DIAGNOSTIC PROTOCOLS FOR REGULATED PESTS

Trogoderma granarium

Introduction

The Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae) is a stored product pest of great importance. Its importance lies not only in the capabilities of causing serious damage to stored dry commodities but also that countries having established populations are facing export restrictions.

The Khapra beetle is thought to have originated from the Indian subcontinent but has become established in a number of Asian, Middle Eastern, African, and some European countries. It is one of the very few stored product pests that has limited worldwide distribution. T. granarium has very limited ability to spread without human aid as it is unable to fly. It is very important to distinguish between those records which relate to introductions and those of established infestations.

The Khapra beetle is established within an area broadly limited north by central Asia, south by the Equator, west by the Sahel of West Africa and east by Myanmar; i.e. the warm dry regions along the Suez route from the Indian subcontinent to Europe. This area includes the southeastern part of the EPPO region. Khapra beetle has been introduced into areas of similar climatic conditions elsewhere particularly the alternative route between India and Europe around Africa. Currently its status in southern Africa is unclear and it may be absent there now. Initially introductions caused severe damage but outbreaks have been local and in most cases have been eradicated. In general, Khapra beetle is only successful in competition with other major stored product pests in conditions of low humidity. It has also established in some areas of unfavourable climate, where it survives only in protected and/or heated environments such as maltings and feedmills, for example in Europe, Korea and Japan. There were multiple introductions to the United States and Mexico but these were eradicated.

Hosts

The Khapra beetle is a significant stored product pest. It may occur in various dry stored products of primarily vegetable origin. Primary hosts are cereals (rice, wheat, oats, barley, sorghum, maize etc.), cereal products (rolled oats and barley, ground corn, corn meal, wheat flour, bran products, pasta, etc.), pulses (chickpea, soybean, and lentil), various vegetable seeds (cucurbit, capsicum, tomato, etc.), herbs, spices and various nuts (peanut, pecan, walnut, almond etc.). It can successfully complete its lifecycle in copra, dried fruits, various gums and many different dried products of wholly or partial animal origin such as skins, dried dog food, dried blood, dead insects and dried animal carcasses. As a pest it is most prevalent under hot dry conditions where very heavy infestations can develop. In cooler and also in hot and humid conditions it tends to be out competed as a pest by other species such as Sitophilus spp. and Rhizopertha dominica. Commodities stored in bags in traditional warehouses are more at risk from this pest than bulk stored commodities.

Biology

There are important features of T. granarium biology which enable the pest to survive in harsh conditions. In unfavourable circumstances it is able to maintain its presence in very low numbers in the stores.

Khapra beetle can have between one and more than ten generations per year depending on food availability and quality, temperature and humidity. A complete life cycle may be as short as 26 days in an optimum environment, or, as long as 220 days or more in a suboptimal environment. In temperate climates larvae become inactive at temperatures below 50C, so the pest will only survive and breed in protected environments. However, there are two genetic variations of larvae: those which are able to undergo facultative diapausa and those that are unable to do so. Larvae of the first type are stimulated into diapausa by adverse conditions such as low or high temperatures, and/or lack of food. During diapausa their respiration drops to an extremely low level. This leads to problems with fumigation. With such a low respiration rate the gas intake is very low, therefore the rate and/or the exposure time must be increased for successful treatment. Diapausing larvae are also cold hardy and may survive temperatures below -80C. Should favourable conditions return the pest is able to multiply rapidly and causing serious damage to the commodity.

For more general information on T. granarium, see Hinton (1945), Lindgren et al. (1955), Pasek (1998), CABI CPC (2005), EPPO/CABI (1997) and Berg (1999).

Identity

Name: Trogoderma granarium Everts, 1898 Synonyms: T. albonotatum Reiche in Mulsant et Rey, 1868 Trogoderma quinquefasciata Leesberg, 1906 Trogoderma khapra Arrow, 1917 Trogoderma afrum Priestner, 1951 Trogoderma granarium ssp. afrum: Attia & Kamel, 1965

Taxonomic position: Insecta: Coleoptera: Dermestidae, Megatominae Bayer computer code: TROGGA Quarantine status: EPPO A2 list: No. 121 COSAVE: quarantine pest not present Other NPPO:

Detection

T. granarium has the following developmental stages: eggs on the surface of grain and other stored products; larvae (5-11 instars) in stored products (larvae in diapausa may be found in packing material or within storage structure); pupae in stored products, in the last larval skin; adults in stored products.

Methods to detect T. granarium infestations include inspection (physical and visual search) and use of food baits or, more importantly, pheromone traps. Often the infested material contains larvae only. There are three reasons for this: (1) adult longevity can vary between 12-25 days on average although it can be as long as 147 days in unfavourable conditions, while larval longevity is 19 –190 days (it can be up to 6 years shoud larvae go to diapausa); (2) most of the Dermestid larvae occurring in the stored product will partially or wholly consume dead adults; (3) adults are usually present when conditions are favourable for population growth. Larval skins are usually not consumed so their presence is a clear indication of a possible active infestation. Larvae are extremely cryptic by nature. It is particularly so in the case of diapausing larvae that can stay inactive in cracks and crevices for periods of time where they are very difficult or nearly impossible to locate.

Searches for this pest are particularly difficult in cases of low level infestations. In addition to the obvious food sources the potential hiding places should also be inspected. In contrast to most other stored product pests the Khapra beetle prefers hot and dry areas. During inspection efforts should be concentrated in these areas. Larvae of Trogoderma species are very crepuscular and populations can persist in small quantities of residues that may occur within a structure or mode of transport. Larvae in diapausa can survive long periods without food. For diapausing larvae it is important to search under flaking paint and rust, piles of dirt, in empty packaging materials such as hessian bags, tarpaulins, and corrugated cardboard. Larvae are often hiding behind wall panelling, under internal lining, between floorboards, under insulation, on dry ledges, electrical cable trays and conduits, switch boxes, stores and kitchens etc. Larval skins become airborne very easily therefore it is always important to check window sills, grilles of venting holes, and spider webs for their presence. Rodent traps containing baits should be always inspected.

