Insect Samples Research Articles

DNA Barcoding and Metabarcoding Protocols for Species Identification.

The article presents the several steps to be performed on a plant, fungal, insect, or soil sample to obtain DNA sequences for DNA barcode analysis. The chapter begins with a description of sample preparation including procedures for cleaning and proceeds to DNA extraction with methods adapted for the specific type of sample. Next, DNA quantification is described so the proper amount is used for the amplification of the selected barcode regions. Information is provided for reaction mixes and amplification conditions for several referenced barcode primer pairs tuned for the individual sample of interest. This is followed by a description of procedures to access the success of amplification, cleanup, and quantification of the product ready for either Sanger sequencing or library preparation for massive parallel sequencing (MPS). Finally, procedures are provided for Sanger sequencing, library preparation, and MPS sequencing. The chapter provides several references of barcode regions for different sample types.

DIVERSITY OF THE ORDER HYMENOPTERA IN RICE PLANTATION IN MANEMBO-NEMBO DISTRICT AS AN ECOLOGICAL INDICATOR

This research aims to determine habitat conditions as an ecological indicator and measure the level of diversity of insects of the order Hymenoptera on rice plantations in the Manembo-nembo sub-district. The method used in this research is quantitative descriptive. Insect samples were collected using the sweeping net and yellow pan trap methods. The Shannon-Wiener diversity index formula, Pielou's similarity index (E), and Simpson dominance index (C) are used to measure the level of diversity. The results of research on rice plantations found insects of the Hymenoptera order as many as 35 genera, 16 subfamilies and 14 families with a total of 470 individuals with normal habitat conditions where Hymenoptera insects can still carry out activities on rice plantations. Wasp insects from 24 genera also have functions as biological control agents, most of which are parasitoid and predatory wasps, which have potential as ecological indicators in rice plantations in the Manembo-nembo sub-district. The level of diversity is included in the medium criteria, as is the evenness index value. In contrast, the dominance index is included in the low criteria even though the Pimplinae family dominates rice cultivation in the Manembo-nembo sub-district.

Insect Diversity of Some Hybrid Rice Genotypes in Sleman-Yogyakarta Irrigated Agroecosystem

Diversity of insect at the rice field gives a main impact to the stability of ecosystem. Index of diversity reflects the relationship of species abundance in the community. This research was aimed to determine the diversity of insect at some hybrid rice varieties in irrigated rice agroecosystem. Research was conducted in Sleman, Yogyakarta Indonesia from April to August 2018. The experiment was conducted using purposive random sampling. Insect samples were obtained using swing net and visual observation. Diversity index was analysed using Shanon (H), while diversity and uniformity of insect was examined using Evennes index (E). Result showed pest insects found during vegetative stage of rice consist of more species, while lower numbers were found at generative stage with less species. They were belongs to 6 orders (more than 10 families) such as Diptera, Lepidoptera, Coleoptera, Orthoptera, Hemiptera and Hymenoptera. The dominant pest was Cicadellidae, followed by Delphacidae and Alydidae. Based on the diversity data analysis (H), pest insects and natural enemies were between moderate and high, with a range between 1.5 to 2.5. It indicates the rice field ecosystem planted with hybrid rice gave good impact and balance species diversity in the agroecosystem of irrigated rice area in Sleman Yogyakarta.

A NEW FIELD EMERGENCE TRAP FOR SAMPLING INSECTS FROM PRECISE AREAS OF LOGS

A new emergence trap was fabricated for collecting saproxylic insects from precise areas of dead and fallen trees. The trap works on the principle of all emergence traps, i.e., organisms emerging from a substrate get attracted towards light, where they encounter a transparent surface and get collected in a collecting tube with preservative fluid. It is one of the most objective methods for sampling saproxylic insects as they emerge from wood substrata. Using many replicates of this trap, the entire heterogeneity of a substratum like dead wood can be easily covered. The trap can provide a higher definition of the sampling area and be used for studying assemblages of saproxylic insects. It is almost cost-effective and user-friendly under field conditions, on logs and snags, and even on live trees. In this study, 102 such field emergence traps were deployed in a conserved area, and 59 species of saproxylic insects belonging to 5 functional groups were collected during a brief sampling period of 3 months. Extensive sampling trials using such traps in more numbers for extended periods can provide insights into species? diversity and host preferences.. KEYWORDS :Emergence Trap, Insect Diversity, Insect Sampling, Saproxylic Insects, Xylophagous Insects

