First observation of Afromorgus chinensis (boheman, 1858) (Coleoptera: Trogidae) on a rabbit (Oryctolagus cuniculus L., 1758) carcass and its implications in forensic entomology.

Beetles (Coleoptera) are known to constitute forensic evidence in medico-legal investigations as their presence can be used to date human remains in almost all decomposition stages. Many forensic studies focus on the successional colonization pattern of flies (Diptera); however, beetles have not so far been studied extensively for this aspect. A beetle of the genus Afromorgus Scholtz, 1986, A. chinensis (Boheman, 1858) (Scarabaeoidea: Trogidae), was found beneath a late decaying rabbit carcass at Paya Indah Wetland, Dengkil, Malaysia, for the first time. Both genus and species are already known to occur in Malaysia from literature.

A new host record of Haemaphysalis semermis (Acari: Ixodidae) from the domestic cat (Felis catus) (Carnivora: Felidae) in Orang Asli village, Pahang National Park Malaysia.

We report the presence of a male Haemaphysalis semermis collected from the domestic cat, Felis catus in an aboriginal village located in Pahang, Malaysia. This paper constitutes a new host record of this tick species, and also the first documentation of the infestation of companion animals other than domestic dogs (Canis lupus) by H. semermis in Malaysia. Additionally, we have included an updated host index of the tick species in Southeast Asia.

Two new records of chewing lice (Phthiraptera: Amblycera) from the Oriental honey buzzard [Pernis ptilorhynchus (Temminck, 1821)] and house crow (Corvus splendens Viellot, 1817) in Malaysia.

We report two new records of chewing lice from avian pets in Peninsular Malaysia: Colpocephalum apivorus Tendeiro, 1958 from an Oriental honey buzzard (Pernis ptilorhynchus (Temminck, 1821)), and Myrsidea splendenticola Klockenhoff, 1973 from an albino house crow (Corvus splendens Vieillot, 1817). The scarcity of louse records from avian pets and wild birds, and the lack of louse research in Malaysia are discussed.

The first record of parasitic mite, Leptus sp. (Acari: Erythraeidae) associated with a necrophagous fly, Chrysomya villeneuvi Patton, 1922 (Diptera: Calliphoridae).

This is the first documentation of parasitic mite, Leptus sp., found on a necrophagous blowfly, Chrysomya villeneuvi collected from a decomposing wild boar carcass placed in Taman Negara (National Park), Kuala Keniam, Pahang, Malaysia. Blowflies around the carcass were captured using an insect net before being examined under a stereomicroscope. Upon microscopic observation, we found a mite attached on the scutellum of C. villeneuvi adult. The mite was carefully removed and preserved in 70% ethanol subsequently. Then, the mite was cleared in lactophenol before being mounted in Hoyer's medium. The morphological identification of the mite was conducted and Leptus sp. was identified. The species belongs to the phalangii species group and the aldonae species subgroup. This study highlights the new association of Leptus sp. and C. villeneuvi for the first time.

Rhipicephalus annulatus, R. australis or R. microplus? Discordance between morphological and genetic data among three cattle tick species.

The taxonomy of ticks of the subgenus Boophilus has been extensively debated and is often complicated by the high intraspecific variation of morphological features between species. Notably, the cattle tick Rhipicephalus microplus is a species complex consisting of Rhipicephalus annulatus, Rhipicephalus australis and the three mitochondrial clades (A-C) of R. microplus. To gain insight into the taxonomic status of this species complex, we performed morphological and molecular analyses on these cattle ticks across four states in peninsular Malaysia. We morphologically identified 60 males and 104 females of R. microplus, 298 males and 374 females of R. australis, and one R. annulatus male in our field collection, of which the latter two species have never been recorded in Malaysia. However, all three morphologically identified species were molecularly assigned as R. microplus clade A based on the barcoding cytochrome oxidase subunit I (COI) analysis. The discrepancy between morphological and genetic data highlights an urgent need for further exploration and in-depth research into the taxonomic status of these sympatric tick species.

Acari community in association with delayed pig carrion decomposition.

