Infestation of Sphingidae (Lepidoptera) by otopheidomenid mites in intertropical continental zones and observation of a case of heavy infestation by Prasadiseius kayosiekeri (Acari: Otopheidomenidae)

Examination of 761 Sphingidae in museum collections revealed infestation of 24 moths by 4 species of the ectoparasitic otopheidomenid mites (Acari: Otopheidomenidae). The infestation rate was 3.2 %. The mites were: Prasadiseius cocytes, P. donahuei, P. kayosiekeri and P. pholusis. Among the sphingid moths, Pachylioides resumens, collected in Guatemala, unusually, was found to be heavily infested with P. kayosiekeri. It had eggs, larvae, nymphs and the adults, many present on the prothorax.

Pachylioides resumens (Walker, 1856) collected in Guatemala was found to be very heavily infested with P. kayosiekeri (Prasad, 1970b). This mite was originally described from moths Enyo lugubris (Linnaeus) collected in Honduras, Pachylia darceta (Druce, 1881) in Peru and Pachylioides resumens (Walker, 1856) in Panama Canal Zone infesting different areas of the moths but not the prothorax.
Unusually, numerous mites were present on the prothorax. The details of the morphology of this mite studied using scanning electron micrographs were published by Prasad and Walker (2011). Here, the results of examination of 761 moths and the case of the heavy infestation are presented.

MATERIALS AND METHODS
The adult sphingid moths collected from many countries and preserved in A. J. Cook Arthropod Research Collection, Department of Entomology, Michigan State University, East Lansing, Michigan, USA, were examined during February 27 -March 27, 2009 and in the Chicago Natural History Museum, Chicago, USA, on September 11, 2009 by the author. In the former place, the moths were examined using a Wild M5-89111 binocular microscope under 48-96x magnification using the light provided with the microscope. The heavily infested moth was borrowed, photographed using a Kodak High Definition Easy Share Z812IS photo camera, a video was made using Sony Hi8 Handycam camera and placed online (www.indirapublishinghouse.com). The mites in the later place were examined under a binocular microscope without noting details of the microscope. All mites were removed from the moths. These were mounted in Hoyer's medium on glass slides (3 x 1" = 75 mm × 25 mm) under a 18 mm zero thickness round cover slip after clearing of mites in lactophenol for 5 -10 minutes for positive species identification. They were dried for 5-7 days on a slide warming hot plate between 45 -47 o C and edges of the coverslip sealed with Glyptal. The mites were finally identified by the author under Accu-Scope 3000 phase contrast microscope (New York, USA). Photographs of eggs and chelicerae  were taken under 100 to 400 magnification using the mounted MicrometricsTM camera and saved in Photoshop CS2. Photographs of the moths with the mites (Figs. 1 -3, 7 -10) were taken under a Bausch and Lomb stereo binocular microscope having total magnification of 25x using a High Definition Kodak EasyShare Z812 IS camera and placing directly on the eye-piece lens. As these photographs were enlarged or cropped further, no correct magnification are given on the figures. All mites collected from the sphingid moths were given collection numbers from VP09-01 to VP09-26 (VP09-13 and VP09-25 had no otopheidomenid mites).
The world data on previous collections of 8 Prasadiseius species known from the sphingid moths are given in Table 1 to compare with the data given in the results that are based on the present work. The voucher specimens of mites are deposited in the collection of MSU and US National Museum, Washington, DC, USA, now housed at USDA, Beltsville, MD, USA. The terms given in the collection data as MSU refers to the Michigan State University and CNHM refers to the Chicago Natural History Museum.

RESULTS
Collection data -The details of all collections are given below and summarized in Tables 2-4. Unless mentioned, all mites were found on dorsum of the moth host. As there is no true tympanum in the sphingid moths as noctuid moths have, these are written as left or right "tympanic area" where mites were found and indicated so in the results and the discussions. All mites were attached with the hairs or found among the hairs [F = Female, M = Male, E = Egg, L = Larva, PN = Protonymph, DN = Deutonymph].
(16) VP09-17 [P. cocytes (Prasad, 1970a)]: Ecuador, Napo -Misahualli, on bank of Napo River, elevation 350 m, low land, moth host: Manduca rustica (Fabricius), coll. M. Churchill, 20 October 2001, over 1000 mites, most heavily infested moth ever seen (Prasad, 2011c) and VIDEO prepared (www.indirapublishinghouse.com), many eggs and other stages on all over body (including many on ventral and lateral side of body) including wings but most on thorax to third abdominal segments, some mites in pair or together and on proximal half of wings, mites collected on 27 March 2009, 17F, 3M, 2L, 7PN, 7DN (= 36 mites), 22 eggs, many mites saved in 70 % ETOH, some mites used for SEM study (T), from MSU collection [Prasad, 2011b, c]. Infestation rate - Table 2 indicates details of 24  infested sphingid moths while Tables 3 and 4 indicate details of 737 uninfested moths. Thus, out of 761 moths examined, an infestation rate of 3.2 % in the museum preserved moths was present. This pooled data result indicates that a researcher has to examine at least 100 moths in order to find 3 infested moths. Table 2, only 15 species of sphingid moths (2 known from genus only) were infested.

