Setting the morphologic quality limits enabling accurate classification of charred archaeological grape seeds.

This study investigates the morphological changes in grape pips resulting from various charring conditions. Employing high-resolution scanning combined with morphometric measurements for morphological analysis, we aimed to understand the effects of charring on grape pips. Our morphometric analysis demonstrated significant alterations in seed shape above 250 °C. The length-width ratio and the occurrence of cracks notably changed, providing a basis for assessing charring conditions. In addition, applying a machine learning classification method, we determined that accurate classification of grape varieties by the morphometric analysis method is feasible for seeds charred at up to 250 °C and 8 h. Integrating the morphometric changes and temperature ranges suitable for classification, we developed a sorting model for archaeological seeds. By projecting length-width ratios onto a curve calculated from controlled conditions, we estimated charring temperatures. Approximately 50% of archaeological seeds deviated from the model, indicating drastic charring conditions. This sorting model facilitates a stringent selection of seeds fit for classification, enhancing the accuracy of our machine learning-based methodology. In conclusion, combining machine learning with morphometric sorting enables the identification of charred grape seeds suitable for identification by the morphometric method. This comprehensive approach provides a valuable tool for future research for the identification of charred grape seeds found in archaeological contexts, enhancing our understanding of ancient viticulture practices and grape cultivation.

Economic Aspects of Bovine Ephemeral Fever (BEF) Outbreaks in Dairy Cattle Herds.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne virus (arbovirus) transmitted by blood-feeding insects (mosquitoes and Culicoides biting midges). While the dispersal of arboviral diseases such as bovine ephemeral fever (BEF) into naive areas is often the result of globalization and animal movement, the endemization and local outbreaks of these diseases are mainly influenced by environmental changes. Climate change affects the activity, distribution, dynamics, and life cycles of these vectors (arthropods), the replication of viruses within their vectors, and weakens animal's immune systems. Although BEF does not currently occur in the Americas and Europe (other than in the western regions of Turkey), the risk of BEFV emergence, spread, and endemization in Europe is real. Over the past two decades, arboviruses such as the bluetongue virus (BTV) and Schmallenberg virus (SBV) have emerged in Europe without warning and caused significant losses to the dairy and meat industries. Since the European cattle population has never been exposed to BEFV, the economic losses to dairy and beef production in this continent due to the reduction in milk production, loss of valuable cows, and abortion, should BEF emerge, would probably be considerable. Moreover, arboviruses can also cause substantial financial damage due to restrictions on animal trade and transportation, like the current EHDV-8 outbreak in the Mediterranean basin. In this study, we used national data stored in the Israeli herd book to examine the economic aspects of BEF outbreaks in affected dairy cattle farms countrywide. Our results demonstrate that BEF outbreaks can have immediate and delayed effects, causing severe economic losses due to culling (loss of valuable cows) and a reduction in milk production that affects dairy farm income for months after clinical diagnosis. To our knowledge, this is the first extensive study on the impact of a BEF outbreak at a population level, enabling to conduct accurate risk assessments in future cases of BEFV emergence and re-emergence.

Applying thermal demagnetization to archaeological materials: A tool for detecting burnt clay and estimating its firing temperature.

