DEVELOPMENT AND REPRODUCTION OF OLLA V-NIGRUM (COLEOPTERA: COCCINELLIDAE) FED ANAGASTA KUEHNIELLA (LEPIDOPTERA: PYRALIDAE) EGGS SUPPLEMENTED WITH AN ARTIFICIAL DIET

Headnote

Abstract

Olla v-nigrum Mulsant (Coleóptera: Coccinellidae) preys on eggs and larvae of Coleóptera 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 kuehni- ella Zeller (Lepidoptera: Pyralidae) eggs supplemented with an artificial diet. Adults of O. v- nigrum 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 im- provements will be necessary for successful mass rearing of this valuable predator.

Key Words: Anagasta kuehniella, biological control, Caesalpinia peltophoroides, ladybugs, mass rearing, predator, prey, reproduction

Resumo

Olla v-nigrum Mulsant (Coleóptera: Coccinellidae) é um predador de ovos e larvas de Co- leóptera e Lepidoptera e de pulgôes em plantas arbóreas. Presa alternativa suplementada com dietas artificiáis pode ser utilizada na criaçào massai de predadores Coccinellidae pre- dadores para programas de controle biológico. Este estudo analisou o desenvolvimento e a reproduçào de O. v-nigrum alimentada com ovos de Anagasta kuehniella Zeller (Lepidopte- ra: Pyralidae), suplementado com urna dieta artificial. Adultos de O. v-nigrum foram cole- tados em Caesalpinia peltophoroides Benth. (Fabaceae) e levados para o laboratorio. Très casais foram alimentados com ovos de A. kuehniella e urna dieta artificial (100 g de levedura, 40 g de mel, 0,5 g de ácido ascórbico e 60 mL de água). Oito casais obtidos a partir desta primeira geraçào foram submetidos ao mesmo procedimento, para estudar os efeitos sobre as geraçôes subséquentes. O número de ovos por postura foi de 11,7 ±0,7 com viabilidade de 54,8 ± 2,9% e período de incubaçào de 3,1 ± 0,02 dias. A duraçào dos I, II, III e IV estádios e os estágios de larva, pré-pupa e de larva a adulto foi de 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 e 17,1 ± 0,5 dias, respectivamente. A viabilidade dos estágios de larva, pré-pupa e pupa desse predador foi de 46,3 ± 4,5%; 90,0 ± 5,0% e 100%, respectivamente. Os métodos utilizados foram adequados para a criaçào em laboratorio de O. v-nigrum, mas devido à baixa viabilidade dos ovos e dos estádios imaturos outras melho- rias seráo necessárias para o sucesso da criaçào massai desse predador.

Palavras chave: Anagasta kuehniella, Caesalpinia peltophoroides, controle biológico, criaçào massai, joaninhas, predador, presa, reproduçào

The mass rearing and release of predators into agricultural and forest areas has been an increas- ing biological control strategy worldwide ( Pervez & Omkar 2004; Mohaghegh & Amir-Maafi 2007; Kutuk et al. 2008). However, the provision of ade- quate space and labor to effectively rear Coccinel- lidae, especially aphidophagous species, presents difficulties (Kalaskar & Evan 2001; Phoofolo et al. 2009; Silva et al. 2013), which could be allevi- ated by using alternative food sources (Dong et al. 2001; Silva et al. 2009; Berkvens et al. 2010).

The nutritional and ecological needs of en- tomophagous insects have been studied in at- tempts to increase their effectiveness against pests (Thompson 1999; Eubanks & Denno 2000; Kagata & Katayama 2006). Studies on the devel- opment, reproduction and behavior of predators, and mass rearing techniques can improve their potential as biological control agents, but the pro- vision 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 mac- ulata DeGeer (Coleóptera: Coccinellidae) being the first one reared in vitro to produce fertile de- scendants (Kariluoto 1980; Kariluoto et al. 1976). This predator achieved a larval stage viability of 86% when fed a diet based on raw pig liver sup- plemented with vitamins on which the adults fed voraciously, maintaining the colony for months without live prey (Attallah & Newson 1966). Di- ets based on fresh pig liver were subsequently used to rear Adalia bipunctata L., Coccinella septempunctata L., Coccinella transversoguttata richardsoni Brown, Hippodamia tredecimpuncta- ta tibialis Say, and Propylea quatuordecimpunc- tata L. (Coleóptera: Coccinellidae) (Sighinolfi et al. 2008; Bonté et al. 2010). Predatory Coccinel- lidae 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 propi- tious 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 (Coleóptera: Coccinellidae) is frequently found on the Brazilian forest plant Caesalpinia peltophoroides Benth. (Fabaceae) and other le- gumes feeding on Psylla sp. (Hemiptera: Psyl- lidae) (Stuart et al. 2002; Michaud 2004; Mizell 2007). This predator occurs in stable populations alongside Diaphorina citri Kuwayama (Hemip- tera: Psyllidae), indicating that it might be a pos- sible 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 com- plete 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 (Mi- chaud 2000; Michaud & Grant 2004).

The objective of this work was to study the de- velopment, reproduction, behavior and character- istics 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) pro- grams.

Material and Methods

The experiment was performed in an incuba- tor 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 La- goas, Minas Gerais State, Brazil. This tempera- ture was chosen because it is suitable for rearing Coccinellidae in laboratory. Moreover, it is near the average temperature in the field in Sete La- goas (Silva et al. 2009).