Infested sphingid moths -As indicated in
Otopheidomenid mites infesting the sphingid moths -Tables 2 shows only 4 species of otopheidomenid mites infesting 24 moths and the new records marked with **.
Geographical distribution of the moths and the mites -All 4 species of the mites mentioned in Table 2 were found infesting moths in different countries of North, Central and South Americas as follow (new records marked with **): Brazil (P. cocytes and P. kayosiekeri), Ecuador (P. cocytes and P. pholusis), Guatemala (P. cocytes, P. donahuei, P. kayosiekeri and P. pholusis), Mexico (P. donahuei) and Peru (P. cocytes). Thus, 3 species of Prasadiseius were present in Guatemala when only one each was found in Mexico and Peru. All of these moths were inhabitants of Neotropical ecozone. Mostly, they were present in South America (Brazil, Ecuador, Peru) but some were present in Central America (Guatemala) while one was present in North America (Mexico). None of the moths from the United States were found to be infested with any of the Prasadiseius species. Of all these, Guatemala and Mexico included new country records for P. donahuei, Brazil for P. kayosiekeri and Ecuador and Guatemala for P. pholusis.
Tympanic areas of the moth host and the mites -Moths of the family Noctuidae have one true tympanum located posteromedial to the hind wing base on lateral side of the metathorax where some otopheidomenid mites have been reported feeding (Treat, 1955(Treat, , 1975. There is no such true tympanum in the moths of the family Sphingidae. However, in that area of the metathorax and wing base, there are several sclerites that support the wings and the thorax (Fig. 3) where otopheidomenid mites are present. This tympanal region has been redefined giving a broad definition that includes the tympanal organ proper as well as functionally related structural modifications of the metathorax and the first abdominal segment (Speidel et al., 1996). Many otopheidomenid mites parasitizing the sphingid moths are found in this area (Figs. 1, 2).
Metathoraxic-first abdominal cavity of the moth host and the mites -In addition to the metathoraxic-wing base-tympanic areas, in Sphingidae, the posterior part of the metathorax that attaches with the first abdominal segment, develops a cavity or space. It is concave posteriorly allowing attachment of the anteriorly rounded first abdominal segment and its free movement from the metathorax. Sometimes, the mites could be seen in this space and among the hairs on top of it. This concavity, depression or space may represent "acarinarium" where these mites harbor, feed and multiply.
What do these mites feed on -Each chelicera in the otopheidomenid mite parasitizing the sphingid moths has a well developed movable digit with several recurved denticles (Fig. 4). These mites have never been observed feeding on live sphingid moths. In fact, these mites were seen alive on live sphingid moths for the first time ever only during an expedition to Peru Amazon Forest in 2010 by this author and the expedition team members but the feeding could not be observed. The closely related Treatiinae otopheidomenid mite having similar movable digit, Hemipteroseius indicus (Krantz and Khot, 1962), an ectoparasite of red cotton bug, Dysdercus cingulatus (Fabricius), has been reported as scraping feeder (Costa, 1968). It is believed by this author that the otopheidomenid mites parasitizing the sphingid moths probably are scraper feeders also. Do these mites feed on some secretion or moth hairs also? This is not known and needs to be investigated.
Unidentified eggs among the mites -The eggs of the Prasadiseius species are typically whitish and oval (Fig. 5). The skin surface of these eggs is glistening without any sculpturing pattern. But, among the eggs of these mites, several eggs were found that had narrow ends having very typical skin sculpturing patterns (Fig. 6). These were identified as eggs of Psocoptera by Mr. Gary Parsons, Collection Manager, A. J. Cook Arthropod Research Collection, Department of Entomology, Michigan State University, East Lansing, Michigan, and considered laid after moths were dead, dried and stored in the collection.

Unusual heavy infestation of Prasadiseius kayosiekeri on Pachylioides resumens in
Guatemala -In the present study, a sphingid moth, Pachylioides resumens Walker (VP09-23), collected in Guatemala was found infested with over 300 mites that were present mostly on dorsal side of the body. Many of these were present on metathorax-first abdominal segment attached with the hairs. However, unusually, many mites were present on prothorax (Figs. 7,8). In addition some mites were present on head, left antenna, around the left eye and one on the proboscis. This kind of presence of so many mites on dorsum of the prothorax had never been seen before. As stated before, some of these mites were also found on dorsal proximal half of the forewing and the hindwing (Figs. 9, 10).