Burnt materials are very common in the archaeological record. Their identification and the reconstruction of their firing history are crucial for reliable archaeological interpretations. Commonly used methods are limited in their ability to identify and estimate heating temperatures below ~500°C and cannot reconstruct the orientation in which these materials were burnt. Stepwise thermal demagnetization is widely used in archaeomagnetism, but its use for identifying burnt materials and reconstructing paleotemperatures requires further experimental verification. Here we present an experimental test that has indicated that this method is useful for identifying the firing of mud bricks to 190°C or higher. Application of the method to oriented samples also enables reconstruction of the position in which they cooled down. Our algorithm for interpreting thermal demagnetization results was tested on 49 miniature sun-dried "mud bricks", 46 of which were heated to a range of temperatures between 100°C to 700°C under a controlled magnetic field and three "bricks" which were not heated and used as a control group. The results enabled distinguishing between unheated material and material heated to at least 190°C and accurately recovering the minimum heating temperature of the latter. Fourier-Transform Infrared Spectroscopy (FTIR) on the same materials demonstrated how the two methods complement each other. We implemented the thermal demagnetization method on burnt materials from an Iron Age structure at Tell es-Safi/Gath (central Israel), which led to a revision of the previously published understanding of this archaeological context. We demonstrated that the conflagration occurred within the structure, and not only in its vicinity as previously suggested. We also showed that a previously published hypothesis that bricks were fired in a kiln prior to construction is very unlikely. Finally, we conclude that the destruction of the structure occurred in a single event and not in stages over several decades.

Invasive parasites and global change: Evidence for the recent and rapid spillover of a potential pathogen of tilapias with a complex, three-host life cycle.

Biological invasions pose a serious threat to local flora and fauna and have negative impacts on ecosystems. Invasive parasites can also cause severe losses in aquaculture. In this article, we provide evidence of the recent spillover of an African parasite with a complex, three-host life cycle that has rapidly and successfully established itself in the Middle East, most likely due to the recent migration of its final hosts (great cormorant) from Africa. This case of parasite introduction into a country with intensive aquaculture is also important from an economic point of view, since large (up to 2 cm long) larvae of this parasite, the cyclophyllidean tapeworm Amirthalingamia macracantha (Cestoda) localised in the liver, can be pathogenic to their fish hosts, including farmed and wild fish, as shown by our histopathological examination of heavily infected fish. Since its first detection in Israel in November 2020, the parasite has spread rapidly and is currently found in both migratory (great cormorant, Phalacrocorax carbo) and non-migratory birds (pygmy cormorant, Microcarbo pygmaeus), as well as in fish intermediate hosts, including farmed tilapia in several farms in Israel and wild cichlids. There are numerous examples of the spillover of introduced parasites, including those that parasitise fish of commercial importance, but have a direct life cycle or use only a single intermediate host. Tilapines are the second most important group of farmed fish in the world after carps and are produced mainly in Southeast Asia, Central and South America. The global spread of great cormorants and the early evidence that pygmy cormorant may also harbour A. macracantha pose the risk of further spread of this invasive parasite to other countries and areas. In addition, global warming and reductions in foraging and resting areas near these waters may allow the parasite to complete its life cycle in new hosts.

Stretching the wings further- susceptibility of Culex pipiens Linnaeus to bovine ephemeral fever virus infection under experimental conditions.

Bovine ephemeral fever (BEF) is a significant viral disease of cattle in the tropical, subtropical, and temperate climatic zones. This disease is also known as three-day sickness due to the spontaneous recovery of the cattle within a short period (usually 3-5 days). Despite its short duration, the disease may have a considerable impact. It can cause heavy economic losses, primarily due to decreased milk production, lowered fertility in bulls, and even fatality in severe cases. The virus is suspected to be transmitted by haematophagous insects (mainly mosquitoes and Culicoides biting midges); however, the identity of a competent vector for BEFV remains a mystery. Here, we investigated whether BEFV may replicate for a short duration in Culex pipiens Linnaeus, 1758, the most prevalent mosquito species in Israel and a potential vector of this virus to Israeli cattle. We applied nested- qPCR to test BEFV abundance in Cx. pipiens every 24 h for 14 consecutive days post-infection. Additionally, we collected eggs laid by BEFV-infected females and investigated BEFV abundance in the different developmental stages of F1 mosquitos. Our results suggest that Cx. pipiens mosquitoes have the potential to act as a vector of BEFV and also indicate that BEFV may be vertically transmitted from Cx. pipiens female parent to her female offspring.