Olla v-nigrum adults were collected in a C. pel- tophoroides field plantation in Sete Lagoas and placed in glass containers ( 12 cm diam x 18 cm high) sealed with PVC film, perforated to allow the passage of air. A 50 mL 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 x 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 ingredi- ents 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 deter- mined 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 fe- male from the formed couples died.

The O. v-nigrum couples selected after copula- tion 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 con- taining 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 Coc- cinellidae 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 con- sisting of a Petri dish with paper filter and a cot- ton ball moistened with distilled water, and cov- ered with PVC film. A3-cm2piece of carton withA. 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 pair- ings and inbreeding, which has been a problem in coccinellid rearing (Morjan et al. 1999; Na- kayama 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; dura- tion of first, second, third and fourth instars; du- ration and viability of the larva stage, pre-pupa stage, pupa stage and the larva to adult period and the sex ratio.

The experiment had an entirely randomized design. Data set were subjected to ANOVA fol- lowed by the test of Tukey (P < 0.05) through the computer program SAS (SAS Institute 1997) (Supplier: Federal University of Viçosa).

Results

Olla v-nigrum has 4 instars. The biological pa- rameters 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 segmenta- tion 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.

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%, re- spectively. 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 fe- males 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 cou- ples 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 via- bility varied considerably, but it was higher in the last egg masses than in the first ones (Fig. IB).

Discussion

The number of instars of O. v-nigrum was similar to that of Coccinella undecimpunctata L. (Coleóptera: Coccinellidae) feeding on Aphis fa- bae Scopoli, Myzus persicae Sulzer (Hemiptera: Aphididae), and Aleyrodes proletella L. (Hemip- tera: Aleyrodidae) (Cabral et al. 2006), and, also of Scymnus (Neopullus) sinuanodulus Yu & Yao (Coleóptera: 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 in- stars. 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 in- sects mass reared for IPM programs.

The oviposition behavior and egg color of O. v- nigrum were similar to those oí Hippoda mia var- iegata Goeze (Coleóptera: Coccinellidae) reared on Dysaphis crataegi Kaltenbach (Hemiptera: Aphididae) (Kontodimas & Stathas 2005) and Rhyzobius lophanthae Blaisdell (Coleóptera: Coc- cinellidae) fed on Aspidiotus nerii Bouché (He- miptera: Diaspididae) (Stathas 2000), showing that these are characteristics of the family Coc- cinellidae.

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-ni- grum larvae were similar to those of C. mandata, Cycloneda inunda Say, Hippodamia convergens Guérin-Méneville, Harmonia axyridis Pallas (Coleóptera: 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 (Hemip- tera: Aphididae) than when fed 5 other species of aphids, which was attributed to L. erysimi hav- ing a higher level of protein ( Omkar & Srivastava 2003). The longer duration of O. v-nigrum larva stage indicates the need to accumulate nutri- ents, 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 & Cop- land 2002). However, differences in the morpholo- gy, behavior and chemical composition of prey can affect the development of these predators, as seen in Propylea dissecta Mulsant (Coleóptera: Cocci- nellidae) 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 complet- ed 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 re- mained 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. How- ever, a high percentage of S. sinuanodulus pu- pae 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).

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 vi- ability of those that fed on recently-hatched S. frugiperda armyworm larvae may be due to nu- tritional factors, because the growth rates, larval survival and reproduction of Coccinellidae are associated with prey quality. The best larval per- formance 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 nega- tivel affected by the artificial diet and A. kuehni- ella 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 va- riety 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 (Mi- chaud 2005). This was observed in Coleomegilla mandata fuscilabris Mulsant (Coleóptera: Cocci - nellidae) whose development was not completed when fed only on T. citricida, but it survived when this prey was supplemented with a suitable arti- ficial 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 advan- tages 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).

Olla v-nigrum is a holometabolic insect with exarate pupae with free and visible appendages. Initially the pupa has a light coloration that slow- ly darkens, acquiring the characteristic spots of the Coccinellinae subfamily (Hagen 1962). Soon after emergence the adult develops a lighter col- or 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. Com- mon characteristics of O. v-nigrum adults are the gray-colored membranous wings, capitada- shaped antennae and black legs. The color pat- tern 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 cou- ples that were formed from the first generation.

Cannibalism was observed in the adult stage; both males and females eat eggs, making it es- sential 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 dif- fered from that of these authors, who obtained 50% O. v-nigrum male or female with black color- ing 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 improve- ments are necessary for successful mass rearing this valuable predator.

Acknowledgments

To Dr. J. P. Michaud (Department of Entomology, Agricultural Research Center, Kansas State Univer- sity, United States) and Dr. S. N. Thompson (Depart- ment of Entomology, College of Natural and Agri- cultural Sciences, University of California, United States) for reviewing the manuscript. To "Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)", "Coordenaçào de Aperfeiçoamento de Pesso- al de Nivel Superior (CAPES)" and "Fundaçào de Am- paro à Pesquisa do Estado de Minas Gerais (FAPE- MIG)" for financial support. To the Group Solución for translating this manuscript. To Asia Science Editing of the Republic of Ireland for reviewing and editing this manuscript.

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