1 Pink Hibiscus Mealy Bug, Maconellicoccus hirsutus Green.

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1 The present investigations on various aspects viz., bionomics of pink hibiscus mealy bug, Maconellicoccus hirsutus Green, bionomics of Australian beetle, Cryptolaemus montrouzieri Mulsant and safety of some chemicals on Australian beetle, Cryptolaemus montrouzieri M. carried out at Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra, India during Aurangabad is situated at latitude 19 53' 47 N, longitude 75 23' 54 E, altitude 582 MSL. Annual mean temperature in Aurangabad ranges from 17 0 C to 33 0 C and most of the rainfall occurs in the monsoon season from June to September with an average annual rainfall is 711 mm. The details of the materials used and methods adopted in this investigation are given here under separately for each aspect. The red pumpkin is such as a host and is the host of choice for an insectary operation for mass rearing of pink hibiscus mealy bug. The method of rearing of mealy bugs on red pumpkin (Cucurbita maxima Duch.)standardized by Chacko et al (1978) and Singh (1978) was followed for rearing of to obtain the culture throughout the period of research. 1 Pink Hibiscus Mealy Bug, Maconellicoccus hirsutus Green. Studies on bionomics of pink hibiscus mealy bug, Maconellicoccus hirsutus Green at DBT-RBC project, NARP Campus, Aurangabad during December August Temperature and relative humidity were recorded daily throughout the course of study. 1.1 Surveying of pink hibiscus mealy bug Starter culture of PHM, M. hirsutusg.were collected from grape orchards near Verul and Andhari village, hibiscus plants in nurseries and cycads in and Aurangabad, Maharashtra, India. PHM, adult females were collected from the fields as these were stable stages for further development of nucleus culture. These adult females were collected in glass petridish (Borosil 75

2 with dimensions 9 cm diameter X 1.5 cm height) with the help of camel hair brush no-1. Adult females collected were separated and the females contains ovisac with eggs discarded and only those females which does not laid the eggs were used for the further studies. Each female were kept separately to prevent cross-contamination. 1.2 Sampling and disinfection of pumpkins To avoid the cross contamination mealy bug culture and predator culture reared in a separate room having dimensions 120 ft 2 (12 ft X 10 ft) each. Each room swept and then mop with 10 percent bleach solution for decontamination. Curtains made up of heavy black cloth this helped to eliminate all light from PHM culture. These curtains prevent light from attracting the crawlers. While working use less light since light attracts the crawlers and crawlers walking off from the red pumpkins. To facilitate easy handling well ripe, medium sized wound free red pumpkins with grooves and ridges with small stalk were purchased from the local market. Due care was taken that these pumpkin fruits were not be green or over ripen as this helps in determining the shelf life of red pumpkins. Normal shelf life of the pumpkins was 8 weeks. Prior to use or placing in storage, each pumpkin washed using a soft bristle brush using water only to remove dirt as well as insect life, followed by a fungicide, Carbendazim 50 % WP. 2gm/liter of water for 10 minutes, to make the pumpkins free from diseases and moulds then soaking in a 5% household bleach solution for 10 minutes and subsequently thoroughly rinsed with tap water and open air dried. Care was taken that the red pumpkins should not touch to each other during storage. Each week all the stored pumpkins were examined carefully for rotting and rotting pumpkins were discarded safely at the disposal site. 76

3 To initiate a pure culture of PHM, single, gravid adult females from the field material transferred to host material i.e. red pumpkins. This transfer took place with the help of camel hair brush no- 1and using 10 X magnifying glass. Dampening the brush with water may help the female mealy bug stick to the brush during the transfer process. These pumpkins were further placed in a wooden cage (30 X 30 X 30 cm) with glass sliding front wooden base and clothes on either sides as suggested by Padmaja et al (1995). Care was taken to close all cracks and crevices with wax to prevent escape of early instars. These cages were kept in a dark place so that further movement of adults was prevented and they were settled at one place. As the mealy bug attracts ants, ant barriers were kept at the bottom. Mealy bugs were typically maintained in closed dark room facilities. This reduces the crawler movement and escape. By utilizing double door screened entrance ways, along with placing fine mesh screening over all room vents and openings, PHM cultures can be maintained free of contaminants. Room temperature and relative humidity were recorded at 8.00 am and 6.00 pm in the evening thus meteorological data were maintained. Average temperature was 25±2 0 C and 65±4% relative humidity during the study period. Daily observations of the female were taken for the eggs laid. Laboratory observations were measured under the microscope with the help of occular micrometer calibrating with stage micrometer. Freshly laid eggs by the female were used for the further study to develop nucleus culture. Pink mealy bug, Maconellicoccus, hirsutus Green passes through incomplete metamorphosis. Old culture and the host material (red pumpkins) properly discarded. All the material kept in heavy black plastic bags, sealed tightly and taken away to the disposal site a safe distance from the insectary facilities. 77