Khapra beetle infestations can be recognised by (1) the presence of the pest (especially feeding larvae and cast skins) and (2) symptoms of infestation. The short-lived adults are sometimes not seen. Damage to the commodities can be a warning sign, but often it is a result of the feeding of other common stored product pests. Early instar larvae of this pest feed on broken or damaged kernels, whereas older larvae feed on whole kernels. Larvae usually feed first on the germ portion of cereal seeds and then on the endosperm. The seed coat is eaten in an irregular manner. Larvae can occasionally be found as deep as 3-6 m in bulk grain. In the surface layers of the infested stored product numerous cast larval skins are present, and these are often covered with long hairs. Infested material is also polluted with frass, larval skins and broken setae.

During stored product inspections of commodities from areas where T. granarium is indigenous or known to be established, particular attention should be paid to the presence of various developmental stages of the pest, cast larval skins and signs of damage. Samples of the suspect products have to be visually inspected in a well-lit area, using a 10x magnification hand lens. If no signs of Trogoderma infestation are found then larger quantities of the product should be passed over sieves with aperture sizes relevant to the particle size of the products. Usually sets of sieves of aperture sizes 1.0, 2.0 and 3.0 mm are used. The sifted material collected on particular sieves should be placed in Petri dishes and examined under at least 10x but preferably greater than 25x magnification through a stereoscopic microscope to detect the pest. This sieving technique allows the detection of various developmental stages of the pest. However, some larvae feeding within grains may remain undetected. Therefore, it may become necessary to heat samples to 40oC to drive pests out of the grains. Visual inspection is preferential because sieving can easily destroy or seriously damage dead adults and larval skins rendering the identification very difficult or impossible.

Insects found during quarantine, routine or monitoring inspections of warehouses, ships and cargo containers should be carefully picked up with small forceps or collected using an aspirator (“pooter”). One of the most important requirements is that the inspector should collect multiple specimens of the pest. Identification of larvae is difficult, and if the dissection of a single specimen is not successful and serious damage occurs to the mouthparts then exact identification is impossible. Specimens should be placed, in a sturdy plastic or glass container or jar (McCartney bottle) containing 70% alcohol. Sending live specimens of any pest through mail services or even courier services is not advisable. It is particularly important in cases of suspected quarantine pests of great pest potential. Sending live insects is acceptable only when the inspector is not able to preserve the specimens correctly because of lack of equipment. Collection data (exact locality, date, commodity, inspector's name, contact details) should be written on a small piece of paper using pencil or alcohol proof pen. The label should be placed in the jar with the insect specimen(s). The jar should be placed in a strong, sealable plastic bag with another label with the same data. alcohol When young larvae are present it may become necessary to rear them out for reliable identification. Such rearing should only take place in a PC3 quarantine facility. If there is no such facility available and/or no safe transport available then the species should be considered as a suspect pest of quarantine importance, and as a precautionary approach the commodity should be fumigated with methyl-bromide at the rate for Khapra beetle

In warehouses, it is possible to monitor the presence of T. granarium using various traps. Traps can be as simple as offering hiding place for the larvae such as pieces of corrugated cardboard or hessian bag placed on the floor. Food baited (oil seeds, peanuts, wheat germ etc.) or attractant (wheat germ oil) traps can be used to attract larvae. These traps need to be left in place for weeks to provide opportunity for the larvae to find it. After finishing the monitoring all the traps should be collected and destroyed. Forgotten traps may offer the pest a breeding place. The most efficient and easiest method to monitor is the use of pheromone traps where the pheromone capsule is combined with a non-drying sticky trap. These traps can be monitored easily on a weekly basis. With the trapping grid design it is always very important to remember that adult Khapra beetles don't fly and larvae are slow moving cryptic insects. It’s also important to keep in mind that the Trogoderma pheromone traps are not species specific and attract many Trogoderma species and also other Dermestid beetles. (Saplina, 1984, Barak, 1989; Barak, Burkholder & Faustini, 1990, Mordkovich and Sokolov, 2000. Traps baited with pheromone and food bait are commercially available for this species.

Adults in good condition can be identified under stereomicroscope using 10 - 100 times magnification. However movement of the stored product -particularly cereals - will damage the dead adults. In Most cases the legs and antennae will break off and also the setae on elytra and pronotum will be rubbed off. In the case of a damaged specimen with missing body parts or morphological features not visible, it is necessary to examine the genitalia. For reliable identification the genitalia should be always examined. Genitalia should be removed (process described later) and mounted on a cavity microscope slide temporarily using glycerol or permanently using Hoyer’s solution. For larval identifications the mouthparts should be dissected out (process described later). The larval skin and dissected mouthparts should be mounted on a cavity microscope slide using Hoyer’s solution (Beal, 1960). Details of mounting procedures are included in the next section. Adult and larval dissection can be performed under 10 - 40 times magnification using stereo dissecting microscope. For the examination of genitals and larval mouthparts - particularly the papillae of the epipharynx - a good quality compound microscope is necessary and must be capable of 400-800 times magnification in bright field and phase contrast. Depending on the microscope use of oil immersion may be necessary for satisfactory resolution.

Identification

The genus Trogoderma includes: according to Mroczkowski (1968) 117 species, Beal (1982) 115 species and according to Háva, 2003, 130 species. Many more remain unknown to science. Great caution needs to be exercised with the synonymies established because few of them are based on detailed comparison of the type specimens. Many other Dermestid species belonging to other genera occur in stored products. Members of Dermestes and Attagenus genera are frequently found feeding on materials of animal origin such as dog biscuits, dried meat, dried blood and also rat, mice and bird carcasses. Anthrenus and Anthrenocerus species can be serious pests of wool and woollen products. In stored products heavily infested by other stored product pests, non-pest Trogoderma will usually feed on these dead insects. Other genera rarely found in stored produce are Reesa, Orphinus, Phradonoma, and Thorictodes.