Gut microbiomes of cycad-feeding insects tolerant to β-methylamino-L-alanine (BMAA) are rich in siderophore biosynthesis

Ingestion of the cycad toxins β-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of selected bacteria. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate selected taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We characterized the biosynthetic potential of four bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages, and discovered a suite of biosynthetic gene clusters notably rich in siderophores. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, including desferrioxamine B, suggesting the occurrence of an unprecedented desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, including a possible role for bacterial siderophores.

Entomophagy practices, use patterns, and factors influencing perception and consumption frequency of edible insects in the Republic of Benin

BackgroundEdible insects are important sources of essential nutrients and have the potential to contribute to malnutrition reduction and food security in the Republic of Benin. However, their consumption is always restricted to a limited number of sociocultural groups. To determine how the consumption of insects could be promoted as an alternative food source, this study documents the endogenous knowledge associated with edible insects and, the main factors that govern their perception and frequency consumption.MethodsA survey was conducted towards 479 rural households consuming edible insects through 91 villages of Atacora, Alibori, Zou, and Plateau departments using individual interviews with a semi-structured questionnaire. The survey was focused on the inventory of edible insects and the documentation of consumers’ acceptance, frequencies and motive reason of consumption, local uses, and accessibility to edible insects. Samples of edible insects were collected and preserved in 70% alcohol for taxonomic identification.ResultsThe majority of surveyed people (79.1%) were consumers of edible insects since many years ago (29.1 ± 17.2 years). Insect species belonging to 17 genera of 7 families and 3 orders of insects were used as food, with Brachytrupes membranaceus Drury being the most widespread and consumed. Six factors affecting edible insect availability were identified with the chemical pollution as the most important. Besides their food use (63.2%), edible insects in the study area were used for several purposes. We find that ethnicity, religion, age, education level, and monthly frequency of insect consumption are the main factors influencing the local perception of edible insects. Indeed ethnic group, religion ethnicity, and market accessibility have a positive influence on edible insect consumption frequency. The Hierarchical Clustering of Principal Components has allowed us to classify the interviewees into 3 groups with different perceptions of entomophagy and their characteristics will make it possible to better orient the strategies for promoting entomophagy in the Republic of Benin.ConclusionsReligion and tradition are among the main factors that influence entomophagy in Benin Republic. The development of a national strategy to promote entomophagy should take into account the recorded insect consumption motivations, and their different uses by each ethnic group, and mainly target young people.

Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Survey of viruses and vectors in tomato plants, alternative hosts and weeds in the state of Santa Catarina, Brazil

AbstractSanta Catarina is an important tomato‐producing state in Brazil, but there is little data regarding the prevalence and characterization of viruses and insect vectors in tomato plants in this state. This study surveyed viruses and their vectors in commercial tomato‐producing areas, as well as alternative hosts and weeds in the surrounding areas. Sixty‐five samples of young leaves were collected between 2019 and 2023, and viruses were detected with generic or specific PCR primers targeting several virus genera. In addition, 397 virus vector insect samples were collected and identified. The orthotospoviruses tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) were the most prevalent and distributed in several regions, whilst the crinivirus tomato chlorosis virus (ToCV) and begomovirus tomato severe rugose virus (ToSRV) were restricted to the metropolitan region of Florianópolis. This is the first report of ToSRV in Solanum americanum in Brazil and of TCSV in zucchini and cucumbers worldwide. A survey of insect vectors found whiteflies, Mediterranean cryptic species of Bemisia tabaci (MED) and Trialeurodes vaporariorum were predominant, while Frankliniella occidentalis and F. schultzei thrips were often found cohabiting in the sampled crops. These results highlight the need to adapt management strategies in Santa Catarina, especially in the Santo Amaro da Imperatriz region, where a greater frequency and diversity of viral species and vectors were found. In addition, both the thrips and whitefly species (especially MED) found are a cause for concern, as they readily transmit viruses that infest crops in the open fields of Santa Catarina.