4 1.3 Bionomics of pink hibiscus mealy bug, Maconellicoccus hirsutus Green Observations were recorded on various biological parameters of PHM on red pumpkinat different conditions of temperature and relative humidity (RH) (Plate-1).Biological parameters of PHM under laboratory condition.the observations were recorded onduration of different developmental stages their colour, shape, and size. The length and breadth of eggs and early instar larvae was measured using precalibrated ocular micrometer Egg Female laid egg sac and these freshly laid eggs were kept separately in glass vial and examined under microscope for studying, size, colour and shape of the eggs. The length and breadth of eggs (N=40) were measured under the microscope and observations were recorded. The day of egg laying by female and the hatching of eggs were recorded, the difference between both was taken as incubation period. The eggs were considered as hatched when nymphs come out of it. Colour of egg sac and the position of egg sac were also recorded. Hatching percentage of the eggs calculated separately by taking number of eggs from one egg sac and the number of eggs hatched from the eggs and this replicated thrice. Plate no-1:-rearing of PHM 78

5 1.3.2 Nymphs To study the number and duration of different nymphs, newly hatched nymphs were transferred separately on red pumpkin with the help of camel hair brush. Morphological characteristics were recorded with the help of microscope. Length and breadth of each nymph recorded separately (N=40). Shedding of exuviae (skin) taken as moult. The duration between first moult and hatching taken as first instar, second moult and first moult as second instar and third moult and second moult recorded as third instar. Duration of first to third instar nymphs in case of female and first to fourth instar nymphs in case of male was recorded Adult Size, shape and colour of the adults were observed under microscope and recorded. The difference between male and female were observed and recorded separately Pre-oviposition and oviposition period Females kept separately and observed for preoviposition and oviposition period. The period between the third instar female nymphs and the commencement of egg laying was considered as pre-oviposition period. The period between commencements of egg laying and ceasing of eggs laying taken as oviposition period Fecundity and Adult longevity and sex ratio Newly emerged virgin females obtained from the developmental study were used to assess reproduction. Male and female were kept separately for copulation. Number of eggs laid by individual female was recorded when egg sac laid by the individual female. Average fecundity 79

6 was calculated throughout the study. Adult longevity of male and female was calculated separately from the date of emergence to the death of adult. To study sex ratio, male and female laboratory reared adults were separated on the basis of their morphological characters and sex ratio was calculated. 2 Australian beetle, Cryptolaemus montrouzieri Mulsant Newly emerged Australian beetle, Cryptolaemus montrouzieri M. (N=30) were brought from the Department of Entomology, Marathwada Agricultural University, Parbhani, Pink hibiscus mealy bug, M.hirsutus G. were provided as a food source to Australian beetle, C. montrouzieri M. Thus Australian beetle reared on pink hibiscus mealy bug, M. hirsutus G. The newly emerged Australian beetle introduced on developed mealy bug colonies of red pumpkin and allowed to develop fully. From this culture, newly emerged adults were paired and used for the further study purpose to develop nucleus culture. Australian beetle, Cryptolaemus montrouzierimulsant passes through complete metamorphosis.observations were also recorded on all biological parameters like incubation, larval, pupal period, pupation, emergence, male/ female ratio; longevity period. Temperature was 25±2 0 C and 65±4% relative humidity (Plate-2). 2.1 Eggs Adult female laid eggs singly or in groups inside the egg sac of mealy bugs. Eggs (N=30) laid by the female were kept in glass vial for hatching and observed for shape, size and colour of eggs under microscope. The length and breadth were measured with the help of occular and stage microscope. The date of egg laid and the date of egg hatching were recorded and it was taken as incubation period of eggs. The eggs were considered as hatched when larvae come out of the 80