Trogoderma species other than Khapra beetle may also be found in stored products, but only some of these feed on them. Among these species the biggest economic losses are caused by T. variabile Ballion that is a recognised quarantine pest in some countries. However, most of Trogoderma species occurring in stored products appear to be scavengers feeding on dead bodies of other insects. During a 12-year survey conducted in California, eight species of Trogoderma were found in stored seeds, animal feed and grocery commodities (Strong & Okumura, 1966). Some species occurring in stored products closely resemble T. granarium.

Identification of Trogoderma eggs and pupae based on external features is currently not possible. Insect eggs and pupae possess very few external features and therefore poorly studied. If in the future there is an available ELISA or PCR test for larval and adult identification it may well be suitable for egg and pupa identification. Larval identification is difficult. It requires experience in identification and also good skills in dissection of small insects. Pupation takes place in the last larval cast. The larval skin can be used for identification but one needs to be more cautious because it is brittle. Adults are the easiest to identify though misidentification by less experienced entomologists is still common.

Many taxonomic revisions, descriptions, and keys have been constructed for various Dermestidae genera but unfortunately so far no key has been published for all known Trogoderma species. Care must be taken when using references because descriptions of new pest species and synonymies continually appear in journals.

Several keys have been published for the economically important species. Most recently Banks (1994) published a key to adults and larvae of the genus Trogoderma associated with stored products, as well as keys to larvae and adults of some species found in warehouses. Mordkovich & Sokolov (1999) published a key for identification of adults of some Dermestid beetles including Trogoderma found during quarantine inspection of plant and animal products and packing material. Beal (1960) has constructed an identification key to larvae of 14 species of Trogoderma from different parts of the world, including stored products pests. Mitsui (1967) published illustrated keys for identification of larvae and adults of some Trogoderma species. Kingsolver (1991) and Barak (1995) published keys to adults and larvae of some Dermestid beetles occurring in stored products, including a few Trogoderma species. Beal (2005) published a key to distinguishing adults of Trogoderma yunnaeunsis Zhang et Liu from other Eastern Palearctic Trogoderma species, including T. granarium.

Hinton (1945), Beal (1954), Varshalovich (1963), Booth et al. (1990), Haines (1991), EPPO/CABI (1997) and Berg (1999) give detailed descriptions of T. granarium. Green (1979) described male and female genitalia and Beal (1960) described the larva.

Procedure for preparation of larva and larval exuviae

For identification larvae should be mounted on a microscope slide using the following method. Before dissection the larva should be examined under stereo microscope. Size, body color, arrangement and colour of setae should be recorded.

Materials

• Hotplate • Insect pins • Micropins 0.015 mm diameter attached to the end of a toothpick with hot-glue, wax or nailpolish • Insect pins (No. 1) and/or micropins with hook or loop end • 10 % KOH solution • Test tubes • Fine dissection forceps, preferably jeweller’s forceps #4 • Eye surgery scissors • Cavity microscope slides • Circular coverslip #1, 19 mm diameter • Cavity glas block (embryo glass) • Fine brush • Distilled water • Hoyer’s solution • 5% Decon 90 Solution

Methods

The preparation method in Hoyer’s solution described below is recommended for current identification of larvae in phytosanitary laboratories. Hoyer's mountant is waterbased therefore can't be considered permanent. Permanent slides can be made using Euparal or Canada Balsam for mounting. Mounting in Canada Balsa requires laborious dehydration technique but the slide will be permanent.

• Place the specimen ventral side up on a microscope slide. Cut open the whole body along mid-line from under the head capsule to the last abdominal segment using eye surgery scissors, to ensure that the specimen can be completely open up for mounting. • Put the larva into a test tube containing 10% KOH solution and heat in boiling water bath. (Using NaOH solution instead of KOH is more time consuming but lessens the possibility of damaging the specimen.) • Rinse thoroughly in warm distilled water. • Remove all the internal tissues using very fine, short hair brush or convex surface of a hooked tip of a No. 1 insect pin, or loop formed of a micropin (0.015 mm diameter). On one side the setae should be removed from the 7th and 8th abdominal segment. • Remove head capsule and put it back in the hot KOH solution for 5 minutes. • Rinse the head capsule in warm distilled water. • Depending on personal preference the dissection of head can be performed in a few drops of Hoyer’s solution or glycerol on a microscope slide or in water in an excavated glass block (embryo glass). • Turn the head ventral side up and fix it to the glass with a blunt No. 1 insect pin. Remove the mandibles, maxillae and labial palpi using jeweler’s forceps #4, and micropins attached to toothpick. Remove the epipharynx and antennae. • Mount the head capsule and the mandibles in the cavity of the microscope slide using Hoyer’s solution. • Mount the cleared skin, fully opened on the flat part of the microscope slide, next to the cavity. It is usually best done ventral side up. Epipharynx, antennae, maxillae and labial palpi should be mounted with the skin under the same coverslip. (If they are mounted in the cavity the parts will rotate rendering the examination difficult or in case of the epipharynx impossible.) All body parts should be mounted on the same microscope slide. • In the case of larval exuviae before proceeding with the dissection the specimen should be soaked in 5% Decon 90 solution for about two hours and thoroughly rinsed in distilled water. Cut the specimen open anteriorly and dissect out the mouthparts. It can be mounted directly in Hoyer’s solution. • Slides should be labeled immediately after mounting the specimen to prevent possible mix-ups. Labels should be written using pencil, pigment pen, Indian ink or printed using laser printer. Fountain pens or Biro should not be used. • The slides should be placed in an oven for a few days at 400C . • After drying slides should be ringed using Glyptol, Brunseal, Euparal or at least two layers of nail polish in order to prevent the Hoyer’s from drying and possibly damaging the specimen.

The identification should be performed using a high powered (at least 400x) compound microscope. Depending on the quality of the microscope oil immersion may need to be used to achieve satisfactory resolution.

