Toxorhynchites species: A review of current knowledge

The increasing global incidence of mosquito-borne infections is driving a need for effective control methods. Vector populations have expanded their geographical ranges, while increasing resistance to chemical insecticides and a lack of effective treatments or vaccines has meant that the development of vector control methods is essential in the fight against mosquito-transmitted diseases. This review will focus on Toxorhynchites, a non-hematophagous mosquito genus which is a natural predator of vector species and may be exploited as a biological control agent. Their effectiveness in this role has been strongly debated for many years and early trials have been marred by misinformation and incomplete descriptions. Here, we draw together current knowledge of the general biology of Toxorhynchites and discuss how this updated information will benefit their role in an integrated vector management program

First isolation of Leishmania from Northern Thailand:case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis

Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3’UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis

Parasitism of Soldiers of the Termite, Macrotermes gilvus (Hagen), by the Scuttle Fly, Megaselia scalaris (Loew) (Diptera: Phoridae)

Termites of the genus Macrotermes (Termitidae: Macrotermitinae) are serious agricultural and structural pests, which also play vital roles in ecosystem functioning, and are crucial for the maintenance of tropical biodiversity. They are widely distributed, mainly in Southeast Asian countries; however, the parasitism of termites has been little researched. This research was conducted to identify and study the ecology of the parasitoids of termites at Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand. Macrotermes gilvus (Hagen) soldier termites were collected from 25 mounds. In four of the 25 mounds, scuttle fly larvae were found inside the bodies of the soldier termites, and adult flies were found in all of the mounds. Some of the larvae successfully developed to pupae under laboratory conditions. The percentages of parasitized major soldier termites collected from the four mounds were 43.79%, 47.43%, 0.86%, and 3.49%, respectively, and the percentages of parasitized minor soldier termites were 0.64%, 0.00%, 0.21%, and 0.00%, respectively. Larvae, pupae, and adult flies were identified using both morphological and molecular identifications. Molecular identification used the partial nucleotide sequences of the mitochondrial cytochrome c oxidase I (COI) gene. The results of both identification methods identified the parasitic Diptera as the scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae). The phylogenetic analysis of the 23 scuttle fly samples (11 larvae, 7 pupae, and 5 adults) classified them into two clades: (1) Those closely related to a previous report in India; (2) those related to M. scalaris found in Asia and Africa. This is the first discovery of M. scalaris in M. gilvus. Further investgation into termite parasitism by M. scalaris and its possible use in the biological control of termites is needed

Molecular Identification of Host Blood Meals and Detection of Blood Parasites in Culicoides Latreille (Diptera: Ceratopogonidae) Collected from Phatthalung Province, Southern Thailand

Five hundred and fifty-nine female biting midges were collected, and seventeen species in six subgenera (Avaritia, Haemophoructus, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and two groups (Clavipalpis and Shortti) were identified. The dominant Culicoides species was C. peregrinus (30.94%), followed by C. subgenus Trithecoides. From blood meal analysis of engorged biting midges, they were found to feed on cows, dogs, pigs, and avians. The majority of blood preferences of biting midges (68%; 49/72) displayed a mixed pattern of host blood DNA (cow and avian). The overall non-engorged biting midge field infectivity rate was 1.44 % (7/487). We detected Leucocytozoon sp. in three Culicoides specimens, one from each species: C. fulvus, C. oxystoma, and C. subgenus Trithecoides. Crithidia sp. was found in two C. peregrinus specimens, and Trypanosoma sp. and P. juxtanucleare were separately found in two C. guttifer. More consideration should be paid to the capacity of biting midges to transmit pathogens such as avian haemosporidian and trypanosomatid parasites. To demonstrate that these biting midges are natural vectors of trypanosomatid parasites, additional research must be conducted with a greater number of biting midges in other endemic regions

Development of loop-mediated isothermal amplification (LAMP) for simple detection of Leishmania infection

Abstract Background Leishmaniasis is a neglected tropical disease that is caused by an obligate intracellular protozoan of the genus Leishmania. Recently, an increasing number of autochthonous leishmaniasis cases caused by L. martiniquensis and the novel species L. siamensis have been described in Thailand, rendering an accurate diagnosis of this disease critical. However, only a few laboratories are capable of diagnosing leishmaniasis in Thailand. To expand leishmaniasis diagnostic capabilities, we developed a simple colorimetric loop-mediated isothermal amplification (LAMP) technique for the direct detection of Leishmania DNA. Methods LAMP was performed for 75 min using four primers targeting the conserved region of the18S ribosomal RNA gene, and the DNA indicator used was malachite green (MG). To simulate crude samples, cultured promastigotes of L. siamensis were mixed with blood or saliva. Also, clinical samples (blood, saliva, and tissue biopsies) were obtained from patients with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). All samples were boiled for 10 min and introduced directly into the LAMP reaction mixture without DNA purification. Results The use of MG resulted in an unambiguous differentiation of positive and negative controls. For L. siamensis, the detection limit was 103 parasites/mL or 2.5 parasites/tube. Saliva, tissue biopsies, and whole blood were indicative of active Leishmania infection, and their direct usages did not adversely affect the detection limit. In addition, this LAMP assay could detect DNA from multiple Leishmania species other than L. siamensis and L. martiniquensis, including L. aethiopica, L. braziliensis, L. donovani and L. tropica. Conclusions The simplicity and sensitivity of LAMP in detecting active Leishmania infection could enable the rapid diagnosis of leishmaniasis, thereby facilitating the survey and control of leishmaniasis in Thailand. However, our limited number of samples warranted a further validation with a larger cohort of patients before this assay could be deployed

First Evidence of Co-Circulation of Emerging Leishmania martiniquensis, Leishmania orientalis, and Crithidia sp. in Culicoides Biting Midges (Diptera: Ceratopogonidae), the Putative Vectors for Autochthonous Transmission in Southern Thailand

Since 1996, autochthonous cases of emerging leishmaniasis caused by Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis have been more frequently reported, especially in the northern and southern parts of Thailand. However, the accurate identification of their natural vectors and reservoirs remains unconfirmed. Previous studies have suggested that these emerging parasites might be transmitted by other non-phlebotomine vectors. Herein, we speculated that Culicoides biting midges might act as the competent vectors responsible for autochthonous leishmaniasis in southern Thailand. In this research, 187 non-engorged, parous and gravid Culicoides females and 47 blood-engorged ones were trapped from the residences of two recently diagnosed visceral leishmaniasis patients in Sadao District and the unaffected site in Rattaphum District, Songkhla Province, southern Thailand. Species diversity and abundance of biting midges varied among the trapping sites. Using ITS1-PCR and BLASTn analysis, L. martiniquensis was predominantly detected in several Culicoides species, including C. peregrinus, C. oxystoma, C. mahasarakhamense, and C. huffi from the vicinity of patients’ houses; and in C. fordae and C. fulvus from the unaffected site. L. orientalis was also co-circulated in C. peregrinus and C. oxystoma caught near the second patient’s house. Additionally, Crithidia sp. were also detected using SSU rRNA-PCR across Culicoides spp. Host blood meal analysis of eight different Culicoides species from the unaffected site also revealed that all trapped Culicoides had fed on cows and goats, indicating the possible role of these mammalian species as reservoir hosts. Essentially, this study is the first entomological investigation, revealing the co-circulation of emerging trypanosomatids among several species of Culicoides biting midges and strongly supporting the potential role of this insect group as the main vectors responsible for the epidemiology of autochthonous leishmaniasis in southern Thailand