7 eggs. Hatching percentage of the egg was calculated separately by taking number of eggs taken for observations and the number of eggs hatched from the eggs. 81

8 Plate no-2 Rearing of Australian beetle 82

9 2.2 Grub (Larva) Newly hatched grubs were kept separately in each petridish (Borosil with dimensions 9 cm diameter X 1.5 cm height). Blotting paper of same diameter kept at the bottom of the petridish and mealy bug crawlers and eggs were provided as a food source in petridish. Everyday this food is changed and increased. Exuivae were removed as and when observed. Day of hatching to first moult is recorded as first instar, first moult and second moult taken as second instar, second moult and third moult taken as third instar and third moult and prepupal stage is taken as fourth instar. Morhometric characteristics were recorded such as length and breadth of grubs. Duration of grubs, colour and shape of the grubs were observed and recorded. 2.3 Pre-pupa and pupa Last instar grub went into prepupal stage when it ceases the feeding procedure and remained inactive and isolated. Duration of prepupal period were recorded. After pre pupal stage pupal period starts it is quiescent stage in the life cycle. Size shape and colour of the pupa observed under microscope and observations were recorded. Duration of prepupal and pupal stage were recorded separately. 2.4 Total developmental period Total development period from eggs laying till adult emergence was computed by combining the data obtained from the observations of incubation period upto the pupal period and it is presented. 83

10 2.5 Pre-mating period and mating period Newly emerged adult beetles from pupae were observed. Male and female were kept separately based on the sexual dimorphism and paired. Each pair kept separately in a plastic jar (9X 11 cm) closed with muslin cloth for easy movement of beetles. Ten such containers were kept under observation for pre-mating and mating period. The period from adult emergence of the female up to its first mating was recorded as pre-mating period and from the same pair duration of mating was counted in minutes for five random mating in each pair from this average pre-mating and mating periods were recorded. 2.6 Pre-oviposition period All the ten pairs confined in containers for studying pre-mating and mating periods were used for recording the observations on pre-oviposition period. Pre-oviposition period is the period from female adult emergence to first egg laying. 2.7 Oviposition The period between first egg laying up to the last egg laying i.e. the period during which female laid eggs was recorded for ten females and the average was worked out. The beetles were provided the enough quantity of prey eggs, nymphs of prey throughout the study period. 2.8 Post- oviposition period The period between egg cease and the adult female mortality for ten females were recorded and average was worked out. This period noted as post- oviposition period of C.montrouzieri. 84

11 2.9 Fecundity To study fecundity, ten pairs of the beetles which were previously used for the study i.e. premating, mating, pre-oviposition and oviposition period were observed and number of eggs laid was recorded daily. The total number of eggs laid by individual female in its life time was recorded. As the female laid eggs inside the PHM egg sac, during this period the pairs were supplied with sufficient quantity of mealy bug egg sacs and every day new egg sacs were offered for oviposition. From the data obtained average fecundity was calculated Adult and its longevity Ten pairs of beetles previously used for the study were observed and the size, shape and colour of adult beetles were recorded. Eggs and nymphs of PHM were provided as food source to the adult beetles. The longevity of male and female adult in each pair was recorded and average period was worked out for these ten pairs Sex ratio Adults emerged from the mother culture of Australian beetle, C.montrouzieri M. maintained on pink hibiscus mealy bug, Maconellicoccus hirsutus G. were sexed and sex ratio was worked out by observing 200 emerged beetles. Sex was judged by observing the differentiation in first pair of legs i.e. female has black and male has orange coloured legs (Babu and Azam 1987b). 85