Cryptosporidium parvum gp60 subtypes in diarrheic lambs and goat kids from Israel.

Cryptosporidium parvum is the second-most prevalent Cryptosporidium species that infects humans worldwide. In European countries, it is the most prevalent species in sheep, suggesting that these animals are a source of zoonotic infection. Preweaned lambs and goats are particularly susceptible to infection by the parasite and may suffer from severe diarrhea whilst excreting large quantities of infectious oocysts. Fifty fecal samples from preweaned lambs and goats with diarrhea from 35 farms across Israel, found to be Cryptosporidium-positive by microscopy, were tested by PCR and sequence analyses to determine the infective species and subtypes. Cryptosporidium parvum DNA was detected in most samples from both lambs and goats (46/50). Cryptosporidium xiaoi DNA was detected in three samples from kids, with co-infection detected in a single sample. Eleven different C. parvum subtypes were found, 10 in lambs and 5 in goats. All subtypes were from the IIa and IId subtype families, with subtypes IIdA20G1 and IIaA15G2R1 being the most prevalent and widespread. These subtypes were previously found in calves and humans in Israel and are considered the most prevalent C. parvum subtypes in small ruminants globally. These results underline the zoonotic potential of C. parvum from small ruminants and the high subtype diversity compared to previous reports from other Middle Eastern countries. In addition, this is the first report of C. xiaoi in Israel.

Insights on Transmission, Spread, and Possible Endemization of Selected Arboviruses in Israel-Interim Results from Five-Year Surveillance.

Outbreaks of arthropod-borne (arbo) viruses that infect livestock impact the health and welfare of domestic and wild animals are often responsible for significant economic losses in livestock production. Surveillance and early warning systems effectively predict the emergence and re-emergence of arboviral disease. This paper presents the interim results of five years monitoring the exposure of sentinel naïve heifers and Culicoides biting midges (Diptera; Ceratopogonidae) to bovine ephemeral fever virus (BEFV), Simbu serogroup viruses, bluetongue viruses (BTV), and epizootic hemorrhagic disease viruses (EHDV). The data were collected from 11 dairy farms situated within eight different geographical regions in Israel. The results indicate that cattle in Israel are affected by all four viruses from the early summer onward. The investigated viruses exhibit unique site-specific profiles in both ruminants and vectors. The potential of several vectors to transmit these viruses and lack of cross-protection to re-infection with multiple serotypes (BTV and EHDV) or species (Simbu serogroup viruses) highlights some likely mechanisms that may play a role in these viruses' maintenance cycle and possible endemization in our region.

Simbu Viruses' Infection of Livestock in Israel-A Transient Climatic Land.

Important lessons have been learned by the Israeli veterinary community regarding Simbu serogroup viruses infections. This serogroup of viruses might cause the births of neonatal malformation in susceptible ruminant's populations. Until 2012, only Akabane virus was connected with the births of malformed ruminants in Israel. However, serological and genomic detection tests, coupled with viral isolations, revealed that more than a single Simbu serogroup serotype could be present concurrently in the same farm or even in the same animal. From 2012 to date, Aino, Shuni, Shamunda, Satuperi, Peaton, Schmallenberg, and Sango viruses have been found in Israel either by serological or genomic investigation. Israel is located in the Eastern Mediterranean Basin, a terrestrial and climatic bridge between the three old continents. The Eastern Mediterranean shores benefit from both the tropical/subtropical and the continental climatic conditions. Therefore, the Eastern Mediterranean basin might serve as an optimal investigatory compound for several arboviral diseases, acting as a sentinel. This review summarizes updated information related to the presence of Simbu serogroup viruses in Israel.

Cryptosporidium parvum subtypes from diarrheic dairy calves in Israel.

