Detection of Khapra Beetle Environmental DNA Using Portable Technologies in Australian Biosecurity.

Khapra beetle, Trogoderma granarium Everts, 1898, is a serious pest of stored grain products globally. Environmental DNA (eDNA)-based methods offer sensitive detection tools used to inform biosecurity officers on the presence of high-risk pests. This study tested laboratory and portable molecular technologies to detect khapra beetle environmental DNA extracted from dust samples collected during biosecurity responses (Tuggeranong and Fyshwick) to khapra beetle incursions in Australia. Airborne and floor dust samples were collected opportunistically using handheld vacuum cleaners and eDNA was extracted using either field or laboratory-based extraction methods and analyzed using laboratory benchtop real time PCR machines and portable machines with two TaqMan and one LAMP-based assay. We successfully collected, extracted, and amplified khapra beetle eDNA from dust samples by qPCR, but failed to amplify T. granarium eDNA using LAMP. The Laboratory qPCR machine showed significantly higher mean Ct values (p < 0.001) and significantly higher positive detections for both assays (p < 0.001) compared to the portable thermocycler. DNA yield was significantly higher in samples extracted using laboratory-based kits compared to field kits (p < 0.001) for both vacuumed and airborne samples (Mean DNA ± S.D. = 5.52 ± 4.45 and 4.77 ± 1.68 ng/µL, respectively), compared to field kits, (1.75 ± 1.17 and 1.36± 1.29 ng/µL for vacuumed and airborne samples, respectively). There were no significant differences in DNA yield between collection methods or differences in amplification associated to extraction or collection methods in either platform tested in this study. Portable technologies tested in this study (Franklin™ Real Time Thermocycler and Genie III) accurately amplified all tissue derived DNA during assay optimisation and field testing, highlighting the capacity of these technologies to complement biosecurity in confirming specimen ID. There was a high incidence of positive detections in field negative controls (Tuggeranong = 12.3 % and Fyshwick = 50 %), mostly attributed to the use of contaminated vacuum cleaners. We discuss suitable methods to minimize sample cross-contamination, the potential of portable molecular technologies as tools for biosecurity applications, and the suitability of eDNA-based molecular detection methods to complement global trade biosecurity for one of the most invasive and important grain pests worldwide.

Parasite Dispersal From the Ornamental Goldfish Trade.

Goldfish, Carassius auratus Linnaeus, 1758, are immensely popular ornamental cyprinid fish, traded in more than 100 countries. For more than 500 years, human translocation has facilitated the spread of goldfish globally, which has enabled numerous and repeated introductions of parasite taxa that infect them. The parasite fauna assemblage of goldfish is generally well documented, but few studies provide evidence of parasite coinvasion following the release of goldfish. This review provides a comprehensive synopsis of parasites that infect goldfish in farmed, aquarium-held, native, and invasive populations globally and summarises evidence for the cointroduction and coinvasion of goldfish parasites. More than 113 species infect goldfish in their native range, of which 26 species have probably coinvaded with the international trade of goldfish. Of these, Schyzocotyle acheilognathi (Cestoda: Bothriocephalidae), Ichthyophthirius multifiliis (Ciliophora: Ichthyophthiriidae), Argulus japonicus (Crustacea: Argulidae), Lernaea cyprinacea (Crustacea: Ergasilidae), Dactylogyrus anchoratus, Dactylogyrus vastator and Dactylogyrus formosus (Monogenea: Dactylogyridae) are common to invasive goldfish populations in more than four countries and are considered a high risk of continued spread. Coinvasive parasites include species with direct and complex life cycles, which have successfully colonised new environments through utilisation of either new native hosts or suitable invasive hosts. Specifically, I. multifiliis, A. japonicus and L. cyprinacea can cause harm to farmed freshwater fish species and are important parasites to consider for biosecurity. These species may threaten other aquatic animal industries given their low host specificity and adaptable life histories. Future attention to biosecurity, management and border detection methods could limit the continued spread of exotic parasites from the ornamental trade of goldfish.