Note: Banks, (1994) recommends placing the larva ventral side up and cutting the head vertically just posterior to the mandibles using a razor blade. Remove the mandibles and the palpi in order to prevent them covering the epipharynx after mounting. Although it is a good way of preparing the larva, if the blade used is not in perfect condition and the person carrying out the dissection is not experienced enough it is very easy to damage or even destroy the epipharynx.

Procedure for preparation of adults

Adult Trogoderma specimens may need to be cleaned before identification. If the specimen was caught in a sticky trap the glue can be dissolved in Limolene, Citricide, Dissolv-It, baby oil or kerosene. The solvent and any dirt can be removed by any laboratory detergent, like Decon 90. Dirty specimens can be cleaned using an ultrasonic cleaner.

In cases of quarantine importance dissection and examination of the genitals is recommended.

There are different ways of processing adults. Banks (1994) recommends complete dissection and mounting of the whole adult on a cavity microscope slide. We believe it is not necessary because mounting will obscure important characters e.g. colour of setae. Also placing a cover slip on the microscope slide will inevitably lead to movement, turning and sometimes congregation of the body parts rendering examination of important features very difficult or impossible.

The procedure for preparation of adults is as follows:

Materials

• Hotplate • Insect pins • Micropins 0.015 mm diameter attached to the end of a toothpick with hot-glue, wax or nailpolish • Insect pins (No. 1) and/or micropins with hook or loop end • 10 % KOH solution • Test tubes • Fine dissection forceps, preferably jeweller’s forceps #4 • Eye surgery scissors • Cavity microscope slides • Circular coverslip #1, 19 mm diameter • Cavity glas block (embryo glass) • Distilled water • Hoyer’s solution • 5% Decon 90 Solution • Chlorazole Black E or Safranin O

Methods

• Soak adults in warm distilled water for about an hour. • Remove abdomen while the specimen is still in the water using fine forceps. • Dry the specimen and mount it either on the tip of a cardboard triangle or preferably laterally on a cardboard rectangle. On a rectangle the specimen is less exposed therefore less likely to get damaged. Gluing them on the side make the specimens accessible for both dorsal and ventral examination. • Cut the abdomen laterally open leaving the last abdominal segment untouched. Place it in 10% KOH or NaOH solution in a hot water bath for about 10 minutes. • Rinse the specimen and carefully remove the genitals using hooked micropins. The abdomen should be glued onto the same cardboard rectangle with the insect, ventral side facing up. Usually the genitals need to be macerated further in the caustic solution. Experienced entomologists should be able to separate the aedeagus from the periphallic tergite and the 9th abdominal segment using micropins. • It might be necessary to stain the genitalia using Chlorazol Black E or Safranin O.

Genitals can be mounted on a microscope slide using Hoyer’s solution. Aedeagus should be mounted on a cavity microscope slide so it is able to retain its shape. Female genitals can be mounted on flat microscope slide.

Slides and pinned insects should be labeled immediately after mounting the specimen to prevent possible mix-ups. Labels should be written using pencil, pigment pen, Indian ink or printed using a laser printer. Fountain pens or Biro should not be used. The slides should be placed in an oven for a few days at 400C. After drying all slides should be ringed using Glyptol, Brunseal, Euparal or at least two layers of nail polish in order to prevent the Hoyer’s from drying and damaging the specimen.

If there is no need for mounting the genitals using a permanent or semi-permanent mounting agent they can be examined in a drop of glycerol on a microscope slide. After the identification the organs can be placed in a microvial in a drop of glycerol or Hoyer’s. Then the microvial can be attached to the pinned body by driving the pin through the stopper of the vial. In case of male – the genitals can be mounted on the cardboard rectangle next to the abdomen

Genera of the family Dermestidae frequently ocurring in stored commodities

Besides Trogoderma spp. other dermestid genera may also be found in stored products such as Anthrenus spp., Anthrenocerus spp., Attagenus spp. and Dermestes spp. The first step of diagnosis of collected specimens is identification to genus. Adults and in some cases larvae of these beetles can be identified using the keys of Kingsolver (1991, 2002), Haines (1991), Banks (1994), Rees (2004) and/or Háva (2004). The following simple key is to aid the quick distinguishing features of Trogoderma from the other four genera.

Larvae

1 Urogomphi present on 9th abdominal segment, 10th segment sclerotised, cylindrical ...... Dermestes spp. No urogomphi present, 10th abdominal segment not sclerotised...... 2

2 Dorsal surface without hastisetae ...... Attagenus spp. Dorsal surface with hastisetae ...... 3

3 Posterior margins of abdominal tergites sinuate, or emarginate, tufts of hastisetae placed on posterior membranous parts of tergites, 8th abdominal tergite without tufts of hastisetae ...... Anthrenus spp. Posterior margin of tergites not sinuate or emarginate, tufts of hastisetae placed on sclerotised tergal plates, 8th tergite with tufts of hastisetae ...... 4

4 Second antennal segment about twice as long as last segment, head of hastisetae at least 3 times as long as wide at the widest point ...... Anthrenocerus spp. Antennal segments are subequal, head of hastisetae less than 3 times as long as wide at widest point ...... Trogoderma spp.

Adults

1 Median ocellus absent ...... Dermestes spp. Median ocellus present...... 2

2 Body covered with scale-like setae, antennal cavity fully visible from anterior view ...... Anthrenus spp. Body covered with simple setae, some of them whitish, flattened (ensiform) but never scale-like...... 3

3 Antennal cavity completely closed behind, antennal club 3-segmented, well defined...... Anthrenocerus spp. Antennal cavity open behind or partially delimited by a posterior carina ...... 4

4 Antennal cavity open behind, posterior margin of hind coxa angulate, first segment of posterior tarsi shorter than second segment...... Attagenus spp. Antennal cavity carinate posteriorly, posterior margin of hind coxa straight, arcuate or sinuate, first segment of posterior tarsi longer than second segment...... Trogoderma spp.

