The cochineal scale insect, Dactylopius opuntiae (Cockerell) (Hemiptera: Dactylopiideae) is an insect used in countries such as Peru, the Canary Islands, and Mexico to produce the natural food coloring carmine. It has also been used as a biological control agent against invasive cactus species in South Africa. In Brazil and Mexico, this insect attacks crops of Opuntia ficus-indica (L.) cactus, causing severe loss of forage and fruit production, resulting in economic losses for farmers (^{Nobel, 2001}; ^{Paterson et al., 2011}; ^{Tiago et al., 2016}).

The control of insect pests is often achieved using chemical products, which it used excessively can cause the contamination of the environment and harm to animal health. However, the use of entomopathogenic fungi can contribute to insect pest control (^{Alves, 1998}; ^{Avery et al., 2013}). The genus Isaria comprises important pathogens that can be used to control insect pests. Isaria farinosa (Holm: Fries) Fries and Isaria fumosorosea (Wize) Brown & Smith were efficient against the mealy bugs Rhizoecus kondonis Kuwana and Planococcus citri (Risso) (^{Zimmermann, 2008}; ^{Demirci et al., 2011}). The combined application of entomopathogenic agents and insecticides can amplify the lethality of natural pathogens (^{Avery et al., 2013}; ^{Silva et al., 2015}). This study analyze the potential of the I. farinosa, I. javanica, and I. fumosorosea in controlling D. opuntiae, and evaluated the effects of the insecticides chlorpyrifos, acetamiprid, thiamethoxam, and lambda-cyhalothrin on these fungi in terms of conidia germination, mycelium growth, and spore formation.

Isaria farinosa (URM5016 and URM5060) and I. javanica (URM4993 and URM4995) were obtained from the URM Culture Collection (WDCM604) of Federal University of Pernambuco (UFPE); I. farinosa (ESALQ1205 and esalq1355) and i. fumosorosea (Esalq1296 and ESALQ1297) were obtained from the ESALQ Collection of Microorganisms - ESALQ of the University of São Paulo (USP). The strains were cultivated in Sabouraud Agar Medium (SAD) for 12 days, and then suspensions (1 × 10⁸ conidia/mL) were made in 10 ml of Tween 80 (0.1%) solution.

The cochineal was collected in O. ficus-indica plantations, and the insects were raised in culture chambers at 28 ± 1^oC, on healthy cactus cladodes. The experiments were performed with three replicates and consisted of nine treatments (eight strains and one control), totaling 150 females and 150 nymphs per treatment. The cactus cladodes infested with nymphs second instar and adult females were sprayed with 10 mL of 1 × 10⁸ conidia/mL of each strain, and for control with Tween 80 (0.1%) solution using a manual sprayer. The cladodes were placed on rectangular plastic trays (30 ×15 × 8 cm) and maintained at 26 ± 1°C, for 10 days; after which 50 adult females and 50 nymphs were collected from each cladode. Confirmation of insect mortality was observed after fungal sporulation by the fungal infection according to ^{Alves (1998)}.

The chemical insecticides chlorpyrifos, acetamiprid, thiamethoxam and lambda-cyhalothrin were tested at three different concentrations based on the recommendations of the manufacturers, with the median concentration (CMed) corresponding to that recommended for field use; the minimum concentrations (CMed/2) and maximum concentrations (CMedx2) (Table 1), according to (^{Lopes et al., 2018}). To evaluate the germination, 0.1 mL of each suspensions with 1 × 10⁸ conidia/mL in Tween 80 (0.1%) solution + insecticide were mixed and after one hour, were inoculated into Petri dishes containing SDA and incubated (26 ± 1°C; 80 ± 10% RH); the control was the fungal suspension without the insecticide. The germination percentage was determined after 16 hours, counting 500 conidia, between germinated and non-germinated by plates, and then the results were given in the formula G = N × 100/500, where N is germinated spore according to ^{Alves and Pereira (1998)}. To evaluate mycelial growth, a filter paper disc (0.3 mm diameter) was soaked with 0.01 mL of 1 × 10⁸ conidia/mL in a Petri dish with SDA + insecticides (as well as control plates without pesticide), and incubated (26 ± 1°C; 80 ± 10% RH) for 12 days. The mycelial growth was determined by measuring the diameter of each colony. To analyze sporulation, colony fragments were transferred to test tubes containing 10 mL of Tween 80 (0.1%) solution, and the number of spores in each suspension was counted. Statistical analyses of the data were performed by analysis of variance (ANOVA) and the means compared using the Tukey test at a 5% probability (^{SAS Institute, 1999-2001}).

Isaria species did not cause efficient mortality averages on the cochineal. Isaria javanica URM4995 and I. farinosa ESALQ1355 caused only 14 and 16% mortality of the nymphs, compared to 5% mortality in the control (p = 0.05) (Table 2). Isaria farinosa ESALQ1205 and I. javanica URM4993 caused the death of 6 and 7% of D. opuntiae nymphs, respectively, while the other strains did not cause nymph mortality (Table 2). None of the Isaria strains caused adult female mortality. This is the first study to report on the pathogenicity of Isaria spp. on D. opuntiae. Dactylopius have protective layers of fatty and waxy substances covering their bodies that repel aqueous solutions (^{Nobel, 2001}; ^{Demirci et al., 2011}). This waxy barrier probably impedes the contact of fungal conidia with the insects' tegument, thus stifling fungal germination. The association of entomopathogens with adjuvants (such as oils, plant extracts, and/or insecticides) could represent viable options for increasing the prevalence of infection with fungal pathogens in this insect (^{Silva et al., 2015}).

The insecticides chlorpyrifos and lambda-cyhalothrin inhibited conidia germination, mycelium growth and a sporulation, principally when used at the maximum concentrations (Table 3). ^{Asi et al. (2010)}, verified that 13 insecticides significantly inhibited the germination and mycelial growth of M. anisopliae and I. fumosorosea, with chlorpyrifos being the most toxic in insecticide. ^{Avery et al. (2013)}, observed that the adjuvant oils significantly inhibited germination and mycelial growth of I. fumosorosea. The insecticides thiamethoxam and acetamiprid caused no negative effects on conidia germination, mycelium growth, or sporulation of the strains (p = 0.05), although the maximum concentrations of the insecticides tested were detrimental to I. fumosorosea ESALQ1297 (Table 3); those insecticides can therefore be considered for combined applications. Similar results were reported by ^{Amjad et al. (2012)}, who found that acetamiprid inhibited germination in I. fumosorosea only at the highest concentration tested, having no effect at lower concentrations. Entomopathogenic fungi and selective insecticides can act synergistically, reducing the of insecticide dosages, which contributes to the preservation of natural enemies and, may reduce the environmental pollution and the appearance of resistant insects (^{Silva et al., 2015}).

Our results indicate that the species of Isaria were not pathogenic to adult females and nymphs of D. opuntiae. Additionally, the insecticides thiamethoxam and acetamiprid demonstrated compatibility with all the Isaria fungal strains tested, suggesting that their use in mixtures could enhance the effectiveness of Isaria in the control of insect pests.