Genetic variability of the pear lace bug Stephanitis pyri F. (Heteroptera: Tingidae) in Krasnodar Territory, Russia

Aim. Study of the genetic variability of the pear lace bug Stephanitis pyri F. depending on geographical location and trophic specialization.Material and Methods. The object of the study was insect samples (n=60) from the natural population of the pear lace bug Stephanitis pyri F. (Tingidae: Heteroptera) in the Krasnodar Territory: in Krasnodar, Kropotkin and Novorossiysk. The collection of S. pyri bugs was carried out on model trees of domestic apple (Malus domestica Borkh) and sour cherry (Prunus cerasus L.). Molecular genetic analysis was carried out in several stages: sample preparation, DNA extraction, and diagnostics based on RAPD (random amplified polymorphic DNA)‐PCR (DNA amplification).Results. With the help of molecular genetic markers, the genetic variability of the pear lace bug in the Krasnodar Territory was studied. The indicators of genetic differentiation between geographic (Gst=0.209) and trophic (Gst=0.049–0.302) intrapopulation groups of S. pyri were calculated. The levels of variability were determined within geographic (79.1 %) and trophic groups (70.0–95.1 %), as well as between them – 20.9 % and 4.9–30 %, respectively. The influence of the food factor on the genetic diversity and differentiation of insect populations is shown through the example of the Kropotkin trophic group of bugs that prefer the sour cherry P. cerasus as the main food plant.Conclusion. A small effect of isolation by distance was noted, which was characterized by a weak dependence of geographical and genetic distances between the population groups of the pear lace bug. The influence of the nutritional factor on the genetic variability of phytophages populations has been established, as is confirmed by other researchers.

Technique for the identification of insect species in processed foods based on three short DNA sequences

For pest control, it is necessary to identify insect species in food. However, it is difficult to identify the insect species in processed food based on their morphological characteristics owing to deformation or fragmentation. DNA sequence-based identification is useful not only to corroborate morphological identification, but also as an alternative identification approach. Therefore, markers are required to obtain the target specific amplicons from insect DNA sequences fragmented by food processing. We developed a DNA sequence-based insect species identification technique that combined two markers in the cytochrome oxidase subunit I (COI) region and one marker in the 16S ribosomal RNA (rRNA) region, with amplicons up to 200 bp in size. The identification results from this technique were consistent with those from the morphological identification of 89% of the 38 insect samples selected from seven orders. This technique identified the insect species in insect samples recovered from model food that were heat-treated at 118 °C for 18 min.

The Existence of Predatory Insects in the Agroecosystem of Potato Plant (Solanum tuberosum L.) Integrated with Refugia Plants

Inappropriate handling of pests can cause substantial losses in both quantity and quality of potato plants. One of the promising alternative methods is by modifying the agroecosystem such as using refugia plants to conserve the diversity of predators in potato plants. This study aimed to determine the effect of refugia plants on the diversity of predatory insects in potato plants. This study used a randomized block design (RBD) with 6 treatments and 3 replications. Sampling of predatory insects was carried out using traps, namely the yellow pan trap and direct (conventional) collection. The results of this study indicated that there were 3 orders of predatory insects from 3 families and 9 species. The most common insects were found in the treatment of marigold and sunflower flowers. The highest diversity index value was 1.33 obtained in the sunflower treatment, which is classified as moderate

Evaluation of the Mangrove Ecosystem Conditions in Wundulako Sub-District and Their Consequences on Insect Diversity