Quick preliminary identification of Trogoderma larvae

• Elongate, cylindrical, hairy larvae • Hastisetae present on sclerotised part of tergites • Pretarsal setae on the ventral side of claws unequal • Antennal segments are subequal

If all of the above listed features can be observed on the larva or cast skin it is very likely that the specimen is a Trogoderma species therefore it is warranted to check the detailed list of features listed below.

Discriminating features of Trogoderma larvae

Adapted from Banks, 1994; Beal, 1954, 1960; Haines, 1991; Hinton, 1945; Kingsolver, 1991; Lawrence 1991a; Lawrence et al. 1999a; Okumura and Blanc, 1955; Peacock, 1993; Rees, 1943.

• body elongated, cylindrical, somewhat flattened, roughly 6 times as long as wide, nearly parallel sided but gradually tapering toward rear; • head well developed, sclerotised, and hypognathous; • three pairs of jointed legs present; • pretarsal setae on the ventral side of claws unequal; • very hairy, being covered with different type of setae; spicisetae, hastisetae and/or fiscisetae; • head of hastisetae not more than 3 times longer than wide; • numerous hastisetae on all nota and tergites with prominent tufts of erect hastisetae inserted on the posterolateral part of the tergal plates of abdominal segments 6 to 8 (in Anthrenus genus the tufts of hastisetae are inserted on the membrane behind the sclerotised part of tergites 5, 6 and 7) • no urogomphi present

Identification of larvae of Trogoderma granarium Everts

The larvae of T. granarium may be separated from other Trogoderma spp. using the following short key. If necessary, larvae of other pest and a few non-pest species can be identified, or at least separated, with reasonable confidence using the keys of Beal (1956, 1960), Banks (1994) and Peacock (1993).

1 Epipharynx with 4 distal papillae, usually in a single sensory cup...... 2 Epipharynx with 6 distal papillae...... 3

2 Tergites uniformly yellowish-brown, without greyish pigmentation at base of large spicisetae; acrotergites weakly sclerotised; antecostal sutura on 8th abdominal segment absent, if present faint and usually broken; setae on basal antennal segments almost completely encircling segment, second antennal segments usually with a single seta, apical segments with sensory pores in basal quarter...... Trogoderma granarium Everts Tergites usually dark greyish-brown, at least at base of major spicisetae; acrotergites brownish, sclerotised; antecostal suture on 8th abdominal segment distinct; second antennal segment without setae...... Trogoderma teukton Beal

3 Setae on basal antennal segment grouped on inner and inner-dorsal side leaving the outer and outer-ventral side glabrous, on fully extended antenna setae on basal segment not reaching apex of the second segment, sensory pore(s) on apical antennal segments not in basal quarter; median small spicisetae on acrotergites not long enough to extend over the antecostal suture; hastisetae very sparse on thoracic and anterior abdominal tergites; tergites with single row of large spicisetae...... Trogoderma variabile Ballion Specimen doesn't possesses the above combination of characters ...... other Trogoderma spp

Notes:

Larval identification can not be accepted as conclusive if it based only on one specimen, or cast skins or worn specimens. In many species the intraspecific variation can be such extent that features considered being specific to the species can't be seen while features specific to other species can be observed. In addition large numbers of non-pest Trogoderma species occur in stored commodities and many of them are unknown to science.

Discriminating features of Trogoderma granarium Everts larvae

• antennal segments subequal; • setae of basal antennal segments almost completely encircling the segment, reaching or surpassing apex of second segment, at least three-fourth as long as second antennal segment; • second segment of antenna of last instar usually with one seta; • last antennal segment with at least one sensory pore in basal quarter; • epipharynx with four papillae in distal sensory cup, usually in a single unit; • no fiscisetae present; • mesally directed tergal setae absent; • at least six small spicisetae on first abdominal tergite posterior to antecostal suture anterior to large spicisetae; • anterior-median small spicisetae anterior to antecostal sutura not long enough to reach over the sutura; • large median spicisetae on first abdominal segment smooth or covered with inconspicuous scales tips smooth for at least 4 times the diameter of seta; • 7th abdominal tergite with suture faint or interrupted; • antecostal suture of 8th abdominal tergite almost always absent but at least faint and interrupted; • no greyish pigmentation on sides of thoracic and other segments, not even at the base of large lateral spicisetae.

The first-instar larva is 1.6-1.8 mm long and 0.25-0.3 mm wide. Body is uniformly yellowish-white, head and hairs are reddish-brown. The mature larva is 4.5-6 mm long and 1.5 mm wide and body is reddish-brown. The larval body is covered with two kinds of hairs: those termed ‘spicisetae’ in which the shaft is covered with tiny, stiff, upwardly directed, pointed scales, and hastisetae in which the shaft is multi-segmented with spear-headed apex. Spicisetae are scattered over the dorsal surface of the head and body segments. Two groups of long spicisetae on the 9th abdominal segment form the tail. Hastisetae are found on all notal and abdominal segments, but on last three or four segments they form distinctive, paired, erect tufts (Beal, 1960, 1991; EPPO/CABI, 1997).

Quick preliminary identification of Trogoderma adults

• Median ocellus present • Antennal cavity well defined by a posterior carina and open laterally • Antennal outline smooth, antennal club at least three segmented • Body hairy, elytra usually with three transverse bands of pale (ensiform) setae (setae of dead adults often rubbed off!)

Discriminating features of Trogoderma adults

Adapted from Banks, 1994; Beal, 1954, 1960; Haines, 1991; Háva, 2004; Hinton, 1945; Kingsolver, 1991; Lawrence & Britton 1991, 1994; Lawrence et al. 1999b; Okumura and Blanc, 1955; Peacock, 1993.