Cryptosporidium are protozoan parasites with worldwide distribution, infecting a wide range of terrestrial and aquatic animals, as well as humans. Cryptosporidium parvum is the most important zoonotic species and is the primary cause of cryptosporidiosis in preweaned calves, a highly prevalent, economically important disease. Extensive subtyping of C. parvum from infected humans and animals has expanded current understanding of the parasites' epidemiology. Israel has a highly developed dairy sector with intensive, zero-grazing operations. While C. parvum has been found in dairy calves throughout the country, and subtype data from human patients have also been published, subtype data from animals, and in particular preweaned ruminants, are lacking. We carried out an initial study of Cryptosporidium species and subtypes from preweaned diarrheic calves. Cryptosporidium species were determined in 71 fecal samples from 43 different dairy farms using 18S rRNA PCR, and subtyping of C. parvum based on the 60-kDa glycoprotein (gp60) sequences was done on one sample per farm. C. parvum was the only species found, with eight different subtypes belonging to the zoonotic IIa and IId families. Subtype IIaA15G2R1 was the most prevalent and widespread, found in 50% of the farms over a wide geographical distribution. Our results confirm the presence of subtypes IIaA15G2R1 and IIdA20G1, which were previously found in human patients in Israel, also in Israeli calves. In addition, subtype IIaA12G1R1 is reported here for the first time in an animal. These findings demonstrate the value of monitoring C. parvum subtypes in animal samples, and suggest that the role of calves as well as other young ruminants in the transmission of zoonotic cryptosporidiosis in Israel should be further studied.

Genomic Detection of Schmallenberg Virus, Israel.

We discuss genomic detection of Schmallenberg virus in both Culicoides midges and affected ruminants during June 2018-December 2019, demonstrating its circulation in Israel. This region is a geographic bridge between 3 continents and may serve as an epidemiologic bridge for potential Schmallenberg virus spread into Asia.

A two-branched upgrade to demonstrate ITV transmission by blood-sucking insects.

The enveloped flavivirus Israel turkey meningoencephalitis virus (ITV) causes a neuroparalytic disease in adult turkeys leading to morbidity and mortality. This study reevaluates the role of blood-sucking insects in the transmission of ITV. We demonstrate the crucial importance of two factors in detecting viruses carried by blood-sucking insects: first, enhanced molecular detection of ITV in insects by nested qRT-PCR and second, collection and maintenance of live insects until their molecular examination. These upgrades allowed overcoming the small virus quantities contained in the insects and detecting ITV for the first time in field-collected Culex pipiens.

Temporal trends of tilapia lake virus disease in Israel, 2017-2018.

Tilapia lake virus (TiLV) is an emerging viral disease that affects several tilapia species in different countries since 2014. In 2017-2018, 129 samples were collected from 14 tilapia farms in Israel. Ninety samples represented mortality events (ME), and 39 were used as control samples (CS). RT-qPCR was performed on 89 and 39 duplicate brain and liver tissue samples from ME samples and CS, respectively. TiLV was diagnosed in 37 (41.6%) ME, while only two of the CS samples (5%) were positive for TiLV (OR = 13.2, 95% CI = 3.0-58.1). Additional RT-PCR was performed on positive samples, and amplified products were sequenced. Maximum likelihood phylogenetic analysis of segment-3 revealed three distinct clades: the first clade (A) includes 25 sequences of TiLV, detected previously in Israel (2011), Ecuador (2012), Egypt (2015), Thailand (2015-2019), India (2017), Peru (2018) and USA (2018-2019) and 11 sequences of TiLV from the current study (2017-2018); the second clade (B) includes only four sequences from Thailand (2018) and Bangladesh (2017 and 2019); and a third clade (C) which includes a single sequence from Bangladesh (2019). Out of the 39 sequences included in clade A, 14 closely related sequences of TiLV from the current study (2018) formed a distinctive sub-clade (IL-2018). Mann-Whitney U test showed differences in the distribution of survival rates between Israeli sequences (from 2011, 2017 and 2018) of clade A (p = 0.004) and Israeli sequences (from 2018 solely) of sub-clade IL-2018. The average survival rates of clade A and sub-clade IL-2018 were 58.1% (SD = 21.5) and 31.2% (SD = 25.6), respectively. This is one of only few field studies which show direct association of TiLV with mortality events in tilapia farms. The decrease in survival rate in the newly evolved clade might raise concern regarding virus evolution towards increased virulence, which should be further explored.

