum to clean. Fig. 2 indicates that conidia are present in these areas, which confi rms it is diffi cult for this insect to clean them.
Differences between the isolates IBCB 367 and IBCB 394 may be related to the genetic variability of the isolates, the specifi c toxins and enzymes produced by each of them and the pathogenicity, plus the germination of the fungus on the surface of the egg and the production of metabolites, are factors that also need to be taken into consideration (Leger et al., 1986;Hajek & Leger, 1994). Thus, in the strategies in which parasitoids and isolates are used, spaced sprays must be taken into account due to the sublethal effects that may occur. However, in this study the effects observed in the interaction between entomopathogenic fungi and T. pretiosum, were minimal and could classify the entomopathogenic fungus I. fumosorosea, isolates IBCB 367 and IBCB 394 as selective. Thus, based on the evaluation of several biological parameters of T. pretiosum it is possible to use these two control agents together.

CONCLUSION
Based on the biological parameters analyzed, I. fumosorosea (IBCB 367 and IBCB 394) can be considered selective to T. pretiosum in the laboratory conditions. However, based on isolates negative effects on females that carried out the parasitism and came in contact with the fungus, as well as emerging females lower longevity following post-parasitism treatments, further studies should be conducted.