• body ovate, densely setose, setae simple, usually 2-3 different type, recumbent, yellowish-white slightly flattened (ensiform) setae present; • median ocellus present; • pronotum without lateral carina; • antennal cavity of antero-ventral surface not, or slightly visible in anterior view • antennal cavity carinate posteriorly at least to half length and open laterally; • prosternum forming a "collar" anteriorly; • mesosternum deeply divided by sulcus; • posterior margin of hind coxal plate curved or sinuate, never angulate; • first segment of hind tarsus longer than second segment; • antennae short, 9-11 segmented, with a 3-8 segmented club, antennal outline usually smooth or rarely flabellate, terminal segment never disproportionately enlarged; • tarsi of all legs 5-segmented.

Identification of Trogoderma granarium Everts adults

The following short key should be used to distinguish adult T. granarium from some other Trogoderma spp. frequently occurring in stored commodities. If necessary, other species can be identified with the keys of Beal (1954, 1956), Banks (1994), Kingsolver (1991) and Mordkovich & Sokolov (1999). These keys include species occurring in stored products and therefore may be used for identification of Trogoderma adults detected in imported consignments.

Mordkovich & Sokolov (1999) mention other Trogoderma species which may be found in stored products. Among them T. longisetosum Chao et Lee has been noted as a stored product pest in China. It is very similar to T. glabrum. Some tropical Trogoderma species may be present in stored products, such as Trogoderma cavum Beal described by Beal (1982) after examination of specimens infesting stored rice in Bolivia. Delobel & Tran (1993) provide an account of tropical species.

1 Dorsal pubescence unicolorous ...... non-pest Trogoderma spp. Dorsal pubescence of whitish, ensiform setae in addition to yellowish- and reddish- brown setae...... 2

2 Elyra without well-defined pattern, unicolorous or vaguely mottled...... 3 Elytral integument with well defined lighter and darker areas...... 4

3 Integument black, rarely with vague brownish maculation, basal loop, submedian and subapical bands formed by yellowish and whitish, ensiform setae; antennae always 11 segmented, male antennal club 5-7 segmented, female 4-5 segmented; setae on 5th sternite of males uniform, recumbent...... Trogoderma glabrum (Herbst) Integument light reddish-brown, often with indistinct lighter maculation, scattered ensiform setae rarely forming 2-3 indistinct bands; antennae usually 11, rarely 9 or 10 segmented, male antennal club 4-5 segmented, female 3-4 segmented; 5th sternite of males with apical patch of dense, coarse, setae...... Trogoderma granarium Everts

4 Elytral integument with distinct light basal loop...... 5 Elytral integument with distinct bands and spots only...... 7

5 Anterior margin of eyes distinctly emarginate ...... Trogoderma inclusum LeConte Anterior margin of eyes straight or slightly sinuate ...... 6

6 Basal loop never connected to the antemedian band...... Trogoderma variabile Ballion Basal loop of elytral maculation connected to the antemedian band by a longitudinal band or bands (T. inclusum with less obvious emargination of eyes may key out here) ...... Trogoderma ornatum (Say), T. simplex Jayne, T. sternale Jayne, T. versicolor (Creutzer)

7 Elytral integument with three well-defined (basal, submedian and apical) fasciae, setae on fasciae largely white, ensiform with very little yellowish recumbent setae...... Trogoderma angustum (Solier) Elytral integument with well-defined basal band and median spot...... Trogoderma variabile Ballion reduced pattern

Notes:

Elytral fasciae usually form a more or less complete basal loop, ante-median and median bands and apical spots. Some specimens have a reduced elytral pattern where the basal loop is indicated by curved anterior band, ante-median and/or median bands by small spots, and apical spots are usually missing.

In the case of abraded or damaged specimens genital dissections should be carried out. This is recommended even on undamaged specimens. The reason is that there is a large number of undescribed Trogoderma species and by examining the genitals, the chances of misidentifications can be significantly reduced.

Matveeva (2001) provides additional discriminating features for separation of adults of Trogoderma granarium Everts from T. variabile Ballion and T. glabrum (Herbst). These species frequently found in stored products imported to Russia. These supplementary characters concern size and morphology of hide wings and can be useful for the identification of damaged specimens and also help to increase the certainity of identification based on oter features. During dissection hind wings should be removed and mounted in glycerol or Hoyer's.

Hind wings of the khapra beetle: are smaller (their mean length is 1.9 mm in T. granarium comparing with 2.5 mm in case of T. variabile and T. glabrum); are paler in colour with less visible venation (unfortunatelly this character might be useful only in case of mixed infestation); number of setae S1 on costal vein is half the number of (mean number 10) of setae on T. variabile and T. glabrum (mean 20-23); number of small setae S2 between costal vein and pterostigma less (mean 2, sometimes absent) than on the other two species in T. variabile and T. glabrum (where mean number 8).

For positive identification all (in case of damaged specimen most) of the features should be observed. Often the material that the discriminating features based on came from a limited source, therefore they are not reflecting the whole range of intraspecific variation. The features were evaluated against Trogoderma species from a restricted geographic area so they may not be proven to be useful on species from other areas. Examination of the genitalia should make identification more reliable without using additional less understood features.

Discriminating features of Trogoderma granarium Everts adults

Adults of T. granarium are oblong-oval beetles, 1.6–3.4 mm long and 0.75 –1.9 mm wide. Head deflexed, head and pronotum darker than elytra, legs and abdomen are brownish. The elytra are brown. Females are slightly larger than males and lighter in colour. To identify the adult stages of T. granarium correctly, specimens should correspond to the characters used to identify the family Dermestidae, the genus Trogoderma and the species granarium. These characters are as follows.

