Standard Operating Procedure for Plant Diagnostic Laboratories. Pink Hibiscus Mealybug Maconellicoccus hirsutus (Green) DRAFT 1.1

Transcription

1 Standard Operating Procedure for Plant Diagnostic Laboratories Pink Hibiscus Mealybug Maconellicoccus hirsutus (Green) DRAFT 1.1

2 Table of Contents Background 3 Protocol 1-Sample Collection and Submission 4 2-NPDN Laboratory Receipt and Examination 4 3-Sample Storage 4 4-Sample Screening Contacts for Advanced Screening and Scale Taxonomy Table 1: Identification Specialists in Scale Taxonomy 4 Table 2: NPDN Advanced Screening Sample Overflow Labs Initial Screening by Triage Labs 7 How to Use the Field Key 7 Field Key to the Most Common Mealybug on Hibiscus in Florida Advanced Screening 16 Slide-Mounting for Specimen Preparation 17 Materials 17 Purpose and Formulas for Some Materials 17 Slide-Mounting Summary 18 Protocol Slide Mounted Key to the Adult Females on Florida Hibiscus 20 Selected References 21 Level 2: Confirmation of Species Identification 21 5-Communication 23 6-Confirmation 23 7-Sample Destruction 23 Appendix I: Documentation and Specimen Submission Forms APHIS-PPQ Form

3 Background: The pink hibiscus mealybug (PHM), Maconellicoccus hirsutus (Green), is an exotic pest species that invaded Hawaii in 1983, California in 1999 and Florida in Worldwide, PHM has been recorded from over 300 host plant species, including citrus, ornamentals, and vegetables. Despite federal (USDA-APHIS) and state (FDACS-DPI) efforts to regulate and control the spread of PHM to other susceptible states, a nursery in Homestead, FL, shipped 900,000 hibiscus plants from potentially infested stock to 36 states in the U.S. from January through July According to USDA-APHIS personnel, 11 of the states that received plant shipments are climatically suitable for establishment of the mealybug. Plants were shipped to numerous national home improvement and supermarket chains. The presence of PHM was confirmed from plant material in Kansas, Louisiana, and North Carolina. Distribution of PHM may have occurred in other states, but were sold and incorporated into landscapes prior to testing for PHM. PHM is believed to be climatically suitable for establishment in the following southern continental U.S. states: California, Nevada, Arizona, New Mexico, Texas, Oklahoma, Louisiana, Arkansas, Mississippi, Alabama, Florida, Georgia, South Carolina, North Carolina, Virginia, Tennessee and Utah. Given the broad host range of PHM, its climatic suitability for potential establishment, and its potential for persistence in more temperate climatic regions on nursery stock in glasshouses, etc., it has been estimated that this pest species could potentially cause economic losses of $750 million per year in the U.S. alone (APHIS-PPQ, 2004). In California and Florida, control methods have primarily consisted of releasing the parasitic wasps, Anagyrus kamali Moursi and Gyranusoidea indica Shafee, Alam & Agarwal (Hymenoptera: Encyrtidae), in order to maintain PHM populations below economically damaging levels. Parasitoids also have successfully controlled the mealybug in Hawaii, Grenada, other Caribbean countries, the Bahamas, Belize, the U.S. Virgin Islands, and Puerto Rico. However, chemical control will likely continue to be emphasized in nursery stock settings that have zero tolerance standards for various pest species. If PHM is able to gain a foothold in a number of climatically suitable states in the U.S. it is anticipated that potentially severe economic damage could follow unless an early detection and rapid response system is established. Early detection techniques supported by a network of professionals would allow for a rapid response to outbreaks of this pest and would likely result in prevention and/or reduction of losses. This is an initial draft for the standard operating procedure for plant diagnostic laboratories for the Pink Hibiscus Mealybug (PHM) Maconellicoccus hirsutus (Green). This manual will be updated periodically as information is available..currently, websites with background information are provided in the following links: DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 2

