Management of insects

During 2013, eggs, pupae and adults of C. carnea were provided by CREROB. After receiving them, eggs and pupae were fumigated as indicated, while adults were given antimicrobials in the water they drank. After fumigation, the eggs were incubated at 28 °C and when hatched the larvae were fed with eggs of Sitotroga cerealella Olivier, provided by the CREROB. The pupae were incubated at 28 °C until obtaining the adult at laboratory temperature (20-25 °C).

The adults were fed a mixture of unrefined sugar, milk powder (Nido^®) and dry beer yeast (Industrial Rosanco®) in a ratio of 2:1:1 w/w/w, prepared with distilled water and heating until the consistency of a jelly. The jelly was stored for 15 days at 0 °C. The adults were fed with jelly, on abate-sterilized tongues (renewed every third day) or on the walls of the confinement cells. To the adults, they were provided in the sterile distilled water with the antimicrobials in the head of sterile swabs, replacing them every third day, the control only the water.

Isolation and identification of Bacillus thuringiensis

The adults of C. carnea were superficially sterilized with 1% sodium hypochlorite and macerated in sterile conditions to subsequently recover the macerate in sterile water. To observe the formation of the parasporal bodies under an optical microscope, bacteria were grown in a nutrient medium until sporulation was induced. After three days of growth, the spores and crystals were recovered and analyzed on an SDS-PAGE gel. This macerate was seeded in Petri dishes containing solid Luria-Bertani medium.

The plates were incubated at 30 °C for 24 h and in these different bacteria were identified, among these two isolates named Cc1 and Cc2 were selected and purified, which presented the typical morphology of bacteria belonging to B. thuringiensis. To verify that these bacteria correspond to this species, PCR amplification of the 16S ribosomal gene was analyzed using specific oligonucleotides designed by (^{Aguino de Muro and Priest, 1993}).

To identify both isolates, they were grown for 12 hours in a plate of nutrient medium (Difco). A small volume of cells was transferred to 0.1 ml of H₂O and frozen at -70 °C for 20 min to be then incubated in boiling water for 10 min to lyse the cells. The resulting cell lysate was centrifuged briefly (10 sec at 10 000 rpm in an Eppendorf centrifuge model 5415C) and 15 μl of supernatant was used as a DNA sample in the PCR.

The amplification was carried out in a Perkin-Elmer model 480 thermocycler. The conditions of the PCRs made with oligonucleotides cry1 (gral-cry1) were the following: a single denaturation step of 2 min at 95 °C, followed by a program of 30 cycles in which each cycle consists of denaturation at 95 °C for one min, hybridization at 52 °C for 1 min and extension at 72 °C for one min. At the end of the 30 cycles an additional extension step was made at 72 °C for five min.

The conditions for the PCRs performed with the other oligonucleotides were similar, except that the hybridization temperatures were modified at 49 °C for oligonucleotides of cry8, at 51 °C for oligonucleotides of cry9, cry11 and cyt1 and at 50 °C for the rest of the oligonucleotides. Once the amplification reaction was performed, it was electrophoresed on a 2% agarose gel in Tris-borate buffer (45 mM Tris-borate, 1 mM EDTA (pH 8) at 250 V for 35 min and stained with ethidium bromide. Finally, with the intention of identifying these proteins, an analysis of possible cry genes present in these strains was carried out using a battery of oligonucleotides (^{Ceron et al., 1995}, ^{Bravo et al., 1998}). These oligonucleotides allow to identify genes cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry13, cry14 and cyt1A.

Pathogenicity bioassays in other species

The activities of the different strains of B. thuringiensis were analyzed in neonatal larvae of Spodoptera frugiperda and Diatraea magnifactella (Insecta: Lepidoptera). Dilutions of spore-crystal suspensions were applied on the surface of the diet to a final concentration of 100 and 1 000 ng cm^-2 (^{Bravo et al., 1998}). Mortality was recorded after seven days.

Egg fumigation bioassays

250 ml glass jars were used to put acetic acid on the bottom as a fumigant, inside this bottle was placed a 25 ml bottle where the eggs of C. carnea were placed. The doses and times of fumigation of the eggs were 0 (control) 0.04 and 0.4 μl ml^-1 acid-air during 8, 16, 24 and 48 h, respectively. In total, four repetitions were used for each dose and time, each repetition was made up of 50 eggs. After the fumigation, the eggs were placed individually in cells of about one cm³, where when the larvae hatched they were fed with eggs of S. cerealella.

