Potentiel de prédation et préférence alimentaire de Cryptolaemus montrouzieri sur Dactylopius opuntiae en laboratoire

Résumé

Récemment, la cochenille à carmin introduite, Dactylopius opuntiae, est l'insecte le plus dévastateur du cactus au Maroc. La cochenille s'est étendue à de nombreuses régions du royaume et a provoqué d'énormes pertes socio-économiques et environnementales. Pour contrôler cette menace, le potentiel prédateur de Cryptolaemus montrouzieri a été étudié en laboratoire à une température de 26±2 °C, 60±10% HR et 12:12 h L: D. Dans cette étude, les expériences ont été menées dans le cadre d’essais d’alimentation en choix (nymphes de deuxième stade, nymphes de premier stade et les œufs de D. opuntiae offerts simultanément au prédateurs). Les adultes de C. montrouzieri et les larves de quatrième stade étaient les prédateurs les plus nourris à différents stades de D. opuntiae. Les valeurs de l'indice de préférence de Manly ont montré que tous les stades prédateurs préféraient les stades plus jeunes de la cochenille (p <0.5) (commutation négative). En outre, des études sur le temps de développement ont montré que les œufs, les larves, La durée des phases prépupale, pupal et adulte était de 3.60±0.67, 4.05±0.90, 4.45±0.85, 5.48±0.91, 8.15±0.36, 1.48±0.60, 10.80±0.65 et 103.28±2.37, respectivement. Les résultats de cette étude ont montré que C. montrouzieri pouvait être exploité comme prédateur de D. opuntiae au Maroc.

Mots-clés: Dactylopius opuntiae, Cryptolaemus montrouzieri, Opuntia  ficus-indica, contrôle biologique, Maroc

Introduction

Dactylopius opuntiae a cochineal very specific to cacti, is a primary pest of Opuntia ficus-indica (L.) Miller in many countries around the word such as Mexico (Portillo and Vigueras, 2006; Vanegas-Rico et al., 2010), Brazil (Oliveira et al., 2013), Spain, Ethiopia, Italy, Turkey, Lebanon and Israel (Portillo, 2009; García Morales et al., 2016; Spodek et al., 2014). This scale insect was recently detected in Morocco in 2014 (Bouharroud et al., 2016). Today the cochineal has spread to other regions such as Rhamna, Bengrir, Abda, Azilal, Benimellal, Taourirt, Haouz and Chaouia where tens of thousands of hectares of cactus are totally destroyed, causing enormous socio-economic and environmental losses. The damage caused by D. opuntiae on O. ficus indica used as forage in Brazil resulted in the loss of more than 100,000 ha, valued at 25 million US dollars (Lopes et al., 2009). Also, in Mexico, the damage to fruit and cladodes caused by D. opuntiae resulted in lower production and higher economic costs (Badii and Flores, 2001; Portillo and Vigueras, 2006). This sap-sucking insect feeds directly on the plant and causes chlorosis and premature dropping of cladodes and fruits. Severe infestations inferior to 75 % of the cladode surface can cause the death of the plant (Mann, 1969; Vanegas-Rico et al., 2010, 2015). Also D. opuntiae is known to have high fecundity, averaging 150–160 eggs (Badii and Flores, 2001). The female life cycle varies from 90 to 128 days and it lives permanently attached to their host plants (Flores et al., 2013), and produces a waxy cottony covering that protects their bodies against predators and reduces the efficacy of chemical control (Badii and Flores, 2001). Since worldwide research on D. opuntiae control strategies is fragmentary and infrequent, Chemical control using organophosphate insecticides has been the main strategy for the control of this mealybug (Badii and Flores, 2001). The insecticides deltamethrin, methidathion and carbaryl have been registered for the control of this pest (Vermeulen et al., 1990). However, the heavy use of these type of pesticides has resulted into the development of resistance of D. opuntiae to many insecticides, and also affects negatively human health and the environment, (Galloway and Handy, 2003; Arias-Estevez et al., 2008). Also, the use of pesticides could limit international trade (Vanegas-Rico et al., 2016). Therefore alternative management strategies, including biological control agents, have been explored to reduce the damage caused by this pest (Vanegas-Rico et al., 2016).