Acari community structure and function associated with delayed pig carrion decomposition has not been examined. In this study, 18 swine carcasses were studied in central Texas, USA, during two consecutive summers (2013 and 2014). Samples of ca. 400 g soil were collected from beneath, aside, and 5 m away from each pig carcass over 180 days. Mites from soil samples were extracted using Berlese funnels and identified to order and family levels and classified according to ecological function. In total 1565 and 1740 mites were identified from the 2013 and 2014 soil samples, respectively. Significant differences were determined for mite community structure at order and family levels temporally on carrion (e.g., day 0 × day 14) regardless of treatments and between soil regions where mites were collected (e.g., soil beneath vs. soil 5 m away from carrion). However, no significant differences were found in mite community structure at the order level between pig carrion with and without delayed Diptera colonization (i.e., treatments). Analysis at the family level determined a significant difference across treatments for both summers. Ecological function of mites did not change significantly following the delayed decomposition of pig carcasses. However, detritivores and fungivores were significant indicator groups during the pig carrion decomposition process. Furthermore, 13 phoretic mite species associated with eight forensically important beetle species were documented. Data from this study indicated that the rate of nutrient flow into the soil impacted associated arthropod communities; however, detecting such shifts depends on the taxonomic resolution being applied.

Juvenile hormone: Production, regulation, current application in vector control and its future applications.

Juvenile hormone is an exclusive hormone found in insects which involves regulating various insect physiology. A total of eight juvenile hormones have been identified in insects which include JH 0, JH I, JH II, JH III, 4-methyl JH I (Iso- JH 0), JHB III, JHSB III, and MF. Corpora allata are the glands responsible for the production and synthesis of these hormones. They are involved in moulting, reproduction, polyethism, and behavioural regulations in different orders of insects. Factors such as diet temperatures, photoperiods, and plant compounds affect the biosynthesis and regulation of juvenile hormones. Juvenile hormones analogue is usually used to disrupt normal regulation of JH and this analogue is categorized as insect-growth regulators (IGRs) and is widely used in pest control as an alternative to chemical insecticides. Other applications of biosynthesis activities of this hormone have not been explored in the area of JHs. In this review, current applications of JHs with an addition of their future application will be discussed.

Soft ticks (Acari: Argasidae) in the island nations of Southeast Asia: A review on their distribution, associated hosts and potential pathogens.

Soft ticks (Acari: Argasidae) are the second major family of the blood feeding metastriates and vectors of a number of viral and bacterial pathogens for both humans and animals. Despite the growing effort on tick surveillance and studies worldwide, there is still limited information on the soft tick distribution in the island nations of Southeast Asia, especially species that are medically and veterinarily important. With the aim to provide an overview of the current status of knowledge on soft tick distribution in the island nations of Southeast Asia (Malaysia, Singapore, Brunei, Indonesia, the Philippines and Timor-Leste), this article reviews the species of soft ticks (Acari: Argasidae) and their associated hosts and pathogens, with the addition of a pictorial summary and list of tick species discovered in this region. The most prevalent soft tick genus is Carios, and the host species most associated with findings of soft ticks in this region are bats, particularly of the Pteropodidae and Vespertilionidae families. Furthermore, the only known pathogen originating from soft ticks in the island nations of Southeast Asia was the Keterah virus, which was isolated from Argas pusillus tick in Malaysia.

Review of Mites Found on Various Animal Hosts and at Different Localities in Malaysia.

Mite biodiversity and distribution in Malaysia is currently understudied. Most previous works on Malaysian Acari have focused on pest organisms of medical, veterinary, and agricultural concern, with a few recent studies centered on mites in forensic contexts. Previous literatures have targeted collection sites in forest reserves and/or mountains in either Peninsular or Malaysian Borneo, though the state of Sarawak had the least publications related to mite species descriptions despite having the highest number of nature parks of any state in the country. Most publications focused on the three states Selangor, Pahang and Sabah. Most of the mite species reported were from mammals (66.3%), with fewer species from birds (21.7%), arthropods (11.2%), and reptiles (0.8%). We believe that further work on the systematic documentation of mite species throughout Malaysia is necessary as it could generate useful tools, such as the use of mites as biogeographical markers or as forensic indicators. Therefore, this review catalogs mite species that have been documented in or on animal hosts in Malaysia and serves as a foundation for future work.

Preliminary observation on the lepidopteran colonization on rat and rabbit carcasses in Malaysia.