Towards modernizing the taxonomy of Mediterranean Culicoides using classical morphology, mtDNA barcoding, and MALDI-TOF MS protein profiling.

Culicoides biting midges (Diptera: Ceratopogonidae) are a highly successful group of small (1-3 mm) hematophagous flies, infamous for the role they play as biological vectors for numerous pathogens of veterinary significance. The principal aim of the national animal disease surveillance program of Israel is to be able to rapidly sort and identify live field-captured insects including Culicoides for arbovirus screening. In this exploratory study, three identification methods-classical morphology, DNA barcoding, and MALDI-TOF MS-were applied simultaneously to individuals of 10 Culicoides species that commonly attack livestock in Israel. The strengths and limitations of the three methods are compared and evaluated. In essence, the CO1 barcoding and MALDI-TOF MS results closely matched those of classical morphology. Furthermore, at a higher level and in strong accordance with recognized subgenera, the 10 species, in the reconstructed phylogenies, coalesced into multiple deeper-branched monophyletic clades. However, some discrepancies between the molecular and protein profiling results did occur and proved difficult to assess in terms of taxonomic significance. This difficulty underscores how tricky it is to establish clear species limits when methods involving borderline cutoff values and similarity indices are used as a taxonomic aid. An added shortcoming of the pluralistic triple-method approach is that a significant percentage of the species-level depositions in the GenBank and BOLD databases are misidentified, hindering structured comparison and interpretation of the morphological and molecular results obtained. Aspects of the unresolved taxonomy of various biting midge assemblages within the Mediterranean basin, including minor changes to the Israeli Culicoides checklist, are discussed in light of the methods applied. It is observed that the direct access that classical morphology provides to the external environment (or species niche) is indispensable to the full and correct interpretation (and application) of concomitant molecular and protein profiling results. The Culicoides taxonomy of the future ought to be fully integrative, during which the assimilation of modern methodological advances should strengthen-rather than undermine-the morphological foundations laid down during the 260-year Linnaean epoch.

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny.

Obligatory hematophagous arthropods such as lice, bugs, flies, and ticks harbor bacterial endosymbionts that are expected to complement missing essential nutrients in their diet. Genomic and some experimental evidence support this expectation. Hard ticks (Acari: Ixodidae) are associated with several lineages of bacterial symbionts, and very few were experimentally shown to be essential to some aspects of tick's fitness. In order to pinpoint the nature of interactions between hard ticks and their symbionts, we tested the effect of massive elimination of Coxiella-like endosymbionts (CLE) by antibiotics on the development and fitness of the brown dog tick (Rhipicephalus sanguineus). Administration of ofloxacin to engorged (blood fed) nymphs resulted in significant and acute reduction of their CLE loads - an effect that also persisted in subsequent life stages (aposymbiotic ticks). As a result, the post-feeding development of aposymbiotic female (but not male) nymphs was delayed. Additionally, aposymbiotic adult females needed a significantly prolonged feeding period in order to replete (detach from host), and had reduced engorgement weight and a lower capacity to produce eggs. Consequently, their fecundity and fertility were significantly reduced. Eggs produced by aposymbiotic females were free of CLE, and the resulting aposymbiotic larvae were unable to feed successfully. Our findings demonstrate that the observed fitness effects are due to CLE reduction and not due to antibiotic administration. Additionally, we suggest that the contribution of CLE is not mandatory for oocyte development and embryogenesis, but is required during feeding in females, when blood meal processing and tissue buildup are taking place. Presumably, under these extreme physiological demands, CLE contribute to R. sanguineus through supplementing essential micro- and macronutrients. Further nutrient complementary studies are required to support this hypothesis.