4 Protocol: Take the appropriate steps, listed below, when plant material suspected of PHM infestation is submitted to your laboratory. 1-Sample Collection and Submission: Sample submission may be directly from a grower questioning the cause of symptomatic plants or from regulatory personnel that have reasons for suspecting a possible PHM infestation. 1. Collected suspect infested plant material must be placed in double ziplock bags. The exterior of the outer Ziploc bag should be disinfested with 70% ethanol. This will kill any mealybug crawlers (immature instars) that may be on the exterior of the bag. It is recommended that a dry paper towel be added to a suspect PHM or any scale/mealybug sample during shipping. Never add water to a sample. The double ziplock bags containing the supect sample should then be placed into a crush proof shipping container and all seams should be sealed. Suspect PHM samples on plant material must be hand delivered or mailed immediately to your local diagnostic clinic, to avoid specimen decay. In some cases, plant pathology, nematology, and entomology disciplines are located in one diagnostic facility, but this is not the case for every state. Please contact your local land grant university cooperative extension service for further submission instructions. 2. Never leave a collected plant specimen in sunlight or a warm location for an extended period of time. If you are collecting or working and cannot immediately return to your office or laboratory, either 1) temporarily place the secure specimen (already double bagged) in a protected, dry cool location, or 2) carefully remove suspect mealybugs and place them in a vial of 70% alcohol solution. It is recommended that you use a small paintbrush to lightly place or brush the specimens into a vial. When shipping alcohol vials, ensure that that are 1) packaged well in a standard mailing tube or strong crushproof container 2) the seal on the vial is secure and is not likely to be disturbed during shipping and 3) host plant information is also included with this sample submission. 3. When collecting PHM, it is important to collect multiple, representative samples. Several other species of mealybugs have been known to occur simultaneously on host plants with PHM infestation. Additionally, specimen quality and life stage can be important for PHM identification, as well as proper arthropod identification in general. Most of the NPDN diagnostic labs will focus on identification of adult females, but identification of other life stages is possible for this pest. For example, male PHM can be relatively easily differentiated from other mealybugs in the U.S. by utilizing morphological characteristics, and NPDN labs will be provided with information on these identifying features. However, collection of male mealybugs is only likely to occur with trapping methods that are currently under investigation by USDA-APHIS personnel. 4. If submitted by regulatory personnel, the inspector will label and complete the appropriate forms. The inspector should record the State, identifier, the grower s license number (if applicable), the host(s), the inspector s initials as well as the location and date DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 3

5 of inspection. The results of all suspect PHM samples will be submitted to the NPDN database and cross-referenced with NAPIS. 5. Many of the NPDN regions have established FEDEX accounts that can be used to ship samples to expert labs. Please check with your regional center before forwarding samples. 2-NPDN Laboratory Sample Receipt and Examination: Upon arrival, contact submitting entity and acknowledge receipt of sample. The sample should be inspected in a clean, workstation that is not contaminated with other samples. The workstation should not contain any alternate, living host plants. Following proper slidemounting of the suspected sample(s), the workstation should be cleaned with 70% alcohol, bleach or a bleach solution, or a standard household cleansing agent. All remaining plant material should be double bagged, frozen, and then discarded. 3-Sample Storage: While examination and specimen preparation is in progress, only the key diagnostician(s) or associated laboratory staff should have access to the living samples. If a sample must be temporarily stored in a refrigerator, it must be access-controlled. 4-Sample Screening: Both initial and advanced screening steps are listed below. Initial screening includes viewing specimens prior to slide-mounting. Initial screening will be conducted by triage laboratories (NPDN land grant university labs). Some triage labs may choose to conduct initial screening with digitally assisted diagnosis. Contact your state s triage lab for more information. Contact your local cooperative extension service for more information. Two levels of advanced screening will occur: Level 1. Slide-mounting of suspected PHM samples and tentative identification. Some NPDN triage labs will conduct Level 1 advanced screening. If a facility cannot perform Level 1 advanced screening, it will be referred to one of the NPDN advanced screening labs (Table 2). Level 2. Final confirmation of species-level identification. At this time, all suspect PHM samples must be confirmed by one of the four identification specialists in scale insect taxonomy. Once an NPDN triage lab has correctly identified at least 10 PHM specimens correctly, they may petition to be listed as an approved PHM identifier. More information on this process will be available at a later date. Plans for testing and/or additional training for other individuals to positively identify PHM are in progress. It is anticipated that several of the NPDN triage labs and other cooperators will be listed as sources for confirming PHM identification during It is also anticipated that regional centers will be able to handle overflow, Advanced Screening Level 1 during Current NPDN sample overflow contact information is listed in Table 2. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 4