Many predators were reported associated with D. opuntiae and other Dactylopius species in many countries around the world (Vanegas Rico et al., 2010), including coleoptera (coccinellidae), dipetra and lipedoptera. Predators are often known as regulators of insect pests in global agroecosystems (Juen et al., 2012; Lu et al., 2012). The coccinellid lady beetles (Coleoptera: Coccinellidae) are often used in biological control programs because of their large body sizes and their predation potential (Obrycki et al., 2009; Hodek et al., 2012; Nawaz et al., 2017). The predator, Cryptolaemus montrouzieri is native to Australia and commonly known as ‘mealybug destroyer’ since both adults and larvae prey on pests completely (Clausen, 1978). Because of its efficiency, this predator has been introduced to many countries for biological control of several mealybug species (Moore, 1988; Solangi et al., 2012). The lady beetle was used as biological control agent of citrus mealybug, Planococcus citri (Singh, 1978), pink mealybug, Maconellicoccus hirsutus (Mani and Thontadarya, 1988), and the cochineal, Dactylopius tomentosus infesting the prickly pear Opuntia dilenii (Baskaran et al., 1999). In Brazil the lady beetle was used for biological control of cassava cochineal infesting cassava and D. opuntiae (Sanches and Carvalho, 2010). Also 100,000 C. montrouzieri were successfully released in the North of Israel for biological control of D. opuntiae infesting cactus crop (Protasov et al., 2017). Recently Bouharroud et al. (2018) reported that C. montrouzieri has the potential to be exploited as biological control agent of D. opuntiae in Morocco. C. montrouzieri is available commercially and has been shown to be an efficient biological control agent as part of IPM strategies (Solangi et al., 2012).

The aim of the present study was to estimate the predation potential of C. montrouzieri in choice feeding tests on different stages of D. opuntiae under laboratory conditions in Morocco.

Materials and methods

Mealybug source

The D. opuntiae colony was established from infested Opuntia ficus-indica cladodes collected from Khemis Zemamra locality (32°37’48” N, 8°42’0” W) in the Sidi Bennour region of Morocco. A modified version of the ‘cut cladode technique’ of Aldama-Aguilera and Llanderal Cazares (2003), reported by Vanegas-Rico et al. (2016) was used to increase numbers, and follow the age of insects. Briefly, each cladode was perforated at the basal end by a wooden stake, left to scar for 24 hours under laboratory conditions and then hanged vertically from metal grids. The gravid D. opuntiae females were isolated from infested cladodes and placed in open waxed paper bag (15 cm2). Then, each bag was attached to the apex of each cladode; the other cladodes were placed horizontally beneath for nymphs, which were not fixed on to the vertical cladodes. The attacked cladodes were maintained in entomological cages at 26 ± 2°C, 60 ± 10 % RH and 12:12 h L:D regime.

Predator source

The C. montrouzieri colony was established from adults imported by the laboratory of entomology at INRA, Agadir-Morocco. Adults were placed in entomological cages (80-80-80 cm) comprised of a wooden frame covered with a mesh fabric to allow ventilation. Access to water was provided via a white cotton inserted into a 25 ml glass vial of water. Cladodes infested with D. opuntiae were introduced weekly into the cages to provide food and substrates for C. montrouzieri females oviposition. To prevent cannibalism and competition for food, C. montrouzieri new larvae were transferred to another cage with same characteristic described above to complete their development. All the assays with C. montrouzieri were conducted at 26 ± 2 °C, 60 ± 10 % RH. In addition, all the ladybird adults, during rearing, were daily offered a complementary diet (a mixture of water, honey and brewer’s yeast in a 20:40:40 proportion) (Vanegas-Rico et al., 2016). The use of honey in the diet was shown to increase longevity of Hyperaspis notata Mulsant (Dreyer et al., 1997).