The immature stages of necrophagous insects such as Diptera and Coleoptera play a vital ecological role in carrion decomposition. These invertebrates reduce the necromass significantly through consumption and recycle nutrients into organic forms which are readily being used by autotrophs or served as an abiotic storage in the soil ecosystem. Fly and beetle larvae are frequently encountered decomposers on ephemeral resource patches; however, lepidopterans associated with carrion decomposition is seldom reported. Here, we report colonization of Monopis sp. (Tineidae) and an unknown species of Psychidae on a rat carcass, and a Lithosiini caterpillar (Arctiidae) on a rabbit carcass in Peninsular Malaysia for the first time. The feeding behaviour and their potential forensic implications are discussed.

Detection of human Sarcocystosis using dried blood on filter papers: An Immunofluorescent Antibody Test.

Sarcocystosis, a parasitic infection caused by a protozoa belonging to the genus Sarcocystis, is found worldwide in both and animals. Sarcocystis spp., require two animal hosts to complete their life cycle. The infection has gathered more global attention after recent outbreaks, especially amongst wester travellers to Malaysia. Other than sporadic cases and the current outbreaks, little information is available regarding human Sarcocystis infection in Malaysia. The present study aims to determine the prevalence of sarcocystosis among humans using an immunofluorescent antibody (IFA) test applied to dried blood on filter papers. A total of 200 blood samples were collected on filter papers from autopsy cases at two Malaysian hospitals: Sungai Buloh Hospital (peninsular Malaysia) and Queen Elizabeth Hospital (Malaysian Borneo). Antigens were prepared from bradyzoites harvested from positive goats' muscle samples. Of the 200 samples, 32 (16%) had Sarcocystis antibodies that showed positive fluorescence reactions on filter papers. There was no significant difference (t-test, p value > 0.05) in prevalence rates between samples collected from autopsies at peninsular Malaysia and Malaysian Borneo. The results demonstrated that the filter paper technique can be used as one of the alternative serological tests in the diagnostic of human sarcocystosis.

First report of Histiostoma sp. (Astigmata: Histiostomatidae) phoretic on a forensically important blowfly, Chrysomya villeneuvi (Diptera: Calliphoridae).

This is the first record of phoretic histiostomatid mites found on a forensically important blow fly species, Chrysomya villeneuvi (Diptera: Calliphoridae), collected from decomposing rabbit carcasses placed in Bukit Lagong Forest Reserve, Sungai Buloh and MARDI Cameron Highlands, Malaysia. The blow flies frequenting around the carcasses were first captured using an insect net. After pinning, they were examined under a stereomicroscope and mites phoretic on their body were carefully removed and preserved in 70% ethanol. Mites were cleared in lactic acid before mounting on slides using Hoyer's medium and identified under a compound microscope. The flies and their mites were identified as C. villeneuvi and deutonymphs of Histiostoma spp. (Astigmata: Histiostomatidae), respectively. This insectmite association may be useful to provide insights regarding the minimum post-mortem interval and the location of death in forensic entomological investigations.

Age estimation of forensically important blowfly, Chrysomya megacephala (Diptera: Calliphoridae) pupae using micro-computed tomography imaging.

Accurate estimation of the minimum post-mortem interval (minPMI) is important in the investigation of forensic cases. Various thanatological methods are being used to estimate this interval. However, entomology approach is the most reliable method for this minPMI estimation especially when death has occurred over 72 hours and involved insects or other arthropods evidence at the death scene. The current methods of age estimation are daunting and destructive especially when dealing with pupal stage. The aims of this study were to characterize the morphological changes during intra-puparial period of Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and their relation with minPMI estimation by using a high resolution micro-Computed Tomography (micro-CT). Gravid C. megacephala were collected from a rural area in Sungai Buloh, Selangor and cultured in the laboratory at 23.83±0.25°C with light: dark hour of 12:12 to initiate oviposition. The resulting larvae were reared until pupal stage. A pupa was collected at first (24 hours), second (48 hours), third (72 hours), and fourth quarter (96 hours) of the intra-puparial period. The pupal samples were placed directly into 70% ethanol for preservation. Micro-CT scanning was employed to acquire microstructural information following pupal sample staining for contrast enhancement. Eight age-informative internal morphological landmarks were mapped from the micro-CT scanning. The present study enhanced the potential value of micro-CT for the estimation of minPMI based on the internal morphological changes of C. megacephala pupae. This novel method is a promising tool for improving medico-legal investigations in forensic entomology.

Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia.

One thousand and forty-five tissue samples of skeletal muscles, tongue, heart, diaphragm and esophagus were collected from 209 animals (43 sheep, 89 goats and 77 cattle) from an abattoir in Selangor between February and October, 2013. Each sample was divided into three pieces with each piece measuring 2-3 mm3. Each piece was then squeezed between two glass slides and examined microscopically at x 10 magnification for the presence of sarcocystosis. Three positive samples from each animal species were then fixed in 10% formalin for histological processing. Seven positive samples collected from each animal species were preserved at -80°C or 90% ethanol for gene expression studies. Microsarcocysts were detected in 114 (54.5%) animals by light microscopy (LM). The infection rates in sheep, goat and cattle were 86, 61.8 and 28.6% respectively. The highest rate of infection was in the skeletal muscles of sheep (64.9%) and goats (63.6%) and in the heart of cattle (63.6%). The cysts were spindle to oval in shape and two stages were recognized, the peripheral metrocytes and centrally located banana-shaped bradyzoites. 18S rRNA gene expression studies confirmed the isolates from the sheep as S. ovicanis, goats as S. capracanis and cattle as S. bovicanis. This, to the best of our knowledge, is the first molecular identification of an isolate of S. ovicanis and S. capracanis in Malaysia. Further studies with electron microscopy (EM) are required in the future to compare the features of different types of Sarcocysts spp.

Morphological descriptions on the larvae of Hypopygiopsis fumipennis (Walker, 1856) (Diptera: Calliphoridae).

The study on biodiversity of forensically important Diptera in the tropical rain forest in Malaysia is scarce. Thus, a preliminary survey was conducted at a jungle fringe near Kampung Bahagia Bukit Lagong, Sungai Buloh, Selangor. A rat carcass was offered to attract carrion flies and we collected an adult female calliphorid, Hypopygiopsis fumipennis (Walker, 1856) during the fresh stage of carcass decomposition. The female fly was allowed to oviposit on chicken liver in a container and the resulting larvae were reared to the adult stage. Along the developmental process, several individuals from each instar were collected and preserved in 70% ethanol and then processed on the slides. We recorded the duration of development for each instar and described its larval features for the first time. The third instar larvae of H. fumipennis showed accessory oral sclerite present, anterior spiracle with 13-15 papillae, intersegmental spines mostly unicuspid with pointed end, and posterior spiracles heavily sclerotized with inter-slit projections. Some larval differences between H. fumipennis and Hypopygiopsis violacea were noted.

A preliminary report on ants (Hymenoptera: Formicidae) recovered from forensic entomological studies conducted in different ecological habitats in Malaysia.

This study reported the ant species that were recovered from monkey carcasses in three different ecological habitats in Malaysia. The study was conducted from 9 May - 10 October 2007, 6 May - 6 August 2008 and 26 May - 14 July 2009 in forested area (Gombak, Selangor), coastal area (Tanjong Sepat, Selangor) and highland area (Bukit Cincin, Pahang), respectively. Monkey carcass was used as a model for human decomposition in this study. A total of 4 replicates were used in each of the study sites. Ants were observed to prey on eggs, larvae, pupae and newly emerged flies. This study found that ant species could be found at all stages of decomposition, indicating that ants were not a significant indicator for faunal succession. However, different species of ants were obtained from monkey carcasses placed in different ecological habitats. Cardiocondyla sp. was only found on carcasses placed in the coastal area; while Pheidole longipes, Hypoponera sp. and Pachycondyla sp. were solely found on carcasses placed in the highland area. On the other hand, Pheidologeton diversus and Paratrechina longicornis were found in several ecological habitats. These data suggests that specific ant species can act as geographic indicators for different ecological habitats in forensic entomology cases in Malaysia.

Human and animal invasive muscular sarcocystosis in Malaysia--recent cases, review and hypotheses.