6 4.1 Contacts for Advanced Screening and Scale Taxonomy Table 1: Identification Specialists in Scale Insect Taxonomy Dr. Greg Evans USDA-APHIS Systematic Entomology Lab, PSI, Building 005, Barc- West Baltimore Ave Beltsville, MD (301) Policies for submission of samples to the USDA, Systematic Entomology Laboratory are available at: Dr. Douglass Miller USDA-ARS Systematic Entomology Lab, PSI, Building 005, Barc- West Baltimore Ave Beltsville, MD (301) Policies for submission of samples to the USDA, Systematic Entomology Laboratory are available at: Dr. Greg Hodges Florida Department of Agriculture, Division of Plant Industry 1911 SW 34 th St Gainesville, FL (352) ext. 183 Dr. Gillian Watson Plant Pest Diagnostics California Department of Food and Agriculture 3294 Meadowview Rd Sacramento, CA (916) NOTE: Identification specialists should be contacted prior to shipping samples. Not all specialists may be willing to receive out-of-state suspect samples. Their willingness and ability to process out-of-state samples may be limited by various factors, including time and personnel cost constraints. At this time, Dr. Greg Hodges has agreed to handle overflow samples for NPDN laboratories. Pending potential sample loads, more locations may be established as overflow labs during All suspect pink hibiscus samples (either positive or negative) must be reported to the NPDN database. THE USDA, SYSTEMATIC ENTOMOLOGY LABORATORY (SEL) HAS DETAILED SAMPLE SUBMISSION INFORMATION REGARDING REGULATORY, UNIVERSITY, AND PUBLIC SUBMISSIONS. A SPECIFIC SAMPLE SUBMISSION FORM IS ALSO AVAILABLE. PLEASE VISIT THE USDA-SEL WEBSITE : FOR MORE INFORMATION. ANY INFORMATION PROVIDED IN THIS MANUAL DOES NOT REPLACE EXISTING POLICIES, RULES, OR REGULATIONS PERTAINING TO SAMPLE SUBMISSIONS TO THE USDA. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 5

7 Table 2: NPDN Advanced Screening Level 1 Sample Overflow (Last Updated June 14, 2005) Greg Hodges Howard Russell Florida Department of Agriculture, Division of Diagnostic Services Plant Industry 116 Integrated Plant Systems 1911 SW 34 th St Michigan State University Gainesville, FL East Lansing MI (352) ext. 183 (517) hodgesg@doacs.state.fl.us bugman@msu.edu DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 6

8 4.2 Initial Screening Field keys are not 100% accurate and should not be relied upon solely as a means of identification. Any identification made by a field key is always tentative. Below is a field key to the most commonly encountered mealybugs found on Hibiscus in Florida. This key is made to help with mealybugs that might be present on suspect plant material originating from Florida. Because many mealybugs are considered polyphagous, it is possible that additional species, beyond what is listed, may be collected. How to use the field key This field key will rely on characters that can be seen with the naked eye or through a hand lense. Characters include: body color; number of lateral wax filaments (Fig. 1); dark stripes on dorsum (Fig. 1); ovisacs (Fig. 2); length of anal lobe wax filaments (Fig. 3). Lateral wax filaments Dark stripe on dorsum Figure 1. Lateral wax filaments, Citrus mealybug (Planococcus citri) (Photo courtesy of Holly Glenn, UF, TREC) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 7

9 Ovisac Fig. (2a-b). Ovisacs produced by Phenacoccus madeirensis. (Photo Courtesy of Avas Hamon, FDACS-DPI) SPECIAL NOTE: There are some scale insects outside of the Pseudococcidae that produce ovisacs and that are sometimes confused with pink hibiscus mealybug. The most commonly confused scales with pink hibiscus mealybug are: Cottony cushion scale, Icerya purchasi Cottony cushion scale adult (Photos courtesy of Avas Hamon, FDACS-DPI) Cottony cushion scale immatures DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 8