Insect diversity is believed to serve as one of the bioindicators for the condition of an ecosystem.In this investigation, data pertaining to the density of mangroves were collected by extracting samples from designated stations. This was achieved by utilizing the transect line and quadrat approach, known as the Transect Line Plot method. The data were gathered through observation plots measuring 10x10 m2, aiming to document the mangrove vegetation categorized as trees.At each station, any insect samples identified were subjected to characterization based on their morphological traits, utilizing an insect identification guide.Based on the research results, there are six mangrove species among themfindings obtained through field research at four observation station sites unveiled the existence of six distinct variations of mangrove forest types within the coastal region of Wundulako District. Among these, four types belonged to the Rhizophoraceae family, specifically Rhizophora apiculata, Rhizophora Mucronata, Ceriopstagal, and Bruguiera Gymnorrhiza, . Additionally, there was one species from the Combretaceae family, Lumnitzeraand Racemosa, and another from the Sonneratiaceae family, Sonneratia Alba. Generally, the density of the mangrove ecosystem was determined to be ranging from moderate to dense.Furthermore, a scrutiny of the insect diversity index yielded values of 1.23 for Station I, 1.39 for Station II, 1.36 for station III, and 1.69 for sStation IV. Based on the findings derived from the research, it is plausible to conclude that the condition of the mangrove ecosystem in Wundulako District across the four stations varies from limited to extensive. Additionally, the outcomes from the insect diversity index suggest a relatively reduced level of insect diversity.

PENGGUNAAN HERBISIDA TRIKLOPIR UNTUK MENGENDALIKAN GULMA PADA PERKEBUNAN KELAPA SAWIT FASE BELUM MENGHASILKAN DAN PENGARUHNYA TERHADAP KEANEKARAGAMAN SERANGGA

Weed is one of the major problems in immature oil palm plantations. Weed control is carried out to optimize the growth of oil palm plants, and the method commonly used in the field is chemical control with herbicides. The active ingredient of the herbicide used in the field is triclopyr, however herbicide application is thought to affect the diversity of insects, especially those found in lower vegetation, at the same time it is necessary to observe the effect of herbicide application on insect diversity. This study aims to obtain an effective dose of herbicide to control weeds in immature oil palm plants and to determine the effect of herbicide application on insect diversity. This study used a Randomized Block Design (RBD) which consisted of 6 treatments and 4 replications. Triclopyr herbicide treatment doses were 864 g.ha-1, 768 g/ha, 672 g.ha-1, 576 g.ha-1, mechanical weeding and control. Insect sampling method uses insect nets and pitfall traps. The data obtained were analyzed using variance or F test at 5% level and further test was carried out by DMRT at 5% level. The results showed that the best dose of herbicide triclopir was 768 g/ha because it was effective in controlling the weeds of Borreria latifolia, Calopogonium mucunoides and Asystasia gangetica. Triclopyr herbicide application affects the diversity of insects and the even distribution of insects.

Phylogenetic analysis among some species of aphids (Homoptera: Aphididae) using DNA sequencing moleculartechnique

The phylogeny of aphids (Hemiptera: Aphididae) has been studied by molecular technique for the first time in Kurdistan, this study comprises relationship between six species of Aphids collected from the leaves of different plants (Chrysanthemum, Oak, Almond, Pine, Asteraceae and Herbaceous) in many localities of Erbil governorate Kurdistan region-Iraq, started in May to July 2022. For understanding about aphid evolution, phylogenetic of aphid is crucial. However, neither the phylogenetic alterations of the Aphid taxa nor their comprehensive definition have been achieved. Therefore, a unique method has been developed in this study to examine COI gene sequencing and infer the relationships between the major aphid taxa. DNA was isolated, and a band of 550 bp of mt COI gene was amplified. Then the amplicons were sequenced. The part of the COI gene of the insect samples are alignment inside of NCBI GenBank by BLAST program, were used to compare our nucleotide have sequenced with other stored species of insect sequences. This research presented that studying phylogeny of aphid species through a sequencing technique can create a phylogenetic tree for the used species with reliable results. So, our species in Kurdistan region clustered in a monophyletic clade with published from most countries with high identic value (100%). The COI sequence of aphid species were submitted to GenBank with six accessions of OP355287 - OP355292. The composition of nucleotides of the sequence of COI gene was low of G-C base pairing. We were successful in showing that the mt-COI gene can be used as a molecular marker for the identification of related species, as shown by the similarities between the phylogenetic association created by COI

MOLECULAR IDENTIFICATION OF THRIPS SPECIES, WHICH INFESTS THE GREENHOUSE VEGETABLES INCLUDING EGGPLANT IN THE CENTRAL AGRARIAN REGION OF MONGOLIA