Development and consumption rate

To obtain an egg cohort, 10 pairs of ladybirds were placed into ten separate petri dishes (14.5 cm in diameter) with different developmental stages of mealybugs. Freshly laid eggs of C. montrouzieri (100) were isolated individually in petri dishes (9.5 cm diameter). The incubation period was recorded. In the free choice feeding test, 40 newly emerged first instars larvae, and considering each larva to be one replicate (Chi and Yang, 2003) were kept individually in petri dishes (9.5 cm diameter) and provided with a known number of all stages of D. opuntiae. To determine the predation potential of first instars larvae, ten eggs, ten first instar nymphs, ten second instar nymphs and three adult D. opuntiae females were provided per petri dish daily. Second instars and third instars larvae were provided with 30 eggs, 30 first instars nymphs, 30 second instars nymphs and ten adult D. opuntiae females per petri dish daily. Fourth instars larvae and adult beetle were provided with 50 eggs, 50 first instar nymphs, 50 second instars nymphs and ten adult D. opuntiae females per petri dish daily. The number of preys consumed was recorded daily during all larval stages until death in the case of adult beetles. Developmental period of each instars of the larva and prepupal and pupal stage as well as adult beetle periods were recorded. The experiment was repeated five times.

Statistical analysis

The difference in the number of different stages of D. opuntiae consumed by different stages of C. montrouzieri was compared by using Tukey’s test (p ≤ 0.05) with the software package SPSS ver. 18.0 (Carver and Nash, 2011).

The preference for various stages of D. opuntiae was determined by calculating the Manly’s preference index (αp) (Manly et al. 1972). Because the number of different stages of D. opuntiae was reduced over time due to feeding by C. montrouzieri, the modified food reduction equation reported by khan et al. (2012) was used to determine the preference index.

The parameters np and nu were the initial numbers of D. opuntiae, respectively; rp and ru were the numbers of D. opuntiae stages consumed during 24 hour period, respectively. The preferences of each stage of development of C. montrouzieri were separately estimated. Manly’s preference index (αp) values ranged from 0 to 1, and if (αp= 0.5) indicate no preference. If index values > 0.5, the predator prefers older stage of prey (positive switching), and while those < 0.5 indicate that the predator prefers early stage of prey (negative switching) (Blackwood et al., 2001, de khan et al. 2012). We tested our null hypothesis of no preference (αp= 0.5) for each instar of C. montrouzieri with a paired samples t-test with the software package SPSS ver. 18.0 (Carver and Nash, 2011).

Results

Development and consumption rate

The C. montrouzieri adults and grubs were active predators on all stages of the mealybug. Also, the choice feeding test results indicated significant differences in the mean consumption of the different development stages of C. montrouzieri in relation with prey stages (Table 1). The number of preys consumed by C. montrouzieri increased significantly with advancement in each development stage. The mean number of preys consumed by first instar larvae of C. montrouzieri on eggs, first instar nymphs, second instar nymphs and adult D. opuntiae females was the lowest (eggs: F= 176545.461, df= 4, P<0.05; first instar nymphs: F= 172073.617, df= 4, P<0.05; second instar nymphs: F=182944.806, df= 4, P<0.05; adult females: F=182944.806, df= 4, P<0.05). The mean consumption among all C. montrouzieri stages on eggs, first instar nymphs, second instar nymphs and adult females of D. opuntiae was the highest for adult beetles followed by fourth, third, and second larval instars respectively. The average number of D. opuntiae adult females consumed by adult beetles was also significantly higher than the other larval developmental stages of C. montrouzieri. The prey consumption (adult female D. opuntiae) of predator showed an increasing trend with the advancement of each development stage of the predator. Fourth, and third larval instars consumed significantly more D. opuntiae adult female as compared with first and second larval instars.

