Olla v-nigrum Mulsant (Coleoptera: Coccinellidae) preys on eggs and larvae of Coleoptera and Lepidoptera and on aphids in arboreal plants. Alternative prey supplemented with artificial diets may be used to mass rearing Coccinellidae predators for biological control programs. This study assessed the development and reproduction of O. v-nigrum fed Anagasta kuehniella Zeller (Lepidoptera: Pyralidae) eggs supplemented with an artificial diet. Adults of O. vnigrum were collected from Caesalpinia peltophoroides Benth. (Fabales: Fabaceae) and taken to the laboratory. Three male female couples were fed on A. kuehniella eggs and an artificial diet (100 g of yeast, 40 g of honey, 0.5 g of ascorbic acid and 60 mL of water). Eight male female couples obtained from this initial generation were subjected to the same procedure in order to assess the effects on subsequent generations. The mean number of eggs per egg mass was 11.7 ± 0.7 with a viability of 54.8 ± 2.9% and an incubation period of 3.1 ± 0.02 days. The durations of instars I, II, III and IV and the durations of the larval, pre-pupal, pupa and larva to adult stages were 2.4 ± 0.3, 1.8 ± 0.2, 2.0 ± 0.3, 5.8 ± 0.1, 12.1 ± 0.3, 1.0 ±0.01, 4.0 ±0.2 and 17.1 ± 0.5 days, respectively. The viabilities of the larval, pre-pupal and pupal stages of this predator were 46.3 ± 4.5%, 90.0 ± 5.0% and 100%, respectively. The diet and other methods employed in this study were adequate for laboratory rearing of O. v-nigrum but, because of the low viabilities of the eggs and immature stages observed with these methods, further improvements will be necessary for successful mass rearing of this valuable predator.
The mass rearing and release of predators into agricultural and forest areas has been an increasing biological control strategy worldwide (Pervez & Omkar 2004; Mohaghegh & Amir-Maafi 2007; Kutuk et al. 2008). However, the provision of adequate space and labor to effectively rear Coccinellidae, especially aphidophagous species, presents difficulties (Kalaskar & Evan 2001; Phoofolo et al. 2009; Silva et al. 2013), which could be alleviated by using alternative food sources (Dong et al. 2001; Silva et al. 2009; Berkvens et al. 2010).
The nutritional and ecological needs of entomophagous insects have been studied in attempts to increase their effectiveness against pests (Thompson 1999; Eubanks & Denno 2000; Kagata & Katayama 2006). Studies on the development, reproduction and behavior of predators, and mass rearing techniques can improve their potential as biological control agents, but the provision of adequate natural diets presents a major problem to rearing Coccinellidae (Specty et al. 2003; De Clercq et al. 2005).
Diets based on pig (Sus sp. L.; Artiodactyla: Suidae) liver with amino acid solutions have been developed for predatory insects (Arijs & De Clercq 2004; Freitas et al. 2006), with Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae) being the first one reared in vitro to produce fertile descendants (Kariluoto 1980; Kariluoto et al. 1976). This predator achieved a larval stage viability of 86% when fed a diet based on raw pig liver supplemented with vitamins on which the adults fed voraciously, maintaining the colony for months without live prey (Attallah & Newson 1966). Diets based on fresh pig liver were subsequently used to rear Adalia bipunctata L., Coccinella septempunctata L., Coccinella transversoguttata richardsoni Brown, Hippodamia tredecimpunctata tibialis Say, and Propylea quatuordecimpunctata L. (Coleoptera: Coccinellidae) (Sighinolfi et al. 2008; Bonte et al. 2010). Predatory Coccinellidae have also been reared on semi-defined, meat free diets, but usually complemented with prey species and other insects (Matsuka et al. 1982; Ware et al. 2008; Silva et al. 2009). The eggs of the Mediterranean flour moth, Anagasta kuehniella Zeller (Lepidoptera: Pyralidae) can be produced easily and at low cost, making this prey propitious for rearing Coccinellidae (Specty et al. 2003; De Clercq et al. 2005; Silva et al. 2009). Anagasta kuehniella can also be used as an alternative host for parasitoids (Oliveira et al. 2000; Eliopoulos & Stathas 2008; Tavares et al. 2009), enabling the mass rearing of a variety of natural pest enemies.