Sarcocystosis, an unusual parasitic zoonotic disease, is caused by coccidian/ apicomplexan protozoa in humans and animals. The parasites usually develop in a heteroxenous predator-prey life-cycle involving final (carnivore) and intermediate (omnivore/herbivore) hosts. Besides the intestinal, non-invasive form of the disease in which humans and animals are the definitive hosts for certain Sarcocystis spp., the invasive form has come to recent attention. In the latter, humans and animals serve as intermediate host harbouring sarcocysts in their muscle tissue. Already in 1991 sarcocystosis was seen as a potential emerging food borne zoonosis in Malaysia, and in 2011 and 2012 the largest cluster of symptomatic human muscular sarcocystosis world-wide was reported from Tioman Island, Pahang state. In this review, we focus on invasive sarcocystosis in humans and animals in Malaysia, review the recorded cases and epidemiology, and present hypotheses.

New locality record of Isomyia paurogonita Fang & Fan, 1986 (Diptera: Calliphoridae) from Peninsular Malaysia and Borneo.

Isomyia paurogonita Fang & Fan, 1986 (Diptera: Calliphoridae), a rare species of the subfamily Rhiniinae (tribe Cosminini) was recorded for the first time in Malaysia. We collected one male and two females during a field trip conducted at Genting Highland, Pahang, peninsular Malaysia in May 2011. A 3-day old cow liver was offered as attractant and dipterans collected were transferred to the laboratory for specimens processing and identification. The adults of I. paurogonita were attracted to the odour and then captured by using a sweep net. Isomyia paurogonita was also recorded from two other localities in Peninsular and Malaysian Borneo, namely Gombak Utara, Selangor and Sibu, Sarawak.

Dermatitis caused by Paederus fuscipes Curtis, 1840 (Coleoptera: Staphilinidae) in student hostels in Selangor, Malaysia.

We report a series of dermatitis cases caused by the staphilinid beetles, Paederusfuscipes Curtis, among university students staying in the residential college in Puncak Alam, Selangor, Malaysia from 1 January to 31 December 2010. A total of 360 cases (6.0%) were recorded in the Student Health Center throughout the year; the majority of patients stayed at a hostel near an oil palm plantation. Skin symptoms included erythema, edema, vesicular papules, painful blisters, burning sensation, pruritus, hyper pigmentation and peeling of skin. The commonly involved sites were the face, neck, shoulders and arms. Most students noticed the symptoms upon awakening in the morning. The patients were treated with fusidic acid cream and the symptoms resolved within 5 days. These beetles are nocturnally active and enter the room whenever a light source is available. The unintentional crushing of these beetles during sleep causes the release of its hemolymph (paederin) which is the cause of the dermatitis.

High prevalence of muscular sarcocystosis in cattle and water buffaloes from Selangor, Malaysia.

The prevalence of sarcocystosis in cattle and water buffaloes from peninsular Malaysia was investigated in abattoirs in Selangor state, February, 2011, to March, 2012. Fresh muscle samples were collected from the tongue, heart, oesophagus, diaphragm and skeletal muscles of 102 cattle and 18 water buffaloes. Each sample was initially screened by light microscopy and then fixed for further histopathological analysis. Out of 120 animals examined, 49 (40.8%) harboured the microscopic type of Sarcocystis spp. The positivity rate for cattle was 36.2% and for water buffaloes 66.7%. In cattle, the organs highly infected were the skeletal muscles and diaphragm (27% each), followed by tongue and esophagus (24.3% each), and the heart (8%). In water buffaloes, the heart was most often infected (66.7%), followed by the oesophagus (50%) and skeletal muscle (33.3%); no sarcocysts were detected in the tongue and diaphragm. The shape of the sarcocyst was fusiform to oval with a mean cyst size of 151.66 x 75.83 µm and wall thickness of 2.47 µm in cattle, and 114 x 50.81 µm cyst size and the wall thickness of 1.11 µm in water buffaloes, consistent with Sarcocystis cruzi and Sarcocystis levinei, respectively. Remaining tissue from cattle was subjected to parasite specific 18S rRNA gene PCR and Sarcocystis cruzi was confirmed, at least exemplarily. The peripheral metrocytes and the banana-shaped bradyzoites (15.23 x 2.2 µm in cattle and 11.49 x 2.45 µm in water buffalo hosts) were easily recognized. In conclusion, a high positivity rate was found in Malaysian meat-producing animals with possible implications for meat consumption and human health.