Regulation of mitochondrial metabolism by hibernating bear serum: Insights into seasonal metabolic adaptations.

Hibernating animals undergo a unique and reversible decrease in their whole-body metabolism, which is often accompanied by a suppression of mitochondrial respiration. However, the precise mechanisms underlying these seasonal shifts in mitochondrial metabolism remain unclear. In this study, the effect of the serum from active and hibernating Japanese black bears on mitochondrial respiration was assessed. Stromal-vascular cells were obtained from bear white adipose tissue and cultured with or without an adipocyte differentiation cocktail. When the oxygen consumption was measured in the presence of bear serum, the hibernating bear serum reduced maximal respiration by 15.5 % (p < 0.05) and spare respiratory capacity by 46.0 % (p < 0.01) in the differentiated adipocytes in comparison to the active bear serum. Similar reductions of 23.4 % (p = 0.06) and 40.6 % (p < 0.05) respectively were observed in undifferentiated cells, indicating the effect is cell type-independent. Blue native PAGE analysis revealed that hibernating bear serum suppressed cellular metabolism independently of the assembly of mitochondrial respiratory chain complexes. RNA-seq analysis identified 1094 differentially expressed genes (fold change>1.5, FDR<0.05) related to insulin signaling and glucose metabolism pathways. These findings suggest that the rapid alterations in mitochondrial metabolism during hibernation are likely induced by a combination of reduced insulin signaling and suppressed mitochondrial function, rather than changes in respiratory complex assembly.

Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF ß-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS).

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and ß-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The ß-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the ß-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus).

Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi, Babesia sp. UR2-like group, Cytauxzoon sp. UR1, Babesia sp. UR1, and Babesia microti) were detected from bear blood DNA samples. B. microti was detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens, H. ursi, Babesia sp. UR2-like pathogens, and Cytauxzoon sp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence of H. ursi were significantly higher in Japanese black bear (0% vs 96.4%) while that of Babesia sp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence of Babesia sp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Mixed Mycobacterium tuberculosis Lineage Infection in 2 Elephants, Nepal.

Tuberculosis in elephants is primarily caused by Mycobacterium tuberculosis. We identified mixed M. tuberculosis lineage infection in 2 captive elephants in Nepal by using spoligotyping and large sequence polymorphism. One elephant was infected with Indo-Oceanic and East African-Indian (CAS-Delhi) lineages; the other was infected with Indo-Oceanic and East Asian (Beijing) lineages.

Heart rate during hyperphagia differs between two bear species.

Hyperphagia is a critical part of the yearly cycle of bears when they gain fat reserves before entering hibernation. We used heart rate as a proxy to compare the metabolic rate between the Asian black bear ( Ursus thibetanus) in Japan and the Eurasian brown bear ( Ursus arctos) in Sweden from summer into hibernation. In the hyperphagic period, black bears feed on fat- and carbohydrate-rich hard masts whereas brown bears feed on sugar-rich berries. Availability of hard masts has quantitative and spatial annual fluctuations, which might require increased activity and result in intraspecific stress. Using generalized additive mixed models we analysed the differences in heart rate between the two species. Black bears had decreased heart rates during summer but had doubled heart rate values throughout the hyperphagic period compared to brown bears. This letter illustrates the different physiological consequences of seasonal differences in food availability in two species of the same genus dealing with the same phenological challenge.

Incidence of Multiple Paternity and Inbreeding in High-Density Brown Bear Populations on the Shiretoko Peninsula, Hokkaido, Japan.

Understanding the breeding ecology of a species is essential for the appropriate conservation and management of wildlife. In brown bears, females occasionally copulate with multiple males in one breeding season, which may lead to multiple paternity in a single litter. In contrast, inbreeding, a potential factor in the reduction of genetic diversity, may occur, particularly in threatened populations. However, few studies have reported the frequency of these phenomena in brown bear populations. Here, we investigated the incidence of multiple paternity and inbreeding in a high-density brown bear population on the Shiretoko Peninsula in Hokkaido, Japan. A total of 837 individuals collected from 1998 to 2017 were genotyped at 21 microsatellite loci, and parentage analysis was performed. Out of 70-82 litters with ≥2 offspring, 14.6-17.1% of litters were sired by multiple males. This was comparable to the rate reported in a Scandinavian population, although population density and litter size, factors that potentially affect the incidence of multiple paternity, differed between the 2 populations. Out of 222 mother-father mating pairs, 6 litters (2.7%) resulted from matings between fathers and daughters. Additionally, 1 (0.5%) and 4 (1.8%) cases of mating between maternal half-siblings and between paternal half-siblings, respectively, were observed; however, no cases of mating between mothers and sons or between full siblings were observed. Our results suggest that male-biased natal dispersal effectively limits mating between closely related individuals (aside from fathers and daughters) in brown bears.

Comparison of feeding habits and habitat use between invasive raccoons and native raccoon dogs in Hokkaido, Japan.