The formed pupae were placed in individual confinement cells of three cm³, where the jelly was previously impregnated on the walls to feed the adults that emerged, also supplying them with sterile water and incubating at laboratory temperature (20-25 °C) for 35 days. The count of living adults without symptoms and dead with symptoms was carried out weekly and daily dead adults were removed to avoid the spread of the infection as much as possible. This and subsequent experiments were repeated twice. Another experiment was performed identically, except that larvae were provided with heat-sterilized S. cerealella eggs.

Fumigation bioassays in pupae

The pupae were fumigated with acetic acid at a dose of 0 (control), 0.04. 0.4 and 4 μl ml^-1 acid-air at fumigation times of 10, 20, 30, 60 and 120 min. Then, they were confined in one liter polyurethane cups, and the emerged adults were kept for two weeks at laboratory temperature (20-25 °C), at which time dead adults with symptoms of the disease and adults without symptoms were counted. The adults were fed the jelly in the abbot-tongues and the water supply in the swabs. Dead pupae and adults were removed daily. In total, four repetitions for each dose and time of fumigation of the pupae were performed, each repetition with 15 pupae.

Bioassays with antimicrobials in adults

In the CREROB, we were given ten units each with 700-800 cells occupied by pupae, from which it was expected to obtain at least 4 200 adults, to evaluate six antimicrobials for three doses each (one control) with four repetitions. Of the units, only 530 pupae were recovered from where the adults would be born. With the available pupae, 21 treatments were established with (25 pupae) without repetitions, six antimicrobial substances with three doses and the control without antimicrobial. After the dead adults were discarded in the first two days, the treatments had a different number of adults (Table 1).

The antimicrobial products of the commercial type that were used were Amylamcin Amk^® Ampicillin Binotal^®; Biosint^® cefotaxime, Ilosone^® erythromycin, Flagelase^® metronidazole, vancomycin Vancocin^® in addition to the control.

Pupae and emerged adults were confined in 2 L polyurethane cups, dead adults were removed daily when there were. The lacewings were given food in abate-tongues with the jelly, while the antimicrobials in the water retained the swabs. The insects were temporarily transferred to another vessel, while the source vessel was cleaned using cotton impregnated with a 0.3% commercial chlorine solution. The first cleaning was done seven days after the adults emerged, the subsequent ones every five days.

Analysis of data

The adult data obtained from the egg spray experiments were analyzed by the chi square test (ꭓ²). The experiment with pupae was analyzed with a completely random design, a factorial arrangement and with the GLM procedure, where the time of fumigation and the dose were the factors; each analysis was performed separately for the incubation times of 7 and 14 d. The experiment of adults treated with antimicrobials was not analyzed statistically due to the lack of repetitions.

Of the adult insects that came from the fumigated eggs, their coefficients of linear determination R² and their slopes were established, this for their survival versus incubation time. R² was also obtained between the slopes obtained for each treatment (in each experiment) including the controls versus the initial number of adult insects. All analyzes were performed with the Statistical Analysis System (^{SAS version 9.1, 2003}).

Symptomatology of the disease

The adults of infected C. carnea slow down their movement, their thorax becomes dark, later also the whole digestive tract; the insect does not eat and lose weight until death (Figure 1 A and B), when this occurs, the decomposition of the insect generates a foul odor. Pupae and larvae also turn dark and die (Figure 1C and 1D).

Figure 1 A-D) signs of infection in Chrysoperla carnea. A) adult with darkened thorax and tight abdomen; B) adult with very darkened abdomen; C) pupae with dead and darkened product; and D) larva dead and darkened.  

Isolation and identification of Bacillus thuringiensis

The bacterial colonies isolated were white, opaque, starry and flat. The PCR product obtained showed the size (data not shown) of the Bacillus spp. The parasporal bodies, unique characteristic of B. thuringiensis (where the Cry or Cyt proteins accumulate) were also observed.

Figure 2 shows that the Cc1 strain produces a 150 kDa protein and the Cc2 strain has a very small crystal, so it is difficult to identify the crystal band on the SDS-PAGE gel, noting only that it has a 65 kDa protein that resembles the weight of other Cry proteins reported in B. thuringiensis.

Figure 2 SDS-PAGE gel showing a different molecular weight of the crystal-forming proteins in the Cc1 and Cc2 strains with respect to the Cry3A proteins of B. thuringiensis. 