DISCUSSIONS
Infestation rate of the sphingid moths by the mites -The present study indicated the rate of infestation in 761 sphingid moths preserved in the museum to be about 3 %. The analysis of all 761 sphingid moths in the present study (Table 4) indicated the following: (1) All 24 infested sphingid moths came from Neotropical ecozone. (2) Remain-          15 1965, 1985, 1998Manduca florestan Stoll 5 1985, 1986 Manduca muscosa (R. and J.) Hyles lineata (F.) * 15 1965, 1972, 1985, 1998 Manduca sexta (L.) 1 1984  Prasad (1976) reported that the otopheidomenid mites were mostly distributed on the sphingid moths in various countries located in between the latitude of 30 o North and 30 o South of the equator. Out of 151 genera and 623 species and subspecies of sphingid moths studied, 30 genera and 47 species and subspecies were found infested with the otopheidomenid mites. Based on past and present study, it is evident that the genus Prasadiseius has primarily Tropical-Neotropical distribution.

Geographical distribution of the mites -
Sex ratio of mites -154 females, 62 males, 69 larvae, 51 protonymphs and 28 deutonymphs on all 24 infested sphingid moths in the present study (Note -Mites saved in ETOH were not included and only the mites randomly mounted on the slides were included in the above count). This brings the sex ratio at 2.5:1.0 or about 3 females to 1 male. This had not been reported before.
Any sex preference of the sphingid moths by the mites -This study on museum preserved or live sphingid moths has never been conducted and needs to be done in future to see if mites prefer the female or the male moth to infest. When and where are the moths infested by the mites -This is not known if an infested female or an infested male transmits the mites to uninfested moth during feeding on flower nectar, resting on the tree trunk or the vegetation, or during flight. Future studies are essential to resolve some of these and many other questions in the native areas in the peak abundance season of the moths.
Is there any re-infestation or multiple infestation of the infested moths present -In almost last over 40 years of studying these mites, this author has never found infestation of a single moth by 2 species of Prasadiseius. So far, only 3 sphingid moths have been found, each infested with several hundred mites, that indicated infestation of moth by a single species only.
Presence of mites on the host -Otopheidomenid mites infesting the sphingid moths are known to be present dorsally among the hairs mostly in the tympanic-metathoracic-first abdominal region of the body. It has been believed by this author that the adult mites invade the new adult sphingid hosts either during feeding on nectar of the flowers, mating or congregation on the tree trunk and climb on the proboscis and head. Then move to the thorax in the tympanic-metathoracicfirst abdominal area where they reside, feed and reproduce. So, the question has been as to what they feed on and as to why they congregate mostly in these areas. Treat (1975) had believed that some food source was present in the tympanic area where Otopheidomenis zalelestes Treat, 1955, was feeding, residing and reproducing on the noctuid moths of the genus Zale. This author had also believed before that probably some secretion was present on the sphingid moths where these mites were feeding. But, now, here, he believes that these areas possibly present different kinds of specialized cavities, spaces or acarinaria allowing mites to reside and possibly feed either by scraping on the host or by feeding on the moth hairs but not on any secretion of the moth. These mites have only the movable cheliceral digit with denticles and possibly serve the purpose of feeding as a scraper on the host tissue. Similar kind of movable cheliceral digit (and reduced fixed digit) is known in Hemipteroseius indicus (Krantz and Khot, 1962) which has been considered a scraper feeder (Costa, 1968).  had studied the biology of this mite in India, had not observed the feeding behavior, but had noted the presence of these mites in different grooves on the dorsum under the wings of the bug. Thus, pres-ence of acarinaria and scrape feeding on the host tissue and or feeding on the moth hairs on the sphingid moths are new considerations. The later could be proven either by studying the feeding habits of live mites on live sphingid hosts or by studying the feces of these mites and noticing if some undigested moth hairs are present. Thus, future studies are necessary to confirm these points of views.
Why P. kayosiekeri on Pachylioides resumens in Guatemala on the head and prothorax -In most cases studied by this author over last 40 years, less than 50 mites were found parasitizing a single noctuid or sphingid moth. These mites included all motile stages. Other than these, eggs were also found among these mites attached to the hairs. As stated before, these mites were present mostly in the rectangular tympanic-metathoracic-first abdominal region of the body (Fig. 1). But, in the present study, Prasadiseius kayosiekeri infesting the sphingid moth, Pachylioides resumens,was present on dorsum of prothorax in large number (Figs. 7,8) along with a few on the proboscis, antenna and the head. This was very unusual. It is believed that, possibly, the moth had been infested on the day it was caught and the mites were still moving upward from proboscis to the head and to the common areas of congregation in the tympanic-metathoracic-first abdominal region when they died. Could these mites represent a migratory phase, cannot be said with certainty as not too many such cases have been studied. This is only the first case of this kind known having so many mites on the prothorax. Prasad (2011b) described an unusual case of very heavy infestation of P. cocytes on a sphingid moth, Manduca rustica (Fab.), collected in Ecuador (VP09-17) but in this case over 1000 mites were present on all over the body (mostly on dorsal side, some on the ventral side as well).
Are these mites present on pupae or larvae of the sphingid moths -Several pupae and skin mounts of the larvae of the sphingid moths preserved in the museum collection were examined in the present study but no otopheidomenid mites were found. Thus, where do these mites live, if they hibernate during winter and in what stage, how do they infest the new hosts, and if they infest the adults during emergence from pupae or in the adult stage only -need to be studies in future to answer these questions.