The C. montrouzieri eggs were deposited singly or in group (clutch) in the colony of D. opuntiae. Incubation period was 3.60 ± 0.67 days (Table 2). The grub had four instars. The duration of the first instar grub was 4.05 ± 0.90 days, the second instar grub developmental period was 4.45 ± 0.85 days. The third instar grub was almost similar to the second instar in shape and took 5.48±0.91 days. The duration of the fourth instar was 8.15±0.36 days and the total development time of grub was completed in 22.1 ± 1.70 days. Before reaching pupal stage, the grub underwent a prepupal stage (1.48 ± 0.60 days) when the grub was immobile and not feeding. The duration of pupa was 10.8 ± 0.65 days. The adult period of C. montrouzieri was 103.3 ± 2.37 days.

Column means with different letters are significantly different according to the Tukey’s test (p ≤ 0.05).

Manly’s preference index (αp)

The predator C. montrouzieri consumed all D. opuntiae developmental stages. But the results of Manly’s preference index (αp) indicated that when younger vs. older stages of prey offered simultaneously, all the predators preferred younger stage (αp < 0.5) and showed negative switching (Figures 1–5). Also the predators showed marked preference for mealybug eggs, first instar nymphs, and second instar nymhs over mealybug adult females (first instar grub: egg vs. adult (t= -143.802, df= 39, P<0.05), 1st vs. adult (t= -118.018, df= 39, P<0.05), 2nd vs. adult (t= -63.218, df= 39, P<0.05); second instar grub: egg vs. adult (t= -43.137, df= 39, P<0.05), 1st vs. adult (t= -30.325, df= 39, P<0.05), 2nd vs. adult (t= -16.478, df= 39, P<0.05); third instar grub: egg vs. adult (t= -144.682, df= 39, P<0.05), 1st vs. adult (t= -119.351, df= 39, P<0.05), 2nd vs. adult (t= -86.273, df= 39, P<0.05); four instar grub: egg vs. adult (t= -90.955, df= 39, P<0.05), 1st vs. adult (t= -73.993, df= 39, P<0.05), 2nd vs. adult (t= -58.593, df= 39, P<0.05); adult beetle: egg vs. adult (t= -101.849, df= 39, P<0.05), 1st vs. adult (t= -83.365, df= 39, P<0.05), 2nd vs. adult (t= -65.409, df= 39, P<0.05)).

Discussion

All C. montrouzieri stages (larval and adult) were able to survive by feeding on carmin cochineal D. opuntiae alone, and their predation potential in free choice feeding test were significantly higher with the advancement in each development stage. The C. montrouzieri fourth instars larva consumed the highest number of all stages of D. opuntiae offered and first instars larvae consumed the lowest number of all stages of scale pest when compared with the other larval stages of the predator. Similar results were reported on Maconellicoccus hirsutus (Mani and Thontadarya, 1987), Planococcus citri (Murali Baskaran et al., 1999; Rosas-Garcia et al., 2009) and Dactylopius tomentosus (Murali Baskaran et al., 1999). All these studies indicated that fourth instars larvae were the most voracious feeders and consumed the total food requirement as compared to the third instars larvae, second instars larvae, and first instars larvae.

The fourth instars of coccinellids generally consumes more quantities of prey than the other larval stages (Fandi et al., 2010; Lucas et al., 2004). The reason for the lower predatory potential of first instars C. montrouzieri might be due to its small size, compared with other developmental stages of the ladybeetle (Rosas-Garcia et al., 2009). Predator adult stage consumed the maximum quantities of D. opuntiae stages compared with larval stages. Similar findings were obtained by Rosas-Garcia et al. (2009) on P. citri. These results may be explained by the fact that C. montrouzieri adult stage have longer longevity compared to their larval stages. Incubation period ranged from 3 to 5 days. This is comparable to the incubation period reported by Mani (1986), Murali Baskaran et al. (1999) and Naik et al. (2003) on other mealybugs. Babu and Azam (1988) reported that the incubation period depended on temperature and prey and showed also that the duration of life stages was shorter during summer and longer during winter. The total larval stage period ranged from 18 to 26 days.