Coccinellid adults may survive on artificial diets with carbohydrate solution, but females will usually not lay eggs (Hagen 1962; Lundgren 2009), showing that this type of diet can affect insect fertility and fecundity. The establishment of effective diets is a pre-requisite for progress in coccinellid rearing (Evans et al. 1999; Cabral et al. 2006; Michaud & Jyoti 2007). Olla v-nigrum Mulsant (Coleoptera: Coccinellidae) is frequently found on the Brazilian forest plant Caesalpinia peltophoroides Benth. (Fabaceae) and other legumes feeding on Psylla sp. (Hemiptera: Psyllidae) (Stuart et al. 2002; Michaud 2004; Mizell 2007). This predator occurs in stable populations alongside Diaphorina citri Kuwayama (Hemiptera: Psyllidae), indicating that it might be a possible biological control agent of this pest (Michaud 2001). Olla v-nigrum is attracted to, and feeds on Toxoptera citricida Kirkaldy and Aphis spiraecola Patch (Hemiptera: Aphididae), but does not complete its larva development when feeding on these prey (Michaud & Browning 1999). While females of this predator can produce fertile eggs when feeding on T. citricida, the mass rearing of this prey is difficult, suggesting that an alternative, more easily bred diet is necessary to improve the number of these predators that can be reared (Michaud 2000; Michaud & Grant 2004).
The objective of this work was to study the development, reproduction, behavior and characteristics of O. v-nigrum reared on a diet of previously frozen A. kuehniella eggs supplemented with an artificial diet to devise a viable laboratory rearing method to generate stock for using this natural enemy in integrated pest management (IPM) programs.
MATERIAL AND METHODS
The experiment was performed in an incubator at 25 ± 1 °C, relative humidity of 70 ± 10% RH and a 14:10 h L:D, in the Laboratory of Insect Rearing (LACRI) of the Brazilian Agricultural Research Corporation (EMBRAPA) in Sete Lagoas, Minas Gerais State, Brazil. This temperature was chosen because it is suitable for rearing Coccinellidae in laboratory. Moreover, it is near the average temperature in the field in Sete Lagoas (Silva et al. 2009).
Olla v-nigrum adults were collected in a C. peltophoroides field plantation in Sete Lagoas and placed in glass containers (12 cm diam × 18 cm high) sealed with PVC film, perforated to allow the passage of air. A 50mL plastic cup, sealed with a perforated acrylic seal, containing a cotton ball drenched in water; 10 g of artificial diet (100 g of yeast, 40 g of honey, 0.5 g of ascorbic acid and 60 mL of water), and a 10 × 10 cm carton containing A. kuehniella eggs (stored as frozen for 6 months) was placed in the bottom of each container. This diet was chosen because it had the basic ingredients for rearing of Coccinellidae in the laboratory (Silva et al. 2009). The eggs of A. kuehniella came from LACRI breeding stock and were fixed in the cartons using a 20: 80 liquid glue: water mixture.
The sexes of O. v-nigrum adults were determined after copulation in the container, because this predator does not show any reliable sexual dimorphism. Three male + female couples were formed from the 10 adults collected and of the 4 remaining those identified as females were kept in the laboratory as replacements in case any female from the formed couples died.
The O. v-nigrum couples selected after copulation were placed in 50 mL plastic cups, and sealed with perforated transparent acrylic lids. A total of 0.5 g of artificial diet and a 3 cm2 carton containing A. kuehniella eggs were placed inside the plastic cups.
Olla v-nigrum eggs were removed daily from the rearing containers to prevent cannibalism, which commonly occurs when eggs and adult Coccinellidae are kept together (Martini et al. 2009; Sato et al. 2009; Ware et al. 2009). The eggs were transferred with a brush to a moist chamber consisting of a Petri dish with paper filter and a cotton ball moistened with distilled water, and covered with PVC film. A 3-cm2 piece of carton with A. kuehniella eggs was added as food for the recently hatched O. v-nigrum larvae.
One day after hatching, O. v-nigrum larvae were individually isolated each in a 50 mL plastic cup with a transparent acrylic lid. Separating the larvae on the first day of life reduces cannibalism in predatory Coccinellidae (Michaud 2000, 2005; Pervez et al. 2006). The O. v-nigrum larvae were fed only A. kuehniella eggs (previously frozen for 6 months) offered in 3 cm2 cartons placed inside the plastic cups, until the emergence of the adults.