Monogenean Parasite Cultures: Current Techniques and Recent Advances.

Global expansion in fish production and trade of aquatic ornamental species requires advances in aquatic animal health management. Aquatic parasite cultures permit diverse research opportunities to understand parasite-host dynamics and are essential to validate the efficacy of treatments that could reduce infections in captive populations. Monogeneans are important pathogenic parasites of captured captive fishes and exhibit a single-host life cycle, which makes them amenable to in vivo culture. Continuous cultures of oviparous monogenean parasites provide a valuable resource of eggs, oncomiracidia (larvae) and adult parasites for use in varied ecological and applied scientific research. For example, the parasite-host dynamics of Entobdella soleae (van Beneden and Hesse, 1864) and its fish host, Solea solea (Linnaeus, 1758), is one of the most well-documented of all monogeneans following meticulous, dedicated study. Polystoma spp. cultures provide an intriguing model for examining evolution in monogeneans because they exhibit two alternative phenotypes depending on the age of infection of amphibians. Furthermore, assessments of the ecological, pathological and immunological effects of fish parasites in aquaculture have been achieved through cultures of Gyrodactylus von Nordmann, 1832 spp., Benedenia seriolae (Yamaguti, 1934), Neobenedenia Yamaguti, 1963 spp. and Zeuxapta seriolae (Meserve, 1938). This review critically examines methods to establish and maintain in vivo monogenean monocultures on finfish, elasmobranchs and amphibians. Four separate approaches to establish cultures are scrutinised including the collection of live infected hosts, cohabiting recipient hosts with infected stock, cohabiting hosts with parasite eggs or oncomiracidia (larvae) and direct transfer of live adult parasites onto new fish hosts. Specific parasite species' biology and behaviour permits predictive collection of parasite life stages to effectively maintain a continuous culture, while environmental parameters can be altered to manipulate parasite generation time. Parasite virulence and biosecurity are vital components of a well-managed culture to ensure appropriate animal welfare and uncontaminated surrounding environments. Contemporary approaches and techniques are reviewed to ensure optimised monogenean cultures, which ultimately can be used to further our understanding of aquatic parasitology and identify mechanisms to limit infestations in public aquaria, ornamental trade and intensive aquaculture.

Tracking transparent monogenean parasites on fish from infection to maturity.

The infection dynamics and distribution of the ectoparasitic fish monogenean Neobenedenia sp. (Monogenea: Capsalidae) throughout its development was examined on barramundi, Lates calcarifer (Bloch) (Latidae), by labelling transparent, ciliated larvae (oncomiracidia) with a fluorescent dye. Replicate fish were each exposed to approximately 50 fluorescent oncomiracidia and then examined for parasites using an epifluorescence stereomicroscope at 10 time intervals post-exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Fluorescent labelling revealed that parasites attached underneath and on the surface of the scales of host fish. Parasite infection success was 20% within 15 min, and peaked at 93% two days post-exposure, before gradually declining between four and sixteen days. Differences in parasite distribution on L. calcarifer over time provided strong evidence that Neobenedenia sp. larvae settled opportunistically and then migrated to specific microhabitats. Parasites initially attached (<24 h) in greater mean numbers on the body surface (13 ± 1.5) compared to the fins (4 ± 0.42) and head region (2 ± 0.41). Once larvae recruitment had ceased (48 h), there were significantly higher mean post-larvae counts on the head (5 ± 3.4) and fins (12 ± 3) compared to previous time intervals. Neobenedenia sp. aggregated on the eyes, fins, and dorsal and ventral extremities on the main body. As parasites neared sexual maturity, there was a marked aggregation on the fins (22 ± 2.35) compared to the head (4 ± 0.97) and body (9 ± 1.33), indicating that Neobenedenia sp. may form mating aggregations.