Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

Vector incrimination and transmission of avian malaria at an aquarium in Japan: mismatch in parasite composition between mosquitoes and penguins.

BACKGROUND: Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultaneous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. METHODS: Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mosquitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confirmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identified using molecular methods. RESULTS: Parasite detection in penguins tended to fluctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plasmodium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. CONCLUSIONS: The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confirm competency and infection dynamics of avian malaria parasites.

A fatal case of a captive snowy owl (Bubo scandiacus) with Haemoproteus infection in Japan.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

A new avian Cryptosporidium genotype in a 1-month-old caged brown wood owl (Strix leptogrammica) with severe dehydration and diarrhea.

A 1-month-old brown wood owlet (Strix leptogrammica) purchased from a wholesaler and housed as a companion bird by an individual owner in Japan showed severe dehydration and anorexia following a week of vomiting and severe diarrhea. A great number of approximately 5 × 4-µm-sized Cryptosporidium oocysts were found in the feces by microscopy. The owlet was administered subcutaneous fluid and intragastric tube feeding for 2 weeks, resulting in improvement of the condition with a decreased number of oocysts in the feces. At days 51 and 119, no oocysts were found in the feces by microscope and PCR detection. These results suggested that this parasite was a possible agent of severe diarrhea in the affected bird. Molecular analysis of DNA extracted from oocysts based on the 18SrRNA loci identified C. avium; however, analysis of actin and hsp (heat shock protein) genes identified a novel genotype indicating a mixed infection with C. avium and a novel genotype.

Detection of Dirofilaria immitis DNA in host serum by nested PCR.

The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.

First isolation and characterization of a mosquito-borne orbivirus belonging to the species Umatilla virus in East Asia.

An orbivirus was isolated from a sample from the ornithophilic mosquito Culex sasai in Japan. The virus, designated Koyama Hill virus (KHV), replicated to high titer in a mosquito cell line and to a low titer in an avian cell line, but the release of progeny viruses was not observed in mammalian cell lines inoculated with KHV. Electron microscopic examination of KHV-infected mosquito cells showed approximately 70-nm virus particles and viral tubules typical of members of the genus Orbivirus, family Reoviridae. KHV efficiently replicated in Cx. sasai mosquitoes, suggesting a potential vector species for KHV transmission in nature. Full-length viral genome sequencing and phylogenetic analysis revealed that KHV is closely related to Umatilla virus (UMAV) and Stretch Lagoon orbivirus (SLOV). This suggests that KHV is a new member of the species Umatilla virus, an orbivirus species not previously observed in East Asia. The KHV genome segment encoding NS1 contains a notable sequence deletion and heterogeneity compared with a prototype UMAV, which may affect its growth properties and pathogenicity in host cells. These results provide new insights into the genetic diversity and geographic distribution of members of the species Umatilla virus.

The apicoplast genome of Leucocytozoon caulleryi, a pathogenic apicomplexan parasite of the chicken.

Leucocytozoon caulleryi, a haemosporidian parasite of the chicken (Gallus gallus domesticus), can be highly pathogenic and often fatal. Although this parasite is extremely relevant to veterinary science, knowledge of its genomic features is limited. To gain information applicable to developing novel control methods for the parasite, we analyzed the apicoplast genome of L. caulleryi. This extranuclear organellar DNA of 85.1% A + T and a unit of 34,779 bp was found to encode almost the same set of genes as the plastid genome of Plasmodium falciparum, including 16 tRNA and 30 protein coding genes, and except for one open reading frame, ORF91 absent in L. caulleryi. As in P. falciparum, the L. caulleryi apicoplast DNA contains two sets of a unique inverted repeat (IR), each one 5,253 bp and encoding genes specifying one large and one small rRNA subunit and nine tRNAs but no protein, and separated by a unique 13 bp sequence. Studies of several haemosporidian apicoplast DNA sequences have identified a corresponding IR region; however, none of these studies has looked at the complete sequence, even for well-studied species such as P. falciparum. Phylogenetic studies using a concatenated amino acid sequence based on the open reading frames confirmed the close relationship between L. caulleryi and Plasmodium spp. In this study, we determined the nucleotide sequence of the entire L. caulleryi apicoplast genome, including the region connecting the two IR units. This is the first report of the complete nucleotide sequence of a haemosporidian apicoplast DNA with a canonical IR.

Species Composition and Feeding Behaviors of Vector Mosquitoes of Avian Infectious Diseases at a Wild Bird Rehabilitation Facility in Japan.