Mani and Krishnamoorthy (1990) reported that larvae of C. montrouzieri completed their development in 17.6 ± 0.89 days when reared on eggs of Chloropulvinaria psidii (Maskel) at 25 ± 2°C and 13.9 days when reared on Chloropulvinaria polygonata Cockerell (Mani and Krishnamoorthy, 1998). In another study on Aleurodiecus disperses Russell by Mani and Krishnamoorthy (1999), under laboratory condition at 25 ± 5°C, the average duration of C. montrouzieri fourth instar and the entire larval stage were 6.4 and 17.2 days, respectively. Murali Baskaran et al. (1999) reported that C. montrouzieri completed incubation and larval period in 4.0 and 12.4 days on P. citri and in 4.23 and 17.7 days when reared on D. tomentasus. Larvae of C. montrouzieri completed their development in 13.9 days when reared on M. hirsutus (Parabal and Balasubramanian, 2000).

The duration of prepaupal and pupal stages ranged from 11 to 14 days. The average of C. montrouzieri pupal stage period was 6.2 days at 30°C, 6.1 days at 27.5°C and 14.3 days at 20°C on M. hirsutus Green of grapevine (Babu and Azam, 1987). Adult period ranged from 102 to 114 days. Under natural conditions Mani et al. (1997) and Al-Khateeb and Raie (2001) reported that the adult period of C. montrouzieri was 52 to 80 and 70.6 ± 6.7 days, respectively. Under lab conditions, Mani et al. (1997) showed that the adult period of C. montrouzieri ranged from 121 to 138 days on P. citri as a prey. Also, Persad and Khan (2002) reported that under laboratory conditions (27 ± 3 °C and 58 ± 3% RH), the average longevity of C. montrouzieri was 98.1 ± 1.6 days on M. hirsutus. özgokce et al. (2006) reported that the average longevity of C. montrouzieri was 120.8 ± 17.4 days under laboratory conditions (25 ± 1°C and 45 ± 5% RH).

Many factors could affect the feeding preference of predators such as (1) the size of prey; for a given predator, there is a size of acceptable prey (Sabelis, 1992); (2) the morphological (Dixon, 2000), physical and chemical characteristics of prey (Omkar et al., 2004); and (3) the interaction between these factors. In our study all ladybird stages preferred D. opuntiae younger stages (first instar nymphs and eggs), then older stage (adult females). Similar results were showed by Khan et al. (2012) on Phenacoccus solenopsis. C. montrouzieri first instar larva, second instar larva, and third instar larva consumed more P. solenopsis first instar nymphs compared to third and second instar nymphs of mealybug (negative switching). Also, we suppose that the most probable reason for preference of mealybug eggs by all predator stages could be due to small size of eggs and the low concentration of carminic acid in eggs (Vanegas-Rico et al., 2016), an anthraquinone produced by D. opuntiae and other camin cochineals, which is toxic to other insects (Eisner et al., 1980).

Conclusion

The predation potential and feeding preference of C. montrouzieri on D. opuntiae were studied under laboratory conditions using free choice feeding test (mixed diet). The mixed diet was the model most closely resembling field conditions where the pest populations overlap, and each development stage is used according to the needs of the predator. The results showed that all C. montrouzieri stages, adults and grubs were active predators on all the stages of the D. opuntiae, and all preferred mealybug younger stages (eggs and first instar nymphs), then older stage (adult females). Fourth instar larvae and adults were the most devastator of mealybug. Our results indicated that C. montrouzieri is a good predator of D. opuntiae, and releases of 4th instar larvae and adults could be used as part of the integrated management strategy for the control of mealybug.

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