Osmium isotope analysis as an innovative tool for provenancing ancient iron: A systematic approach.

The innovation of iron production is often considered one of the greatest technological advances in human history. A reliable provenancing method for iron is instrumental for the reconstruction of economic, social and geo-political aspects of iron production and use in antiquity. Although the potential of osmium isotopes analysis for this purpose has been previously suggested, here we present for the first time the results of osmium isotope analysis of ores, bloom and metal obtained from a set of systematic, bloomery iron-smelting experiments, utilizing selected ores from the Southern Levant. The results show that the 187Os/188Os ratio is preserved from ore to metal, with no isotopic fractionation. In addition, enrichment/depletion of osmium content was observed in the transition from ore to metal and from ore to slag. This observation has potential significance for our ability to differentiate between the various processes and sheds light on the suitability of various production remains for this method, which emerges as a robust and promising tool for the provenancing of archaeological ferrous metals.

What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem.

Many insects feed on a low-nitrogen diet, and the origin of their nitrogen supply is poorly understood. It has been hypothesized that some insects rely on nitrogen-fixing bacteria (diazotrophs) to supplement their diets. Nitrogen fixation by diazotrophs has been extensively studied and convincingly demonstrated in termites, while evidence for the occurrence and role of nitrogen fixation in the diet of other insects is less conclusive. Here, we summarize the methods to detect nitrogen fixation in insects and review the available evidence for its occurrence (focusing on insects other than termites). We distinguish between three aspects of nitrogen fixation investigations: (i) detecting the presence of potential diazotrophs; (ii) detecting the activity of the nitrogen-fixing enzyme; and (iii) detecting the assimilation of fixed nitrogen into the insect tissues. We show that although evidence from investigations of the first aspect reveals ample opportunities for interactions with potential diazotrophs in a variety of insects, demonstrations of actual biological nitrogen fixation and the assimilation of fixed nitrogen are restricted to very few insect groups, including wood-feeding beetles, fruit flies, leafcutter ants, and a wood wasp. We then discuss potential implications for the insect's fitness and for the ecosystem as a whole. We suggest that combining these multiple approaches is crucial for the study of nitrogen fixation in insects, and argue that further demonstrations are desperately needed in order to determine the relative importance of diazotrophs for insect diet and fitness, as well as to evaluate their overall impact on the ecosystem.

Seasonal and spatial variation in Culicoides community structure and their potential role in transmitting Simbu serogroup viruses in Israel.

Culicoides biting midges (Diptera: Ceratopogonidae) are a successful group of small (1-3 mm) haematophagous flies, some species of which are biological vectors of veterinary arboviruses, such as bluetongue virus, epizootic haemorrhagic disease virus, African horse sickness virus and Simbu serogroup viruses. In this study, we examine seasonal and spatial effects on the presence and distribution of Culicoides communities associated with ruminant and equine farms in Israel, and their infection with Simbu serogroup viruses. Our results demonstrate that both the vectors and the viruses are widely spread in Israel, including regions that were previously considered Culicoides-free. Moreover, our results show that although seasonality affects infection with Simbu serogroup viruses, both viruses and potential vectors can be found year round, suggesting continuous circulation of Simbu serogroup viruses in Israeli livestock farms. Finally, this study provides novel and basic information on Simbu serogroup-infected Culicoides in Israel: it demonstrates that Sathuperi, Shuni and Peaton viruses were circulating in Israel in 2015-2017 as they were found in C. imicola and C. oxystoma, both potential vectors of these viruses, and supplies the first-ever genomic detection of Sathuperi in Israel. Consequently, the data emerging from this study are of importance in understanding the epidemiology of arboviruses in Israel and are of relevance to the potential spread and possible future outbreaks of different Simbu serogroup viruses within the Mediterranean region and Europe.