Although wild bird rehabilitation facilities are important for the conservation of wild species, individuals may be kept within the facilities for long periods, consequently posing a risk for the bird to be infected with pathogens to which they are not naturally exposed. In turn, novel pathogens may be introduced through rescued migratory species. Avian malaria and West Nile fever are important avian diseases transmitted by mosquitoes. To understand the transmission dynamics of such diseases at rehabilitation facilities, the ecology of vector mosquitoes, including species composition, seasonality, and feeding behaviors, were explored. Mosquitoes were collected at a wild bird rehabilitation facility and wildlife sanctuary in Japan from 2019 to 2020 using mouth aspirators, sweep nets, and light traps. A total of 2,819 mosquitoes of 6 species were captured, all of which are potential vectors of avian diseases. Culex pipiens pallens and Cx. pipiens form molestus were the dominant species (82.9% of all collected mosquitoes). Density and seasonality differed between sampling locations, presumably because of differences in mosquito behaviors including feeding preferences and responses to climatic factors. Blood-fed Culex mosquitoes fed solely on birds, and many mosquito species are thought to have fed on birds within the facility. Particularly, Cx. pipiens group probably fed on both rescued and free-living birds. The rehabilitation facility may be an important site for the introduction and spread of pathogens because 1) numerous mosquitoes inhabit the hospital and its surroundings; 2) blood-fed mosquitoes are caught within the hospital; 3) there is direct contact between birds and mosquitoes; 4) both birds within the hospital and wild birds are fed upon. Furthermore, blood-fed Cx. pipiens form molestus were observed in the winter, suggesting that pathogens might be transmitted even during the winter when other mosquito species are inactive.

Complete mitochondrial genome of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) (Suliformes: Phalacrocoracidae).

We determined the complete mitochondrial DNA sequence of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) using long PCR and primer walking methods. The mitochondrial genome was 19,020 bp in length and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and two control regions. It is basically consistent with the characteristics of the mitochondrial genomes of other Suliformes species. Phylogenetic analysis using 12 species of Suliformes based on the sequences of 13 concatenated protein-coding genes confirmed the monophyly of P. carbo ssp.

The first detection of avian haemosporidia from Culicoides biting midges in Japan, with notes on potential vector species and the transmission cycle.

BACKGROUND: Biting midges (Ceratopogonidae) are capable of transmitting a variety of pathogens including viruses, trypanosomes and haemosporidia. The majority of Haemoproteus parasites are transmitted by biting midges predominantly of the genus Culicoides and are known to cause significant physical and reproductive impacts on both wild and domestic birds. In Japan, Haemoproteus had been detected from various avian hosts, but not from arthropod vectors. In this study, we investigated the prevalence of avian haemosporidia at an educational forest in central Japan in attempt to reveal possible vector species of Haemoproteus, which would help to better understand the transmission cycle of Haemoproteus within Japan and to develop preventative measures for captive and domestic birds. METHODS: Biting midges were caught using UV light traps from 2016 to 2018. The collected samples were morphologically identified, and haemosporidian parasites were detected using PCR-based methods. The detected lineages were phylogenetically analyzed and compared with lineages previously detected from birds. Bloodmeal analyses were also carried out for part of the blood-fed individuals. RESULTS: Six Haemoproteus lineages were detected from 17 of 1042 female Culicoides (1.63%), including three species (C. sigaensis, C. arakawae, and C. pictimargo) in which Haemoproteus was detected for the first time. All detected lineages were placed in the subgenus Parahaemoproteus clade and were previously detected from crows of central Japan, strongly suggesting that parasites of these genetic lineages are transmitted between Culicoides and crows. Two Plasmodium lineages were also detected but are thought to be transmitted between Culex mosquitoes and birds of the educational forest based on previous detections. No amplifications were seen in bloodmeal analysis, possibly due to insufficient amount of blood, denaturation via digestion, or insufficient detectability of the used protocol. CONCLUSION: Haemoproteus DNA was detected from Culicoides for the first time in Japan, suggesting that transmission is possible within the country. These findings highlight the necessity to investigate Culicoides populations and Haemoproteus infections dynamics in Japan. However, vector competence could not be confirmed in this study and further studies are anticipated.

A widespread survey of avian haemosporidia in deceased wild birds of Japan: the hidden value of personally collected samples.