10 Felt scales, Eriococcus species Eriococcus azaleae (Photos courtesy of Lyle Buss, UF) Eriococcus quercus Soft scales: Philephedra, Neopulvinaria, Pulvinaria, Protopulvinaria Philephedra tuberculosa (photo: Whitney Cranshaw CSU) Neopulvinaria innumerabilis (photo: Ray Gill, CDFA) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 9

11 Pulvinaria acericola (photo Demian Kondo, UC-Riverside) Protopulvinaria pyriformis (Photo DPI) Dactylopius species (DPI) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 10

12 Anal lobe filament Fig. 3a. Short anal lobe wax filament (Phenacoccus madeirensis) (Avas Hamon, FDACS-DPI) Anal lobe filament Fig. 3b. long anal lobe wax filament (Pseudococcus longispinus) (Photo courtesy of Avas Hamon, FDACS-DPI) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 11

13 I. Field Key to the most common mealybugs found on Hibiscus in Florida 1. Body color red, brown or pink Body color gray or other than above.4 2. With 1-2 pairs of lateral filaments present (usually at posterior end of body); Body fluid reddish brown; host plant material distorted and/or appearing as bunchy top Maconellicoccus hirsutus (pink hibiscus mealybug) Pink hibiscus mealybug (Lyle Buss, UF) Bunchy top (Hibiscus rosa-sinensis) (DPI) - With many pairs of lateral filaments present; host plant material not appearing as bunchytop Dorsum with dark stripe located medially; anal lobe wax filaments short; body fluid clear..planococcus citri (Citrus mealybug) Citrus mealybug (Photo courtesy of Lyle Buss, UF) Citrus mealybug (Photo courtesy of Holly Glenn, UF) - Dorsum without dark stripe; anal lobe wax filaments long (1/3 to ½ as long as body).. Pseudococccus maritimus and Pseudococcus viburni (slide mounting required to differentiate species) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 12

14 4. With 1 pair of lateral filaments present; crystalline rods arising from dorsum Ferrisia virgata (Photo courtesy of Holly Glenn, UF) - With many pairs of lateral filaments present; crystalline rods absent from dorsum 5 5. Body color green to yellow..6 - Body color gray 7 6. Body color yellow; ovisacs with numerous yellow eggs. Paracoccus marginatus (Papaya mealybug) Papaya mealybug (Photos courtesy of Avas Hamon) - DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 13

15 Body color green; ovisacs not as above; two dark stripes located on abdomen..phenacoccus solani (Solanum mealybug) Solanum mealybug (Photo courtesy of Lance Osborne, UF-Apopka) 7. Body with anal lobe wax filaments as ½ to as long as body Body with anal lobe wax filaments short Body with 1 stripe on middle of back; anal lobe wax filaments as long or longer than body; second pair of wax filaments also long; without ovisac...pseudococcus longispinus Longtailed mealybug, Pseudococcus longispinus(photo courtesy of Avas Hamon) -. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 14

16 Body without stripes along middle of back; anal lobe wax filaments ½ length of body; second pair of wax filaments shorter (1/4 length of body)...pseudococcus jackbeardsleyi (Photo Courtesy of Ray Gill, CDFA) 9. Wax dull gray, 2 dark stripes on abdomen....phenacoccus maderiensis (Madeira mealybug) Madera mealybug (Photo courtesy of Avas Hamon, DPI) (Photo courtesy of Holly Glenn, UF) DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 15

17 - Wax white, two small dark stripes on abdomen, 2 small dark stripes on head region; wax along midline appearing up-raised.. Phenacoccus solenopsis (Solenopsis mealybug) Solenopsis mealybug (Photo courtesy of Lance Osborne, UF) 4.3 Advanced Screening Level 1: Slide Mounting and Tentative Identification General Morphology Species determination of mealybugs will require slide mounting specimens to look for the following morphological characters being present: ostioles; cerarii; one or more circuli; swirled type trilocular pores; translucent pores on the hind legs; 2 pores on each surface of the trochanter. It should be noted that all of these characteristics may not be present in every mealybug species. Some will lack a circulus whereas others may have three, some have translucenct pores on the hind legs and others do not. There is almost always a situation where a qualifying word like usually mostly but except can be applied to a given species of mealybugs. Body shape The body shape of mealybugs can vary depending on the species. Many species are oval in appearance (Citrus mealybug to Pink hibiscus mealybug). Others may be elongate (Rhizoecus species, Trionymus species) or occasionally round (Nipaecoccus species and Hypogeococcus species). General Scale and Mealybug Information A variety of scale/mealybug information is available on scalenet at: DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 16