Descendants from this first generation were paired. However, each individual of these pairs originated from parents different from those of other individuals. This eliminated sibling pairings and inbreeding, which has been a problem in coccinellid rearing (Morjan et al. 1999; Nakayama et al. 2010). This allowed the formation of eight couples, with adults of yellow-straw color, black color and black female + yellow-straw male pairs and black male + yellow-straw female pairs (Table 1). The parameters evaluated in this first generation were: total eggs per female and per egg mass; incubation period; egg viability; duration of first, second, third and fourth instars; duration and viability of the larva stage, pre-pupa stage, pupa stage and the larva to adult period and the sex ratio.
COLOR (YELLOW-STRAW OR BLACK) OF THE FIRST GENERATION OF MALE + FEMALE COUPLES OF OLLA V-NIGRUM AT 25 ± 1 °C, 70 ± 10% RH AND 14:10 H L:D.
The experiment had an entirely randomized design. Data set were subjected to ANOVA followed by the test of Tukey (P < 0.05) through the computer program SAS (SAS Institute 1997) (Supplier: Federal University of Viçosa).
Olla v-nigrum has 4 instars. The biological parameters of the progeny from the 8 couples are summarized in Table 2, and those of the progeny of each couple are summarized in Table 3. The total egg masses numbers laid per female were 10.1 ± 2.0 with 11.7 ± 0.7 eggs each. Eggs were laid in a single layer, occasionally dispersed and were elliptical in shape and light yellow in color, until near hatching, when they turned grayish. The smallest egg mass contained one egg and the largest, 33 eggs (Table 2).
The embryonic period of O. v-nigrum was 3.1 ± 0.02 days and its egg viability was 54.8 ± 2.9% (Table 2).
Larvae of O. v-nigrum are campodeiform with an elongated body, distinct abdominal segmentation and well developed legs. Soon after hatching, they remained immobile and aggregated around the eggs, feeding on the infertile ones and the remainder of the chorion. As they approached instar change they stopped feeding, and fixed themselves by the last abdominal segment onto the surface of the acrylic lids on the sides of the 50 mL plastic cups, to await ecdysis.
BIOLOGICAL PARAMETERS (MEAN ± STANDARD DEVIATION) AND RANGE OF OLLA V-NIGRUM MALE + FEMALE COUPLES FED EGGS OF ANAGASTA KUEHNIELLA (FROZEN FOR 6 MONTHS) SUPPLEMENTED WITH AN ARTIFICIAL DIET AT 25 ± 1 °C, 70 ± 10% RH AND 14:10 H L:D.
The durations of the first, second, third and fourth instar were 2.4 ± 0.3, 1.9 ± 0.2, 2.0 ± 0.3 and 5.8 ± 0.1 days, respectively. The duration of the pre-pupal stage was 1.0 ± 0.01 day, the pupal stage was 4.0 ± 0.2 days, and the period from egg to adult was 20.2 ± 0.8 days (Table 2).
The viability of the larva, pre-pupa and pupa stage was 46.3 ± 4.5, 90.0 ± 5.0 and 100.0%, respectively. The embryonic period was 3.1 ± 0.02 days and the viability of the eggs was 54.8 ± 2.9% (Table 2).
The sex ratio of the hatched larvae was 0.61 ± 0.01 (Table 2). Of the emerged adults, 77% had black coloration (55% being females and 45% males) and 23% were yellow-straw (80% being females and 20% males). Black colored O. v-nigrum adults were obtained from all couples formed and in higher numbers than those of yellow-straw color (Table 3).
Total eggs per egg mass was higher for couples with a black male and a yellow straw female (Table 3).
The first egg mass was smaller and a tendency was found towards an increasing number of eggs in those egg masses laid later (Fig. 1A). Egg viability varied considerably, but it was higher in the last egg masses than in the first ones (Fig. 1B).