• elytral cuticle uncoloured, usually light brown or reddish brown, or vaguely mottled without a clearly defined pattern; • elytral setae predominantly brown; yellowish or white hairs forming no clearly defined banded pattern may also be present; these hairs are gradually rubbed off as the beetle moves around and the adult develops a shiny appearance; • antennae with 9 –11 segments; male antennal club with 4–5 segments; female antennal club with 3–4 segments; • inner eye margin straight or sinuate; • male abdominal tergum 8 more or less evenly sclerotized, with setae along its margin sometimes tending to be grouped medially; tergum 9 with proximal margin of broader section almost U-shaped; tergum 10 with many long setae; • serrate sclerites of bursa copulatrix of female small, not longer than corrugated part of spermatheca, with 10-15 teeth; • male genitalia with bridge straight, and evenly wide, broader at connections to the parameres

Detailed description of Trogoderma granarium Everts adults

Adult male Body: Length 1.4-2.3 mm, width 0,75 1,1 mm, ratio of length to width about 2.1:1. Head and pronotum dark reddish-brown; elytra reddish-brown, usually with indistinct lighter reddish- brown fasciae. Venter of thorax and abdomen reddish-brown; legs yellowish-brown. Setae: Dorsal surface with evenly distributed, coarse, semi-erect, yellowish-brown and few, scattered, dark reddish-brown setae, colour of setae follows the colour of cuticles; pronotum medially and laterally with indistinct patches of yellowish-white, ensiform setae, elytra with two or three indistinct band of yellowish-white, ensiform setae. Ventral surface with dense, simple setiferous punctures, which are denser on ventrites, setae fine, short, recumbent, yellowish-brown. Head: Punctures large, largest anteriorly, ocellate, separated by a distance of about the diameter of one to five punctures, surface between them shiny. Antennae yellowish-brown, 9,10 or 11 segmented with 4- or 5-segmented club. Eyes medially straight, or sometimes slightly sinuate. Thorax: Anterior margin of pronotum with row of yellowish-brown, coarse setae pointing to middle of anterior margin, setae on anterior half of disc pointing backward, on posterior half setae point to the scutellum. Punctures slightly larger and more dense along anterior and lateral margin, and medially, otherwise small, simple on disc and separated by about 2-4 diameters. Antennal fossa shallow loosely filled in by antenna. Posterolateral end smooth, shining, otherwise very finely and densely punctured. Prosternum densely punctured, sides of posterior process straight and gradually tapering to apex. Elytra densely punctured by setiferous punctures, punctures small, denser laterally, on disc separated by 2-4 diameters, laterally by 1-2 diameter. Hind wings with vague venation; mean number of larger setae S1 on costal vein is 10, mean number of small setae S2 between costal vein and pterostigma 2, sometimes missing Tibiae with small spines along outer edge. Proximal segment of hind tarsus abut same length as second; distal segment about twice as long as fourth segment. Abdomen: First ventrite with or without weak femoral lines. Ventrites covered by fine, yellowish-brown, recumbent setae, posterior half of penultimate ventrite with very dense, coarser, semi-erect, dark yellowish-brown setae. Genitalia: Distal end of median lobe of aedeagus shorter than apices of parameres. Parameres wide, with sparse, short setae on inner and outer margin, setae extend to half of aedeagus length. Paramere bridge is located at about third of total length from distal end, straight distally and proximally, bridge is as wide or wider than adeagus at crossing, basal process is tapered.

Adult female Body: Length 2.1 - 3.4 (2.81 mm); width 1.7 - 1.9 (1.64 mm); ratio of length to width about 1.6:1. Antennal club 3-4 segmented, number of segments sometimes less than 11. Posterior half of penultimate ventrite without a dense fringe of semi-erect, yellowish-brown, coarse setae. Genitalia: Bursa copulatrix with two small, dentate sclerites, length of sclerites equal to or shorter than the length of the corrugated part of spermatheca.