BACKGROUND: In Japan, invasive raccoons cause severe ecological and social problems by transmitting pathogens to humans, livestock, and native species, causing substantial crop damage, and competing with native species. Possible competition between invasive raccoons and native raccoon dogs is of concern in Japan because Japanese raccoon dogs have a limited distribution and are native only to Japan and the two species have similar characteristics. We assessed potential competition between raccoons and raccoon dogs by comparing feeding habits and habitat use. RESULTS: Both species were captured in Hokkaido, Japan from 2004 to 2017. More raccoons were captured close to agricultural land at the forest periphery (70.1%, 358/511); conversely, more raccoon dogs were captured in the forest core (74.9%, 253/338). Feeding habits were then examined by fecal analysis and stable isotope analyses. Fecal analysis revealed both species to be opportunistic omnivores that consumed easily found food items. However, raccoon feces contained more crops, whereas raccoon dog feces contained more insects, reflecting the different locations in which the species were trapped. Moreover, stable isotope ratios were significantly higher in raccoons than raccoon dogs (Corn has the highest carbon stable isotope (δ13C) value, and amphibians and reptiles are high in nitrogen stable isotope (δ15N); forest resources such as insects and wild fruits are low in δ13C and δ15N). CONCLUSIONS: We conclude that both species ate similar food types, but their food preferences appeared to differ. Raccoon and raccoon dog habitat use also differed, possibly because the two species inhabited areas where they could easily obtain their preferred foods. Therefore, the current feeding habits and habitat use of raccoons do not appear to overlap sufficiently with those of raccoon dogs to impact the latter. The results of this study, particularly the stable isotope data, may provide a useful precedent for future studies of competition in medium-sized mammals, particularly canids.

First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan.

Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.

Accessory corpora lutea formation in pregnant Hokkaido sika deer (Cervus nippon yesoensis) investigated by examination of ovarian dynamics and steroid hormone concentrations.

Generally, sika deer conceive a single fetus, but approximately 80% of pregnant females have two corpora lutea (CLs). The function of the accessory CL (ACL) is unknown; moreover, the process of ACL formation is unclear, and understanding this is necessary to know its role. To elucidate the process of ACL formation, the ovarian dynamics of six adult Hokkaido sika deer females were examined ultrasonographically together with peripheral estradiol-17ß and progesterone concentrations. ACLs formed in three females that conceived at the first estrus of the breeding season, but not in those females that conceived at the second estrus. After copulation, postconception ovulation of the dominant follicle of the first wave is induced by an increase in estradiol-17ß, which leads to formation of an ACL. A relatively low concentration of progesterone after the first estrus of the breeding season is considered to be responsible for the increase in estradiol-17ß after copulation.

Changes in the immunolocalization of steroidogenic enzymes and the androgen receptor in raccoon (Procyon lotor) testes in association with the seasons and spermatogenesis.

The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3ßHSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3ßHSD expression, suggesting that 3ßHSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.

Areolae of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.

Epidemiology and molecular characterization of Mycobacterium tuberculosis including a drug-resistant strain associated with mortality of Asian elephants in Nepal 2019-2022.

Tuberculosis (TB) is an emerging threat to the survival of elephants in Nepal. We investigated the lung tissue samples from nine elephants that died from 2019 to 2022 in Nepal using culture, conventional PCR, and loop-mediated isothermal amplification (LAMP) and then performed genotyping of five PCR-positive isolates to understand the possible transmission dynamics of Mycobacterium tuberculosis (Mtb). Results showed that two-thirds (6/9) of elephants were confirmed to be infected from Mtb by LAMP, 5/9 by PCR, and 4/9 by culture. Genotyping of Mtb isolates showed that elephants were infected with the Indo-Oceanic and Beijing lineages including an isoniazid-resistant Beijing lineage. MIRU-VNTR-based phylogeny, gyrA, and katG sequencing showed the possibility of ongoing transmission of Indo-Oceanic lineages and likely transmission of the drug-resistant Beijing lineage from human to elephant. Implementation of comprehensive surveillance and preventive measures are urgently needed to address this zoonotic disease and protect elephants from TB in Nepal.

Detection and characterization of vector-borne parasites and Wolbachia endosymbionts in greater one-horned rhinoceros (Rhinoceros unicornis) in Nepal.

Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required.

Environmental and host factors underlying tick infestation in invasive raccoons (Procyon lotor) in Hokkaido, Japan.

Revealing interactions between ticks and wild animals is vital for gaining insights into the dynamics of tick-borne pathogens in the natural environment. We aimed to elucidate the factors that determine tick infestation in wild animals by investigating ticks on invasive raccoons (Procyon lotor) in Hokkaido, Japan. We first examined the composition, intensity, and seasonal variation of ticks infesting raccoons in six study areas in Hokkaido from March 2022 to August 2023. In one study area, ticks infesting tanukis (raccoon dog, Nyctereutes procyonoides albus) were collected in May to July in both 2022 and 2023, and questing ticks were collected from the vegetation by flagging every other week in the same period. Next, we screened 17 environmental and host variables to determine factors that affect the number of ticks infesting raccoons using generalized linear (mixed) models. From 245 raccoons, we identified a total of 3,917 ticks belonging to eight species of two genera: the most prominent species were Ixodes ovatus (52.9 %), followed by Haemaphysalis megaspinosa (14.4 %), Ixodes tanuki (10.6 %), and Ixodes persulcatus (9.5 %). Ixodes ovatus was also predominant among questing ticks and ticks infesting tanukis. Although I. tanuki was frequently collected from raccoons and tanukis, it was rarely collected in the field. The variables that significantly affected the infestation on raccoons differed by genus, species and developmental stage of the tick. For instance, the infestation of adult I. ovatus was significantly affected by four variables: night-time temperature during nine days before capturing the raccoon, the size of forest area around the capture site, sex of the raccoon, and sampling season. The first two variables were also responsible for the infestation on raccoons of almost all species and stages of ticks. Our study revealed that the number and composition of ticks infesting raccoons can be affected not only by landscape of their habitats but also by weather conditions in several days before capturing.

Structure and steroidogenesis of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.

Timing of puberty and its relationship with body growth and season in male raccoons (Procyon lotor) in Hokkaido.

The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.

The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo.

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia-positive and Borrelia-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis, Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.