12 3 Safety of some chemical insecticides to Australian beetle, Cryptolaemus montrouzieri Mulsant. To test the toxicity of insecticides against laboratory reared eggs, larvae and adults of C.montrouzieri, an experiment was conducted in the laboratory under controlled conditions 25±2 C and 60±4% RH. Insecticide solutions were prepared in tape water at the rate of 500 liters per hectare. Insecticides were tested at their recommended dose per hectare. 3.1 Experimental details Toxicity of six commercial formulations tested against eggs, grubs, pupae and adult of C.montrouzieri under the laboratory conditions. Laboratory maintained stock culture was used for bioassay study. Desired concentrations of each insecticide were prepared by diluting the commercial formulations with distilled water. The desired concentration of test insecticide was taken in 100 ml capacity. A laboratory trial was conducted during 2010 in completely randomized block design with six insecticides and one untreated control. The details of the formulations used are presented in Table No-4. Desired strength Quantity of insecticide formulation used = x Quantity of spray material Percentage of insecticides formulation Measured quantity of pesticides were taken and dissolved in small quantity of distilled water after which the remaining water was added to make up the volume and this solution stirred thoroughly for uniform mixing of the pesticides. All the equipments were thoroughly cleaned before using and also using it for the next treatment. The percentage hatching 86

13 /mortality/emergence of various developmental stages in the treatments was calculated at different intervals. 3.2 Toxicity to grubs and adults Fresh spray solution was prepared as and when required healthy and broad leaves of hibiscus (Hibiscus rosa-sinensis) plant were taken and cleaned with distilled water and dried in the shade. Three such leaves were dipped in the concentration prepared of insecticide for one minute. These leaves were then air dried in the shade for the removal of excess moisture. To maintain turgidity of the leaves their stalks were swabbed with wet cotton. Each leaf was then taken in separate petridish (10 x 2 cm). These leaves were used for the exposure of grubs and adults. Second instar grubs (N=20) exposed to the treated leaves and grubs fed with mealy bugs.mortality of the grubs was recorded after 24 hrs of exposure. Larval mortality was determined by the failure to move when larvae were probed by a smooth camel hair brush. Twenty adults of three days old were feed with mealy bugs and 50 percent honey solution was provided as supplementary food. These adults were exposed to the treated leaf surface in each petridish. When probed with camel hair brush, if movement was not noticed, it was considered dead. Observations were made on mortality of adults after 24 hrs after the treatment. 3.3 Toxicity to eggs and pupae To study the toxicity of chemical insecticides to the eggs and pupae of C.montrouzieri Green dipping method was used. In all the treatments, fresh and two to three days old eggs and two days old pupae were randomly selected for the experiment from the laboratory culture. Three days old eggs and two days old pupae were dipped in insecticide solution for 10 seconds. Eggs or pupae were put with the help of fine camel hair brush into a metal mesh net container having a 87

14 rod for handling or dipping.insecticide solution was prepared in glass beaker. After treatment, eggs and pupae were kept in shade for drying after complete drying single egg was placed in single plastic transparent tube sealed from both ends with impulse sealer. Data were recorded on their hatching percentage and compared with control after 3-4 days. Adult emerged from pupa was recorded. This experiment was replicated thrice with twenty eggs and pupae in each replicate. Table 4 Details of the pesticides used for the experiment Common Trade Chemical Name Name of Name Insecticide Chlorpyrifos Dursban 0,0-diethyl 0- (3,5,6-trichloro - 2pyridyl) phosphorothioate Dichlorvos Nuvan 2,2-Dichlorovinyldimethyl phosphate Acetamiprid Pride N[6-chloro-thiazol- 5-ylmethyl]- N 2 Cyano-N 1 - methyl acetamide Fipronil Regent (±)-S-amino-1-(2-6-dichloro-à,à, à- trifluro-p-totyl)-4 trifuromethyl sulfinyl pyrazole-3- carbonitrile Imidacloprid Confidor 1-(6-chloro-3- pyridymethyl)- N=nitromidazolidin -2-ylidene Methomyl Lannate S-methyl N- (methylcarbamoyloxy) thioacetamide Formulati on Concentration (%) Source 20 %EC 0.05 Dow Agro Sciences Mumbai 76 %EC 0.05 Syngenta India Limited Pune 20 %SP 0.05 Dow Agro Sciences Mumbai 5%SC 0.01 Bayer India Limited Mumbai 17.8 %SL Bayer India Limited Mumbai 40 %SP 0.05 EI Dupont India Limited New Delhi 88

15 3.4 Statistical Analysis The average per cent emergence /mortality of each stage like eggs, grubs, pupa and adult was worked out for each treatment. The emergence /mortality data thus obtained were further corrected by using Abbott s formula (1925). The data obtained were subjected to analysis of variance (ANOVA) after transformation of data through CPCS-I software and as per procedure suggested by Gomez and Gomez (1984). 89