18 Slide-Mounting for Specimen Preparation: Materials 10% KOH Distilled H 2 O 70% ethanol 95% ethanol Clove oil Canada balsam Xylene Micro-spatula Minutin pin tool Micro-scissors Syringe (optional, depending on method used) Wilkey s Double or Triple Stain Small Petri dishes (~2 inches) Hotplate Watchglass Slides and cover slips Slide oven or slide warmer Slide labels Purpose and Formulas for Some Materials Modified from information provided by the Florida Department of Agriculture, Division of Plant Industry (FDACS-DPI), Gainesville, FL. A. KOH: This solution starts the clearing process for insects. A 10% solution is prepared for this process. B. Distilled H 2 O + stain: Stains are allowed to adhere to all sclerotized structures and key identifying characters can be observed. Specimens may remain in this solution for 15 minutes to overnight. C. Wilkey s Double Stain: See Wilkey (1962) D. Triple Stain (method used by FDACS-DPI): A combination of 3 for-sale stains. a. Ingredients i. Lignin pink (2%) 10 ml (40 drops) ii. Acid fuchsin (4%) 5 ml (20 drops) iii. Erythrosin (2%) 10 ml (40 drops) iv. Remember to use distilled H 2 O to mix your solutions of Lignin pink, Acid fuchsin, and Erythrosin. b. The above ingredients are added to 100 ml of Essig s Aphid Fluid (EAF). This is used as the stock staining solution. One or two drops is added to approximately half a casserole of EAF to actually stain specimens. c. Essig s Aphid Fluid (modified from Wilkey, 1962): Protect from light. i. Lactic acid (85%) 20 parts ii. Phenol (liquefied) 2 parts iii. Glacial acetic acid 4 parts iv. Distilled H 2 O 1 part E. 95% ETOH: Removes any excess stain on the specimens and helps with the dehydration process. The 70% ETOH step allows the specimen to more gradually dehydrate. Sometimes, the 70% ETOH step may be skipped for faster sample processing. You may leave specimens in the alcohol steps from 2-15 minutes, but remember that alcohol will evaporate. Do not leave in this step overnight. You may also use this step to finish clearing the specimen by pumping it with a micro-spatula. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 17

19 F. Clove Oil: Completes the dehydration process. Specimens may remain in clove oil from 10 minutes to days. Urgent samples should be processed as immediately as possible. Wash hands after using clove oil. G. Canada Balsam: Mounting media. A small drop will be placed on the slide, and the specimen should be pushed all the way to the bottom of the slide. If specimens are allowed to remain suspended in the media, they may be difficult to identify. Slide Mounting Summary A. KOH-Distilled H 2 O + Stain-95% ETOH-Clove Oil-Canada Balsam B. Re-Mounting: Sometimes, it is necessary to remount a specimen if a quality mount is produced. The need to re-mount a specimen may depend on the urgent status of the specimen. a. A slide may be broken, and the basic slide-mounting steps may be followed backwards. b. Slide-Clove Oil-ETOH-Distilled H 2 O-KOH. This will destain the specimen. Once in KOH, allow to sit stay in solution overnight and begin the process from the first step again. Note: Mealybug slide-mounting video tutorial will be available during Protocol Several slide-mounting procedures are available for mounting scales and mealybugs. The following procedure provides two options, a slower and a faster method. Both methods may produce quality slide mounts with sufficient practice. Note: Individuals should practice slide-mounting on other common scales and mealybugs before attempting to slide mount suspect PHM samples. Modified from a procedure used by Greg Evans, USDA-APHIS A. Select the adult females from sample (i.e. the largest individuals except for those with wing pads). B. You may choose to mount specimens by utilizing either a slow or faster process. a. Slow process i. Place specimens in a vial with 10% KOH over night. ii. Puncturing the specimen before placing it in KOH allows the KOH to enter more readily and speeds up the process. iii. Place a label on the vial with the sample number. b. Fast process i. Place specimens in 10% KOH in a watchglass and heat for approximately 10 minutes on low heat (~200 F) on a hotplate. ii. In order to facilitate transfer of specimens, you may want to place the watchglass on one end of a slide, and leave the other end of the slide off of the hotplate. This will allow you to pick up the slide and move it when you are ready to transition to the next step. iii. Write the sample number on another slide, and place it on top of the watchglass so that it almost covers the well. This will allow you to avoid quick KOH evaporation, and it will also ensure that the sample number stays with the specimen. C. Pour or transfer the specimen(s) to a small Petri dish (~2 inch diameter). You may want to have a small amount of distilled water on the bottom of the Petri dish to soak the specimen and have enough space for the next step. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 18