The number of instars of O. v-nigrum was similar to that of Coccinella undecimpunctata L. (Coleoptera: Coccinellidae) feeding on Aphis fabae Scopoli, Myzus persicae Sulzer (Hemiptera: Aphididae), and Aleyrodes proletella L. (Hemiptera: Aleyrodidae) (Cabral et al. 2006), and, also of Scymnus (Neopullus) sinuanodulus Yu & Yao (Coleoptera: Coccinellidae) on leaves of Tsuga canadensis L. (Pinaceae) infested with Adelges tsugae Annand (Hemiptera: Adelgidae) (Lu et al. 2002). These comparisons show that the number of instars is not dependent on the diet. This suggests that our artificial diet plus A. kuehniella eggs were adequate for the development of O. v-nigrum instars. It is essential to hold the O. v-nigrum larvae singly in order to avoid cannibalism by larvae and adults, which is common in many Coccinellidae species (Michaud 2000, 2005; Pervez et al. 2006). This measure can increase the total number of insects mass reared for IPM programs.
The oviposition behavior and egg color of O. vnigrum were similar to those of Hippodamia variegata Goeze (Coleoptera: Coccinellidae) reared on Dysaphis crataegi Kaltenbach (Hemiptera: Aphididae) (Kontodimas & Stathas 2005) and Rhyzobius lophanthae Blaisdell (Coleoptera: Coccinellidae) fed on Aspidiotus nerii Bouché (Hemiptera: Diaspididae) (Stathas 2000), showing that these are characteristics of the family Coccinellidae.
The low viability of the O. v-nigrum eggs and larvae from Sete Lagoas, Minas Gerais State, Brazil may be explained by inbreeding, because eggs from healthy and heterogeneous couples may have higher viability and more eggs per egg mass (Morjan et al. 1999; Nakayama et al. 2010). Indeed a total of 40 eggs per egg mass with 94.5% viability was reported for healthy O. v-nigrum females from Polk County, Florida (Michaud & Grant 2004).
The behavior and characteristics of O. v-nigrum larvae were similar to those of C. maculata, Cycloneda munda Say, Hippodamia convergens Guérin-Méneville, Harmonia axyridis Pallas (Coleoptera: Coccinellidae) (Cottrell 2007) and S. sinuanodulus fed on A. tsugae (Lu et al. 2002), showing that the characteristics documented are those common to coccinellid predators.
The durations of the instars and of larval stage of O. v-nigrum were comparable to other Coccinellidae, but these parameters may vary with the prey provided. The duration of larval development of C. septempunctata was shorter when fed Lipaphis erysimi Kaltenbach (Hemiptera: Aphididae) than when fed 5 other species of aphids, which was attributed to L. erysimi having a higher level of protein (Omkar & Srivastava 2003). The longer duration of O. v-nigrum larva stage indicates the need to accumulate nutrients, perhaps because of increased metabolism in the following stage (Scriber & Slansky Jr. 1981; Thompson 1999). Food quality may affect the development and survival of coccinellid larvae (Kalushkov & Hodek 2001, 2004; Isikber & Copland 2002). However, differences in the morphology, behavior and chemical composition of prey can affect the development of these predators, as seen in Propylea dissecta Mulsant (Coleoptera: Coccinellidae) and C. septempunctata fed on different aphid species (Omkar & Srivastava 2003; Omkar & Mishra 2005).
The durations of the pre-pupal, pupal and egg to adult stages of O. v-nigrum were considered satisfactory compared to other coccinellids. The pre-pupal stage started when the larvae completed their development and stopped feeding and fixed themselves by the last abdominal segment to the surface of the acrylic lids or on the sides of the 50 mL plastic cups. In these positions they remained immobile, but when taunted they reacted with abrupt movements. The pre-pupa stage of O. v-nigrum presents a different pattern than that of S. sinuanodulus, whose pre-pupal stage starts when the larvae stop feeding, release a great quantity of liquid from the anal region and become immobile for 1 to 2 days (Lu et al. 2002). This behavior was displayed by some O. v-nigrum larvae, but S. sinuanodulus larvae may display yet another different behavior (Lu et al. 2002) in that after the immobile period, they crawl and only after this period are the pupae formed. However, a high percentage of S. sinuanodulus pupae showing this behavior died soon after (Lu & Montgomery 2001; Lu et al. 2002). The crawling behavior of S. sinuanodulus larvae is associated with a dispersal mechanism to seek places to feed and to hide for pupation (Lu et al. 2002).