Strain Cc2 presented amplification with the oligonucleotides of the cry3 gene. However, no protein of similar weight was observed as observed with the control strain expressing Cry3Aa. Strain Cc1 did not amplify with any of these oligonucleotides. These data suggest that these strains could contain genes different from those analyzed by means of these PCR assays or could contain a possible new cry gene.

Pathogenicity bioassays in other species

The toxicity of strains of Bacillus spp. including two strains of B. thuringiensis tested against S. frugiperda and D. magnifactella showed that they are not toxic to these insect species, although the toxicity of B. thuringiensis strains was not tested against larvae and adults of C. carnea.

Egg fumigation bioassays

In the first and second experiments, the control treatments without fumigation and fumigated were recovered 19, 17 and 8, 8% of adults, respectively (Figure 3 left). The differences between the treatments were significant (ꭓ²= 74.6, gl= 8, p< 0.001) for the first experiment and (ꭓ²= 109.6, gl= 8, p< 0.001) for the second experiment. After five weeks, the survival of the adults that came from fumigated eggs was double that of the control egg not fumigated (Figure 3 right) in the first experiment (ꭓ²= 36.9, gl= 9, p≤ 0.001) and (ꭓ²= 26.6, gl= 9, p≤ 0.001) in the second.

For each curve (linear) of survival versus initial number of healthy adults, we obtained their slopes, R² and their goodness of fit (Table 2), there it is observed that in both experiments the fumigated egg treatment with 0.4 μl ml^-1 acid-air for 16 h was not significant goodness of adjustment of its slopes, the rest of the treatments and control were significant.

Figure 3 Survival of live and dead adults of C. carnea. Left, live or dead adults that came from 1 600 fumigated eggs or 200 undamaged control eggs in two experiments. Right, adults alive or dead after five weeks of incubation.  

In relation to the R2 obtained between the initial number of adults of C. carnea versus its survival slope for both experiments, the treatment 0.04 μl ml^-1 acid-air for 8 hours (of the second experiment) had the steepest slope (-1.033) followed by the control treatments -0.833 and -0.988 of the first and second experiments, respectively (Table 2 and Figure 4).

Figure 4 Coefficients of determination between the initial number of C. carnea adults and their survival slope. The open and closed squares indicate the control or non-fumigated treatments of the first and second experiments, respectively; the open and closed circles represent the fumigation treatments of the first and second experiments, respectively. 

In the two experiments, in which the larvae were fed as heat-sterilized eggs of S. cerealella, they did not form any pupae.

Fumigation bioassays in pupae

The time and dose of fumigation were significant p≤ 0.001, this occurred for the pupae that remained 7 or 14 days, as they increased the time and dose of fumigation of the pupae their formation of adults decreased.

The adults formed after fumigating the pupae were not in agreement with the doses and times of fumigation, for example, in the incubation times 7 or 14 days, 30 min of fumigation and doses of 4 μl ml^-1 acid-air were not generated adults, but in times of fumigation less than or greater than 30 min (Table 3). The survival of fumigated pupae that was reflected in the emergence of adults, suggests that acetic acid does not cross the outer layer of the pupa, because if it did kill or disinfect it.

Bioassays with antimicrobials in adults

Only the treatment of cefotaxime at a dose of 1 200 μg ml^-1 allowed all adults to survive until day 51. The 267 adults without symptoms at the beginning of the application of antimicrobial substances, represents less than 5% of pupae that formed adults. In contrast, of the initial number (267) of adults treated with antimicrobial substances including the control survived 183, after 51 days, that is, around 69%, although 80% of the treatments with erythromycin and metronidazole survived.

Fumigation of the egg

Fumigation with acetic acid has been found to kill pathogenic microorganisms of bees and insect pests (^{Van Engelsdorp et al., 2008}; ^{Randall et al., 2011}). Although fumigation of lacewings improved survival, it was not enough to completely eliminate the infection, since 100% of adults did not survive.

The infectious agent of C. carnea was manifested in larvae, pupae and adults. Commonly, the toxins of B. thuringiensis or the bacteria itself infect only larval stages of the insects causing their death from 2 to 5 and from 7 to 10 days, respectively (^{Broderick et al., 2006}).

Of the non-fumigated eggs, twice as many adults were born as fumigated eggs (Figure 3. left), which suggests that the fumigation favors the mortality of the eggs and at the same time a selection of adults that will survive longer and in proportion (Figure 3, right). However, the greater the initial number of adults who were confined in individual cells, the death rates were more accentuated (Table 2, Figure 4).

A possible cause of the infection that explains the death of lacewings that remained in individual cells after two weeks, could be the food contained in the cells, where the possible infectious agent could reproduce and reinfect the adults. Consider that this food was not replaced during the five weeks in which the adult insects remained under observation.