20 D. The next step involves removing the internal gut contents of the mealybug. This is necessary in order for you to view the specimen s skeletal structure. Gut contents must be removed in order for identifying characteristics of the mealybug to be apparent. a. Using micro-scissors, you may cut half-way across the specimen between legs 2 and 3. i. In order to flush out the internal material, fill a syringe with water and shoot the water into the cut. ii. You may have to repeat the above process several times in order to properly flush out the sample. b. You may also pump the internal material out of the specimen. i. Pumping the internal material out of the specimen may be accomplished by a cutting a small hole or a puncture in the specimen. In the middle of the specimen s body, between legs 2 and 3 would also be a good location for a small cut or puncture. ii. You may then gently press the specimen with a micro-spatula in order to pump out the gut contents. E. In the next step, you will carefully go inside the specimen with a minutun pin tool. a. Carefully scrape off the tracheae. b. Carefully pull out any other internal material. F. Place specimen in Wilkey s Double or Triple Stain for at least 15 minutes. G. Place specimen in 70% ethanol to wash out excess stain. You may need to gently pump the specimen with a micro-spatula to remove excess stain. H. Place specimen in 95% ethanol to dehydrate specimen in preparation for clove oil. Leave in 95% ethanol for 2-3 minutes. I. Place specimen in clove oil for 2-3 minutes or until in looses its sheen. J. Place a small drop of Canada Balsam at the center of the slide and dilute it with a drop of xylene if necessary. K. Place specimen in balsam drop, head towards you and dorsal side up. L. Spread out legs and antennae. M. Place a cover slip on balsam drop, and gently press on the top until the balsam come to the edge of the cover slip. If necessary, you can add a drop of xylene to the edge, but too much xylene will ruin the mount and cause it to be diluted and runny. N. Place sample number on the slide with a Sharpee pen, and place on slide tray. O. Maintain slide in slide oven or slide warmer for approximately 2 weeks. P. Label. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 19

21 Slide mounted key to the adult females of common mealybugs found on Hibiscus in Florida Greg Hodges, FDACS-DPI NOTE: This key includes the most common mealybugs associated with plant material originating from Florida on hibiscus. Other mealybug species may occasionally occur on hibiscus in Florida and other species may be more prevalent in other U.S. locations. For more detailed, complete mealybug taxonomic keys, refer to the selected references section. 1. Anal bar present Anal bar absent With 18 pairs of cerarii; oral rim ducts absent With 4-6 pairs of cerarii present; oral rim ducts present..maconellicoccus hirsutus 3. With oral rim ducts present; anal bar short..paracoccus marginatus 3 Without oral rim ducts; anal bar long and pronounced. Planococcus citri 4. With oral rim tubular ducts; claw without denticle Without oral rim tubular ducts; claw with denticle Sclerotized area with associated setae surrounding oral rim tubular ducts; with only one pair of cerarii; cerarii without auxiliary setae.ferrisia virgata 5 Area surrounding oral rim ducts not sclerotized and without setae; with 17 to 18 pairs of cerariian setae; cerariian setae anterior to anal lobe with hair-like auxillary setae pseudococcus species 6. Multilocular disc pores present on dorsum and venter in rows across abdominal segements; circulus mushroom shaped, with quinquelocular pores present on venter....phenacoccus madeirensis 6 Multilocular disc pores absent from dorsum but present in venter in rows across the abdominal segments; circulus round to oval; quinquelocular pores absent Circulus small and oval; antennae normally 8-segmented; multilocular disc pores present on abdominal segments IV-VIII Phenacoccus solani 7. Circulus flaccid; antennae normally 9-segmented; multilocular disc pores present on abdominal segments VI-VIII... Phenacoccus solenopsis DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 20