Widespread surveys of avian haemosporidia (Plasmodium, Haemoproteus, and Leucocytozoon) in wild birds have substantially advanced information on the haemosporidian fauna of Japan. However, many areas and bird species remain insufficiently investigated. Bird carcasses collected for personal specimen collection seldom reach academic audience particularly in the veterinary field. The presence of avian haemosporidia was investigated in these personally collected bird carcasses, in order to better understand the avian haemosporidian fauna in Japan. Bird carcasses were donated through personal contact upon approval of the study. Tissue samples were collected from the birds and examined for haemosporidian parasites using nested-PCR targeting the cytochrome b gene. One hundred and forty-three birds of 85 species were donated, including 34 species and two subspecies that were molecularly or collectively investigated for the first time in Japan. Avian haemosporidian DNA was detected from 37 of the 134 tested birds (27.61%). In 8 bird species, avian haemosporidia was detected for the first time. Twenty-nine lineages were detected, including 8 novel and 9 known lineages detected in Japan for the first time. Furthermore, 16 lineages were detected from novel host species. While information that could be drawn was limited and risk management of zoonotic diseases needs re-consideration, these findings expanded information on the host range and distribution of several lineages. Collectively, this method of investigation using personally collected bird samples can provide important additions to more fully understand the avian haemosporidian fauna of Japan, as well as other areas with limited investigations.

Complete mitochondrial genome of the Japanese Cormorant Phalacrocorax capillatus (Temminck & Schlegel, 1850) (Suliformes: Phalacrocoracidae).

The complete sequencing of mitochondrial DNA of the Japanese Cormorant Phalacrocorax capillatus was performed using long PCR and primer walking methods. The assembled genome was 19,105 bp in length. It contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and two control regions. The phylogenetic analysis using the obtained sequence showed that P. capillatus is closest to P. carbo.

A long-term field study on mosquito vectors of avian malaria parasites in Japan.

Avian malaria is a mosquito-borne disease of birds caused by avian Plasmodium spp. in worldwide scale. Some naïve birds show serious symptoms which can result in death. Surveillance of vectors and parasites are important to understand and control this disease. Although avian malaria has been found in Japan, detailed prevalence and dynamics remained understudied. We aimed to observe annual changes in the abundance of mosquitoes and the prevalence of avian Plasmodium parasites in Japan. Mosquitoes were collected using dry ice traps over a 10-year period, at a fixed research area located in Kanagawa prefecture. Collected mosquitoes were investigated for the species composition, population size and prevalence of avian Plasmodium by PCR. Mosquitoes belonging to 13 species in 7 genera were collected (n=8,965). The dominant species were Aedes (Ae.) albopictus and Culex (Cx.) pipiens group (gr.). Seven avian Plasmodium lineages, all of which were previously known, were detected from Cx. pipiens gr., Ae. albopictus, and Tripteroides bambusa. Three genetic lineages were dominant and were probably transmitted by Cx. pipiens gr. whose could be the primary vector of these parasites. Annual variations in the seasonal prevalence of mosquitoes and avian Plasmodium were revealed for the first time during recent 10 years in Japan. Namely, avian Plasmodium occurrence in the vector population peaked often in June to July and September to October when the density of the vector population was presumably high enough for the transmission of avian Plasmodium upon appearance of infected birds.

Uptake dynamics of scrapie agent in the intestinal villous epithelium of suckling and weanling Syrian hamsters.

In mice, the number of intestinal villous columnar epithelium cells that incorporate abnormal prion protein (PrP(Sc) ) decreases significantly after weaning. In this study, the dynamics of PrP(Sc) uptake during the growth of hamsters were investigated by inoculating scrapie 263K agent orally into suckling and weanling Syrian hamsters and estimating the number of PrP(Sc) -positive villous epithelium cells immunohistochemically. The number of PrP(Sc) -positive cells declined significantly as the hamsters aged. The present results suggest that a tendency toward decline of PrP(Sc) -positive cells with increasing age might be a common phenomenon among the superfamily Muridae.

Entomological study on transmission of avian malaria parasites in a zoological garden in Japan: bloodmeal identification and detection of avian malaria parasite DNA from blood-fed mosquitoes.

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.

Blood meal identification and prevalence of avian malaria parasite in mosquitoes collected at Kushiro wetland, a subarctic zone of Japan.

In Japan, the prevalence of avian Plasmodium in birds and mosquitoes has been partially examined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zone have not been adequately investigated. In this study, mosquito collections and avian Plasmodium detections from the mosquito samples were carried out to demonstrate the avian Plasmodium transmission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan. A total of 5657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight species was collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika Deer and one unfed Culex pipiens group were found to be positive for avian Plasmodium by polymerase chain reaction. This is the first report of the detection of avian Plasmodium DNA from mosquitoes distributing in the subarctic zone of Japan. The blood meals were successfully identified to captive or wild animals, including seven mammalian species, four bird species, and one amphibian species. These results indicated that infected birds with avian Plasmodium inhabited and direct contacts occurred between the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.