Tilapia lake virus disease: Phylogenetic analysis reveals that two distinct clades are circulating in Israel simultaneously.

Tilapia lake virus (TiLV) is an emerging viral disease that affects several tilapia species in different countries since 2014. In 2017-2018, 129 samples were collected from 14 tilapia farms in Israel. Ninety samples represented TiLV-suspected cases (TSC), and 39 were used as control samples (CS). RT-qPCR was performed on 89 and 39 duplicate brain and liver tissue samples from TSC samples and CS, respectively. TiLV was diagnosed in 37 (40.1%) of TSC, and two of the CS samples (5%) were also positive for TiLV. Additional validation RT-PCR was performed on positive samples, and amplified products were sequenced. Maximum-likelihood phylogenetic analysis of segment-3 of 25 selected sequences revealed two distinct clades: one virtually identical to sequences from India and the second closely related to isolates from Ecuador, Thailand, Egypt and Peru, apparently imported to Israel from Thailand. Thus, our results indicate that at least two distinct clades of TiLV are circulating in Israel simultaneously. As of today, the number of TiLV sequences available in free publicly accessible databases is limited. Nevertheless, our study provides new molecular epidemiology baseline for further epidemiological studies of TiLV.

Serological evidence suggests that several Simbu serogroup viruses circulated in Israel.

Viruses of the Simbu serogroup are arboviruses that are known to cause outbreaks of abortion, stillbirth and congenitally deformed neonates. This study presents the results of antibody screening of Simbu serogroup viruses in heifers born in Israel after October 2013, and in adult milking cows born before May 2012. Thirteen dairy cattle farms in five regions, and one sheep flock, entered this study. Serum samples that were found to be positive by ELISA were further tested by specific virus- neutralization test against a panel of Simbu serogroup viruses including Akabane, Aino, Sathuperi, Shamonda, and Peaton viruses. Antibody detection in lactating adult cows revealed that several viruses were circulating in Israel between 2008-2014. Moreover, during autumn 2014 the heifers became serum-positive after being exposed to more than one Simbu serogroup virus concurrently. The results of this study shed new light on Simbu virus infections in Israel, and may contribute to the epidemiology of the Simbu serogroup around the Mediterranean Basin in general.

Gut bacteria of weevils developing on plant roots under extreme desert conditions.

BACKGROUND: Many phytophagous insects, whose diet is generally nitrogen-poor, rely on gut bacteria to compensate for nutritional deficits. Accordingly, we hypothesized that insects in desert environments may evolve associations with gut bacteria to adapt to the extremely low nutrient availability. For this, we conducted a systematic survey of bacterial communities in the guts of weevils developing inside mud chambers affixed to plant roots in the Negev Desert of Israel, based on 16S rRNA gene amplicon sequencing. RESULTS: Our analyses revealed that gut bacterial communities in weevil larvae were similar across a wide geographical range, but differed significantly from those of the mud chambers and of the surrounding soils. Nevertheless, a high proportion of bacteria (including all of the core bacteria) found in the weevils were also detected in the mud chambers and soils at low relative abundances. The genus Citrobacter (of the Enterobacteriaceae family) was the predominant group in the guts of all individual weevils. The relative abundance of Citrobacter significantly decreased at the pupal and adult stages, while bacterial diversity increased. A mini literature survey revealed that members of the genus Citrobacter are associated with nitrogen fixation, recycling of uric acid nitrogen, and cellulose degradation in different insects. CONCLUSIONS: The results suggest that although weevils could potentially acquire their gut bacteria from the soil, weevil host internal factors, rather than external environmental factors, were more important in shaping their gut bacterial communities, and suggest a major role for Citrobacter in weevil nutrition in this challenging environment. This study highlights the potential involvement of gut bacteria in the adaptation of insects to nutritional deficiencies under extreme desert conditions.