22 Selected References for additional Pseudococcidae taxonomy Ferris, G.F Atlas of the Scale Insects of North America. (ser. 5) [v. 5]. The Pseudococcidae (Part I). Stanford University Press, Palo Alto, California. 278 pp. Ferris, G.F Atlas of the Scale Insects of North America, v. 6, The Pseudococcidae (Part II). Stanford University Press, Palo Alto, California. 506 pp. Kosztarab, M In:, Scale insects of Northeastern North America. Identification, biology, and distribution. Virginia Museum of Natural History, Martinsburg, Virginia. 650 pp. McKenzie, H.L Mealybugs of California with taxonomy, biology, and control of North American species (Homoptera: Cooccoidea: Pseudococcidae). Univ. Calif. Press, Berkeley. 526 pp. Miller, D.L Identification of the pink hibiscus mealybug, Maconellicoccus hirsutus (Green) (Hemiptera: Sternorryncha: Pseudococcidae). Vol. 13 (3-4): Williams, D.J. & Granara de Willink, M.C Mealybugs of Central and South America. CAB International, London, England. 635 pp. Williams, D.J Mealybugs of southern Asia. The Natural History Museum, London. 896 pp. Level 2: Confirmation of Species Identification Only identification specialists in scale insect taxonomy may positively confirm the identity of the pink hibiscus mealybug, Maconellicoccus hirsutus. Also, Dr. Greg Hodges and Howard Russell are the official contacts for overflow advanced screening in the NPDN at this time. This does not include submitting samples for general state-specific or national surveys for mealybugs or scales. This only applies to suspect pink hibiscus mealybug samples submitted through the extension service or collected by agricultural inspectors in potential suspect plant shipments. Pending sample loads, other labs will potentially be added as overflow sources during DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 21

23 5-Communication: If field screening by an NPDN triage labs results in a suspect PHM sample, follow this communications protocol. A. Notify the appropriate NPDN Regional Center of the suspect sample and the location that the sample is being shipped. Contact the lab that you are sending the sample to prior to sample submission. Indicate the level of screening that has occurred (i.e. field screening or advanced slide-mounted screening). Regional Center Director: Address: Address: Phone Number: Cell Phone Number: Fax Number: Regional Center Diagnostician: Address: Address: Phone Number: Cell Phone Number: Fax Number: B. Contact the State Plant Health Director (SPHD) and the State Plant Regulatory Official (SPRO) in the sample state of origin, State Plant Health Director: Address: Address: Phone Number: Fax Number: State Plant Regulatory Official: Address: Address: Phone Number: Fax Number: DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 22

24 Fax the following to the SPHD at the PPQ regional office: 1. A copy of the updated form 391 with the preliminary diagnosis and the responsible diagnostician s contact information, 2. A copy of the overnight delivery form used to submit the sample to the regional center, 3. A copy of the state inspector s sample card information submitted with the sample. C. Alert the laboratory receiving the sample of shipment details. Provide them with sample shipment time, delivery method, tracking number, sample number. 6-Confirmation: A. State regulatory officials will be notified of the results. Once confirmation is made, state and federal regulatory officials will handle any actions dealing with containment and eradication (if applicable. PHM management strategies may differ by state). B. Diagnosticians will be notified of the results by state regulatory officials. Communication between diagnosticians and state and federal regulatory officials is permitted after confirmation is received. C. Notify the Regional NPDN Director of confirmed results. 7-Sample Destruction: Once the specimens have been properly preserved and obtained from a sample, the sample should be destroyed. This can be accomplished by freezing the plant material for at least 48 hours. DRAFT 1.1 June 14, 2005, Hodges, A. C. and G. S. Hodges PHM Page 23

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