Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals.

BACKGROUND: Molecular analysis of blood meals is increasingly used to identify the hosts of biting insects such as midges and mosquitoes. Successful host identification depends on the availability of sufficient host DNA template for PCR amplification, making it important to understand how amplification success changes under different storage conditions and with different durations of blood meal digestion within the insect gut before being placed into the storage medium. METHOD: We characterised and compared the digestion profile of two species of Culicoides over a 96-h period using a novel set of general vertebrate primers targeting the 16S rRNA gene. A set number of individuals from each species were killed over 13 time points post-blood feeding and preserved in 95% ethanol. Samples were stored either at ambient room temperature or in a - 20 °C freezer to examine the effect of storage condition on the PCR amplification success of host DNA. RESULTS: We found that amplification success across the 96-h sampling period post-feeding was reduced from 96 to 6% and 96% to 14% for Culicoides nubeculosus and Culicoides sonorensis, respectively. We found no effect of storage condition on PCR amplification success, and storage in 95% ethanol was sufficient to maintain high rates of amplifiable host DNA for at least 9 months, even at room temperature. CONCLUSIONS: These findings highlight the limited time frame during which an individual may contain amplifiable host DNA and demonstrate the importance of timely sample capture and processing post-blood feeding. Moreover, storage in 95% ethanol alone is sufficient to limit host DNA degradation. These results are relevant to the design of studies investigating the biting behaviour and disease transmission potential of Culicoides and other biting Diptera.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Functional response of the hypopharyngeal glands to a social parasitism challenge in Southern African honey bee subspecies.

Hypopharyngeal gland (HPG) development in honey bee workers is primarily age-dependent and changes according to the tasks performed in the colony. HPG activity also depends on colony requirements and is flexible in relation to the need for feeding brood. Very little is known about HPG development in the honey bee subspecies found in Southern Africa. We examined HPG development in Apis mellifera scutellata and A. m. capensis, including A. m. scutellata colonies infested with an invasive parasitic clonal lineage of A. m. capensis known to manipulate food provisioning to the parasitic larvae by their A.m. scutellata hosts, under natural in-hive conditions in bees aged 0 to 14 days using light microscopy. We found marked differences in acini size (berry-like clusters of secretory cells) and the age at which maximum HPG development occurred between the subspecies and in the presence of the parasite. In A. m. scutellata workers, acini reached maximum size at 6 days. The acini of A. m. capensis workers were larger (up to double) than those of A. m. scutellata and reached maximum size at 8 days, while the HPG acini in A. m. scutellata workers infested with A. m. capensis clones reached development sizes similar to those of A. m. capensis at day 10 and were 1.5 times larger than those of uninfested A. m. scutellata. This provides foundational insights into a functional response affecting the development of the HPG most likely associated with brood pheromone composition and how this is altered in the presence of a social parasite.

Quantifying and Modeling the Acquisition and Retention of Lumpy Skin Disease Virus by Hematophagus Insects Reveals Clinically but Not Subclinically Affected Cattle Are Promoters of Viral Transmission and Key Targets for Control of Disease Outbreaks.

Lumpy skin disease virus (LSDV) is a vector-transmitted poxvirus that causes disease in cattle. Vector species involved in LSDV transmission and their ability to acquire and transmit the virus are poorly characterized. Using a highly representative bovine experimental model of lumpy skin disease, we fed four model vector species (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans, and Culicoides nubeculosus) on LSDV-inoculated cattle in order to examine their acquisition and retention of LSDV. Subclinical disease was a more common outcome than clinical disease in the inoculated cattle. Importantly, the probability of vectors acquiring LSDV from a subclinical animal (0.006) was very low compared with that from a clinical animal (0.23), meaning an insect feeding on a subclinical animal was 97% less likely to acquire LSDV than one feeding on a clinical animal. All four potential vector species studied acquired LSDV from the host at a similar rate, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in this study were combined with data from studies of LSDV transmission and vector life history parameters to determine the basic reproduction number of LSDV in cattle mediated by each of the model species. This reproduction number was highest for Stomoxys calcitrans (19.1), followed by C. nubeculosus (7.1) and Ae. aegypti (2.4), indicating that these three species are potentially efficient transmitters of LSDV; this information can be used to inform LSD control programs.IMPORTANCE Lumpy skin disease virus (LSDV) causes a severe systemic disease characterized by cutaneous nodules in cattle. LSDV is a rapidly emerging pathogen, having spread since 2012 into Europe and Russia and across Asia. The vector-borne nature of LSDV transmission is believed to have promoted this rapid geographic spread of the virus; however, a lack of quantitative evidence about LSDV transmission has hampered effective control of the disease during the current epidemic. Our research shows subclinical cattle play little part in virus transmission relative to clinical cattle and reveals a low probability of virus acquisition by insects at the preclinical stage. We have also calculated the reproductive number of different insect species, therefore identifying efficient transmitters of LSDV. This information is of utmost importance, as it will help to define epidemiological control measures during LSDV epidemics and of particular consequence in resource-poor regions where LSD vaccination may be less than adequate.

Prisoners receive food fit for a queen: honeybees feed small hive beetles protein-rich glandular secretions through trophallaxis.

The honeybee nest parasite Aethina tumida (small hive beetle) uses behavioural mimicry to induce trophallactic feeding from its honeybee hosts. Small hive beetles are able to induce honeybee workers to share the carbohydrate-rich contents of their crops, but it is not clear whether the beetles are able to induce to workers to feed them the protein-rich hypopharyngeal glandular secretions fed to the queen, larvae and other nest mates. Protein is a limiting macronutrient in an insect's diet, essential for survival, growth and fecundity. Honeybees obtain protein from pollen, which is consumed and digested by nurse bees. They then distribute the protein to the rest of the colony in the form of hypopharyngeal gland secretions. Using 14C-phenylalanine as a qualitative marker for protein transfer, we show that small hive beetles successfully induce worker bees to feed them the protein-rich secretions of their hypopharyngeal glands during trophallaxis, and that females are more successful than males in inducing the transfer of these protein-rich secretions. Furthermore, behavioural observations demonstrated that female beetles do not preferentially interact with a specific age cohort of bees when soliciting food, but males tend to be more discriminant and avoid the more aggressive and active older bees.