References

Banks, H.J. (1994): Illustrated identification keys for Trogoderma granarium, T. glabrum, T. inclusum and T. variabile (Coleoptera: Dermestidae) and other Trogoderma associated with stored products, CSIRO Aust. Div. Entomol. Tech. Pap. No.32, 1-66 pp. Barak, A.V. (1989): Development of new trap to detect and monitor khapra beetle (Coleoptera: Dermestidae), Journal of Economic Entomology, 82, 1470-1477 pp. Barak, A.V. (1995): Chapter 25: Identification of common dermestids, [In:] Krischik, V., Cuperus, G. and Galliart, D. (eds.). Stored Product Management Oklahoma State University, Cooperative Extension Service Circular No. E-912 (revised), 187-196 pp. Barak, A.V., Burkholder, W. E. and Faustini, D. L. (1990): Factors affecting the design of traps for stored-products insects, Journal of the Kansas Entomological Society, 63 (4) 466-485 pp. Bealj R. S., Jr. (1954): Biology and taxonomy of nearctic species of Trogoderma. University of California Publications in Entomology, 10 (2) 35-102 pp. Bealj R. S., Jr. (1956): Synopsis of the economic species of Trogoderma occurring in the United States with description of new species (Coleoptera: Dermestidae), Annals of the Entomological Society of America, 49 559-566 pp. Bealj R. S., Jr. (1960): Descriptions, biology and notes on the identification of some Trogoderma larvae (Coleoptera, Dermestidae), Technical Bulletin, United States Department of Agriculture, No. 1226, 26 pp. Bealj R. S., Jr. (1982): A new stored product species of Trogoderma (Coleoptera: Dermestidae) from Bolivia, The Coleopterists Bulletin, 36(2) 211-215 pp. Bealj R. S., Jr. (1991): Dermestidae (Bostrychoidea) (including Thorictidae, Thylodriidae). [in:] Stehr FW (ed.) Immature Insects, Michigan State University, 434-439 pp. Bealj R. S., Jr. (2005): Status of Trogoderma yunnaeunsis Zhang and Liu, Description of its larva, and distinction from other Eastern Palearctic Trogoderma (Dermestidae), Coleopterists Bulletin 59(2): 157-165 pp. Berg, G. H. (1999): Trogoderma granarium Everts [in:] Hojas de Datos sobre Plagas y Enfermedades de Productos Almacenados de Importancia Cuarentenaria y/o Económica para los Países Miembros del OIRSA. OIRSA. El Salvador. 6. 120 - 145 pp. Booth, R. G., Cox, M. L. and Madge, R. B. (1990): IIE guides to insects of importance to man. 3. Coleoptera, CAB International, Wallingford, UK, 1-384 pp CABI Crop Protection Compendium 2005 ed. CD ROM Delobel, A. and Tran, M. (1993): Les coléopterés des denrées alimentaires entroposeés dans les régions chaudes. Faune tropicale XXXII, ORSTOM, Editions Paris, FR. EPPO/CABI (1997): Trogoderma granarium. In: Quarantine pests for Europe, 2nd edition. (Ed. by Smith, I.M., McNamara, D.G., Scott, P.R, & Holderness, M.) CAB International, Wallingford, UK. Green, M. (1979): The identification of Trogoderma variable Ballion, T. inclusum and T. granarium Everts (Coleoptera, Dermestidae), using characters provided by their genitalia, Entomologist’s Gazette, 30 199-204 pp. Haines, C. P. (1991): Insects and arachnids of tropical stored products: their biology and identification (a training manual), Natural Resources Institute, Chatham Maritime, UK, 1-246 pp. Háva, J. (2003) World Catalogue of the Dermestidae (Coleoptera). Studie a zprávy Okresního muzea Praha-východ, Supplementum 1 1-196 pp. Háva, J. (2004): World Keys to the Genera and Subgenera of Dermestidae (Coleoptera) with Descriptions, Nomenclature and Distributional Records. Acta Musei Nationalis Pragae, Series B, Natural History 60 (3-4) 149-164 pp. Hinton, H.E. (1945): A monograph of the beetles associated with stored products, vol. 1. British Museum (Natural History), London, UK. Kingsolver, J. M. (1991): Dermestid beetles (Dermestidae, Coleoptera) [in:] Gorham JR (ed.) Insect and Mite Pests in Food. An Illustrated Key, USDA ARS and USDHHS, PHS, Agriculture Handbook No. 655, Washington, DC, Vol. 1, 115-136. Kingsolver J.M. 2002. Dermestidae. pp. 228-232. In Arnett, R.H. Jr., Thomas, M.C. Skelley, P.E. & Frank, J. H. eds. American Beetles. Vol. 2. CRC Press: 861 pp Lawrence, J.F. (1991): Order Coleoptera. pp. 144-658 in Stehr, F.W. (ed.) Immature Insects. Dubuque, Iowa : Kendall/Hunt Vol. 2 xvi 975 pp. Lawrence, J. F. and E. B. Britton (1991): Coleoptera (beetles). In C.S.I.R.O. (ed.), Insects of Australia. 2nd Edition. Volume 2, 543-683. Carlton: Melbourn University Press. Lawrence, J.F. & Britton, E.B. (1994): Australian Beetles. Melbourne : Melbourne University Press x 192 pp. Lawrence, J. F., A. M. Hastings, M. J. Dallwitz, T. A. Paine and E. J. Zurcher (1999a): "Beetle Larvae of the World: Descriptions, Illustrations, and Information Retreival for Families and Subfamilies." CD-ROM, Version 1.1 for MS-Windows. Melbourne: CSIRO Publishing. Lawrence, J. F., A. M. Hastings, M. J. Dallwitz, T. A. Paine and E. J. Zurcher (1999b): "Beetles of the World: A Key and Information System for Families and Subfamilies." CD-ROM, Version 1.0 for MS-Windows. Melbourne: CSIRO Publishing. Lindgren, D.L., Vincent, L.E. and Krohne, H.E. (1955): The Khapra beetle, Trogoderma granarium Everts, Hilgardia, 24 (1) 1-36 pp. Matveeva, V. I. (2001): [Identification of the Khapra beetle], Zashchita i Karantin Rastenii, 4, 31 (in Russian). Mitsui, E. (1967): [On the identification of the Khapra beetle]. Rep. Japan Food. Res. Inst. Tokyo, 22, 8-13 (in Japanese). Mordkovich, Ya. B. and Sokolov, E. A. (1999): [Keys for identification of quarantine and other harmful pests of the raw material, stored products and sowing material], Kolos, Moskva (RU) (in Russian). Mordkovich, Ya. B. and Sokolov, E. A. (2000): [Detection of khapra beetle in warehouses], Zashchita i Karantin Rastenii. 2000, 12, 26-27 (in Russian). Mroczkowski, M. (1968): Distribution of the Dermestidae (Coleoptera) of the world with a catalogue of all known species, Annales Zoologici, 26(3) 1-191 pp.. Okumura, G. T. and Blanc, F. L. (1955): Key to species of Trogoderma and to related genera of Dermestidae commonly encountered in stored grain in California., In: California Legislature Joint Interim Committee on Agricultural and Livestock Problems, Special Report on the Khapra Beetle, Trogoderma granarium, Sacramento, California, 87-89 pp. Pasek, J. E. (1998): Khapra Beetle (Trogoderma granarium Everts): Pest-Initiated Pest risk Assessment, USDA 1-46 pp. Peacock ER (1993) Adults and larvae of hide, larder and carpet beetles and their relatives (Coleoptera: Dermestidae) and of derontid beetles (Coleoptera: Derontidae). Handbooks for the identification of British Insects No 5. Natural History Museum, London (GB) 1-144 pp. Rees, B. E. (1943): Classification of the Dermestidae (Larder, Hide, and Carpet Betles) based on larval characters, with a key to the North American Genera, USDA Misc. Publ. No.511, 18 pp. Saplina, G. S. (1984): [Inspection of storage premises using traps]. Zashchita Rastenii, 9, 38 (in Russian). Strong, R. G. and Okumura, G. T. (1966): Trogoderma species found in California, distribution, relative abundance and food habits, The Bulletin, Department of Agriculture, State of California, 55 1 23-30 pp. Varshalovich, A. A. (1963): [Khapra beetle – a serious stored products pests], Selskhoizdat, Moskva (RU), 52 pp. (in Russian).