BIOLOGICAL PARAMETERS (MEAN ± STANDARD DEVIATION) OF OLLA V-NIGRUM MALE + FEMALE COUPLES FED ANAGASTA KUEHNIELLA EGGS (FROZEN FOR 6 MONTHS) SUPPLEMENTED WITH AN ARTIFICIAL DIET AT 25 ±1 °C, 70 ± 10% RH AND 14:10 H L:D.
The greater viability of the larval, pre-pupal and pupal stages of O. v-nigrum that fed on an artificial diet and A. kuehniella eggs than the viability of those that fed on recently-hatched S. frugiperda armyworm larvae may be due to nutritional factors, because the growth rates, larval survival and reproduction of Coccinellidae are associated with prey quality. The best larval performance of Coccinellidae is achieved with prey having a high level of protein and that are eaten in large quantities (Omkar & Srivastava 2003; Zhang et al. 2007). The viabilities of pre-pupal and pupal stages of O. v-nigrum were not negativel affected by the artificial diet and A. kuehniella eggs, in contrast to the reduced viability of eggs, and larvae. This suggests that additional nutrients are needed in this artificial diet, such as essential amino acids and dietary minerals, so as to reflect the outcome of the generalist feeding behavior of Coccinellidae in the wild, where a variety of prey and pollen grains are eaten (Isikber & Copland 2002; Ragkou et al. 2004; Berkvens et al. 2008). Coccinellid larvae rarely complete the first instar with inadequate and/or of poor quality food, such as when lacking a specific nutrient (Michaud 2005). This was observed in Coleomegilla maculata fuscilabris Mulsant (Coleoptera: Coccinellidae) whose development was not completed when fed only on T. citricida, but it survived when this prey was supplemented with a suitable artificial diet fed to the first instar (Michaud 2000). Anagasta kuehniella eggs, previously frozen for 6 months, may have lost some of their nutritional value thus compromising the development of O. v-nigrum. However, this food has great advantages in insect rearing programs due to its ease of storage and lower associated costs compared to alternative fresh prey (Adams 2000; Mohaghegh & Amir-Maafi 2007).
OUa v-nigrum is a holometabolic insect with exarate pupae with free and visible appendages. Initially the pupa has a light coloration that slowly darkens, acquiring the characteristic spots of the Coccinellinae subfamily (Hagen 1962). Soon after emergence the adult develops a lighter color and remain immobile within the exuvia until the normal color develops. Black colored adults continue to darken to a glossy black, the macula of their elytra becoming orange. Adults with the yellow-straw coloration increase the light tone of their maculas on the black-colored elytra. Common characteristics of O. v-nigrum adults are the gray-colored membranous wings, capitadashaped antennae and black legs. The color pattern of O. v-nigrum does not differ between the sexes, but this species is classified as dichromatic, due to sex-linked frequencies of the alleles of a single gene. This was observed for H. axyridis in which color variations are caused by 5 alleles of a single gene (Morjan et al. 1999).
Olla v-nigrum females were on average more robust than males and this feature could be used to sex individuals, but more robust males are not uncommon. Copulations were frequent, despite the different color combinations of the eight couples that were formed from the first generation.
Cannibalism was observed in the adult stage; both males and females eat eggs, making it essential to keep individuals separated or isolated. Adults were also observed to cannibalize other adults with physical anomalies. The sex ratio of O. v-nigrum was similar to those reported for H. convergens and C. septempunctata fed on Myzus persicae nicotinae Sulzer (Hemiptera: Aphididae) (Katsarou et al. 2005).
The total percentage of O. v.nigrum with black or yellow-straw coloration was similar to that of Psylla sp. (Kato et al. 1999), but the sex ratio differed from that of these authors, who obtained 50% O. v-nigrum male or female with black coloring and 46.7% of males and 53.3% females of this predator with straw-yellow color.
The diet and other methods employed in this study were adequate for the laboratory rearing of O. v-nigrum, but because of the low viabilities of the eggs and immature stages, further improvements are necessary for successful mass rearing this valuable predator.
To Dr. J. P. Michaud (Department of Entomology, Agricultural Research Center, Kansas State University, United States) and Dr. S. N. Thompson (Department of Entomology, College of Natural and Agricultural Sciences, University of California, United States) for reviewing the manuscript. To “Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)”, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)” for financial support. To the Group Solucion for translating this manuscript. To Asia Science Editing of the Republic of Ireland for reviewing and editing this manuscript.