The food could favor the development of microorganisms directly or indirectly related to the infection and death of the lacewings. This is suggested by the survival of almost 100% of adults at 16 days in 18 of 21 treatments where antimicrobials were evaluated (data not shown) and where there was food replacement and asepsis of the vessels where the lacewings remained. Although in this work, the presence of B. thuringiensis cannot be attributed as a cause of infection of adults or another stage of C. carnea, it is known that this bacterium alone or in the presence of enteric bacteria can kill lacewing larvae (^{Broderick et al., 2009}; ^{Johnston and Crickmore, 2009}).

Within eggs, larvae and adults of insects including Chrysoperla spp., they inhabit yeasts, bacteria that help them digest their food (^{Chen et al., 2006}), supplement their diet or nutritional deficiencies (^{Gibson and Hunter, 2005}; ^{Ben-Yosef et al., 2010}). In sum, there are at least ten extrinsic factors that affect the selectivity and dose-response relationship of B. thuringiensis toxins to insects, such as their microbiota, nutritional factors, immune response, among others (^{Lawo et al., 2010}; ^{Then, 2010}; ^{Grenier, 2012}). The eggs of S. cerealella used to feed the larvae that came from the fumigated eggs could also harbor infectious inoculum or microorganisms that favored the infection.

In this work, an attempt was made to eliminate the potential infection by sterilizing the eggs of S. cerealella to feed the larvae of C. carnea, but these did not survive with this food. Results obtained by ^{Fletcher (2016)} show that C. carnea larvae were infected and died by feeding on eggs of the insect Adalia bipunctata L. infected with the pathogen Nosema adaliae Steele & Bjørnson. The lacewings can also become infected and die when in contact with bacteria, for example, Chrysoperla externa Hagen died from bacteria isolated from Phyllocnistis citrella Stainton (prey of lacewings) (^{Meca et al., 2009}), while C. carnea died by pathogenic bacteria of humans Pseudomonas aureoginosa and P. putida (^{Fernández et al., 2015}).

Fumigation of pupae

Some pupae that were fumigated up to two hours, which was the maximum time and at the dose of 4 μl ml^-1 acid-air survived, suggesting that some tolerate fumigation. In contrast, the marked emergence and survival variability of the lacewings from the pupae exposed to different doses-time of fumigant suggests that they had a marked deterioration and/or death before applying the treatments (Table 3).

The vertical transmission (adult→egg→larva→pupa→adult) of beneficial or harmful microorganisms of the Chrysoperla species is known (^{Sung-Oui et al., 2004}; ^{Hemalatha, 2015}), this could explain the transmission of the infectious agent with the subsequent mortality of eggs, pupae, larvae and adults.

Application of antimicrobial substances in adults

The asepsis and the elimination of lacewings that died during the first two days during the treatments of antimicrobial substances, possibly allowed keeping C. carnea with the higher survival rates than in the previous experiments. Even in the treatment with 1 200 μg ml^-1 of cefotaxime the death of adults or symptoms of infection did not manifest themselves. Antibiotics have been used to eliminate bacteria and at the same time, determine their role including endosymbiotic bacteria in insects; the doses used that affected the insects were from 10 μg ml^-1 to 5 mg ml^-1 (^{Broderick et al., 2006}; ^{Ben-Yosef et al., 2010}; ^{Lin et al., 2015}).

Entomophagous insects such as Chrysoperla spp., have negative effects on their life cycle when they feed on their prey if the latter have fed on transgenic crops that express toxins of B. thuringiensis (^{Hilbeck et al., 1998}; ^{Dutton et al., 2003}). In contrast, other studies indicate that Chrysoperla is not affected by preying on prey that have consumed transgenic plants (^{Carvalho et al., 2012}; ^{Tian et al., 2013}; ^{Meissle et al., 2014}), therefore, it is not entirely clear B. thuringiensis paper on the lacewings.

It iss also do not know the role of B. thuringiensis found in dead adults of C. carnea, but knowing this could help clarify whether the bacteria could be pathogenic or not on these insects from egg to adult. The tolerance of eggs and pupae to fumigation with AA and that of adults to antimicrobials could be evaluated in the future to eliminate undesirable organisms in the mass production of beneficial insects other than lacewings.

Our quantitative ignorance of the infection and its lack of control in egg, pupa and adult generated an inconsistency dose-times of fumigation and dose of antimicrobials versus survival of adults.