Pigeon louse fly, Pseudolynchia canariensis (Diptera: Hippoboscidae), collected by dry-ice trap.

During a mosquito collection, a female of the pigeon louse fly, Pseudolynchia canariensis (Diptera: Hippoboscidae), was collected by a mosquito trap baited with dry ice in Ishigaki-jima, Yaeyama Islands, Japan. This is the 1st record of P. canariensis from Yaeyama Islands.

First records of prevalence and diversity of avian haemosporidia in snipe species (genus Gallinago) of Japan.

Migratory birds are important carriers of pathogens such as viruses, bacteria and protozoa. Avian haemosporidia have been detected from many wild birds of Japan, but the infection status of migratory birds and transmission area are still largely unknown. Gallinago snipes are long-distance migratory shorebirds, and five species migrate to or through Japan, including Latham's snipe which is near threatened. Haemosporidian parasites in four snipe species were investigated to understand the role of migratory birds in the transmission of avian haemosporidia. Namely, this study aimed: i) to investigate differences in parasite prevalence and related factors explaining infection likelihood among these migratory species, ii) to explore the diversity in haemosporidian lineages and possible transmission areas, and iii) to assess the possibility of morphological effects of infection. Blood samples were collected from snipes caught in central and southwest Japan during migration. Parasites cytb gene DNA were detected via PCR-based testing, and detected lineages were phylogenetically analyzed. Additionally, factors related to prevalence and morphological effects of infection were statistically tested. 383 birds from four Gallinago snipe species were caught, showing higher overall prevalence of avian haemosporidia (17.8 %) than reported in other wader species in previous studies. This high infection rate is presumably due to increased contact with vector insects, resultant of environmental preferences. The prevalence of Plasmodium spp. Was higher in Swinhoe's snipes, while Haemoproteus spp. Was higher in Latham's snipes. These differences are thought to be related to ecological factors including habitat use, distribution and migratory route. Six lineages detected from juveniles indicate transmission between the breeding and sampling area. Contrary to expectations, a direct link between morphological features and haemosporidian parasite infection were not detected. These findings provide valuable information for conservation of this endangered migratory bird group. Further studies linking biological and parasitological research are anticipated to contribute to conservational actions.

Contaminations contaminate common databases.

The polymerase chain reaction (PCR) is a very powerful method to detect and identify pathogens. The high sensitivity of the method, however, comes with a cost; any of the millions of artificial DNA copies generated by PCR can serve as a template in a following experiment. If not identified as contaminations, these may result in erroneous conclusions on the occurrence of the pathogen, thereby inflating estimates of host range and geographic distribution. In the present paper, we evaluate whether several published records of avian haemosporidian parasites, in either unusual host species or geographical regions, might stem from PCR contaminations rather than novel biological findings. The detailed descriptions of these cases are shedding light upon the steps in the work process that might lead to PCR contaminations. By increasing the awareness of this problem, it will aid in developing procedures that keep these to a minimum. The examples in the present paper are from haemosporidians of birds, however the problem of contaminations and suggested actions should apply generally to all kinds of PCR-based identifications, not just of parasites and pathogens.

Use of flow cytometry to separate Leucocytozoon caulleryi gametocytes from avian blood.

The highly pathogenic avian protozoan Leucocytozoon caulleryi infects host chicken cells, and interference by the host genome results in difficulty in obtaining protozoal DNA for genetic analysis. We used flow cytometry analysis to separate expelled L. caulleryi gametocytes from infected chicken blood and to analyse cell populations and sorting by FACS efficiency. Infected blood cells stained with SYTO-24 showed a specific area on 2-dimensional scattergrams compared to uninfected blood. The specific area was sorted, and approximately 85% of the sorted cells were identified as L. caulleryi gametocytes by microscopic observation. DNA was also extracted from the sorted fraction, and a clear increase in polymerase chain reaction (PCR) amplification of protozoal DNA was observed compared to infected blood without sorting. Host-derived DNA was also detected by PCR; however, its amplification was decreased compared to that in unsorted infected blood. This is the first report of the separation of L. caulleryi gametocytes from infected host blood using flow cytometry. This method may be applied to further genetic analyses such as studies of the dynamics of stage-specific L. caulleryi gene expression.