In Mongolia, the first record of onion thrips was informed in vegetable fields by D. Tsedev (Tsendsuren et al., 1979), since that has no detailed research materials relating to the identification of thrips and the Thysanoptera group observed in different crop fields. In 2017–2019, we have taken the thrips samples from the following areas, which spread on eggplant, cucumber, tomato, paprika, and beans grown in the greenhouses of the Agropark research and training center, the greenhouses of the Bornuur soum in the Tuv province and the Mandal soum of Selenge province and onion field accordingly. The mitochondrial COI gene of selected insect samples has been used for species identification. For the nucleotide sequence of the mitochondrial COI area of the insects, we amplified with universal LCO1490 and HCO2198 primers. We have illustrated the COI gene fragments of thrips samples. For building a phylogenetic tree was constructed using the Neighbor-Joining method. A bootstrap test with 1000 replications was carried out. The evolutionary distances were calculated using Tamura’s three-parameter method. A phylogenetic tree was built for 14 sequences with a total length of 681bp nucleotides. When conducting phylogenetic analysis using the MEGA X program, it was displayed the phylogenetic origin of our samples in all inferred trees is the same as the Chinese species; Thrips tabaci (China – Zhejiang), which registered with MN036455 accession number in the gene bank of NCBI. According to these results, the sampled thrips were collected from various vegetables including eggplants in Mongolia identified as Thrips tabaci and we have registered it in the GenBank of NCBI under OP288232 accession number. Keywords. Eggplant damage, thrips species identification, COI gene, phylogenetic origin, PCR.

Entomopathogenic fungi and their biological control of Tetranychus urticae: Two-spotted spider mites

The two-spotted spider mite (TSSM) Tetranychus urticae, is regarded as one of the most dangerous pests responsible for great losses in most of agricultural crops. It is a persistent pest in Saudi Arabia, especially in greenhouses where T. urticae is primarily controlled by chemical pesticides. The main problem for the two-spotted spider mites is its high resistance to pesticides and high fertility rate. In the long term, chemical pesticides cause health problems and economic losses, so it was necessary to search for a safe alternative method for human health and the environment. One of these alternative methods was the selection of plant varieties resistant to the TSSM, in addition to biological control that includes mites or predatory insects and entomopathogenic fungi. The growth, reproduction, and life-table parameters of T. urticae were examined in a laboratory setting with a 16L:8D photoperiod at 28 ± 1 °C and 65 ± 5% RH, in the presence of three major members of Family: Solanaceae tomato, eggplant, and pepper. Pepper was shown to be less conducive to T. urticae growth and reproduction compared to eggplant and tomato. Tetranychus urticae proceeded through all five stages of its life cycle (egg, larva, protonymph, deutonymph, and adult) on tested solanaceous plants, and these plants significantly influenced its growth, reproduction, and Life-table parameters. Additionally, entomopathogenic fungi have been used against insects that have proven highly effective in controlling and reducing the density of two-spotted spider mites. Eight fungi were isolated from 80 insect and mite samples collected from several Saudi Arabia regions. Analysis of the 18S rRNA sequences revealed that the fungal strains identified as Beauveria bassiana, Fusarium sp. F. equiseti, F. oxysporium, Scopulariopsis brevicaulis1, S. brevicaulis2, Aspergillus sclerotiorum, and Penicillium citrinum. The ability of isolated fungi to secrete enzymes degrading the two-spotted spider mite cuticle, namely lipase, protease, and chitinase, were studied.

The Water‐Exclusion Trap (WET): A 3D printable window trap collector that prevents DNA degradation

Abstract Sampling is one of the most important and fundamental processes in field biology. Among many sampling methods in entomology, window traps have long been considered one of the most effective and standardized methods. Unfortunately, the DNA quality of samples collected by window traps is typically low due to incursion by water, mainly via rainfall. Water cannot be avoided in long‐term surveys, even with a roof, due to wind and condensation. As we move into the genomics era, this shortfall in DNA quality has become problematic for molecular biomonitoring, and solutions are urgently needed. In this study, we developed the Water‐Exclusion Trap (WET), a 3D printable long‐term storable bottom collector for window or pitfall traps. The collection medium in WETs does not dilute because water is excluded and evaporation is limited, maximizing DNA preservation. Our field tests show that the WET outperforms conventional traps in preserving DNA quality. Insect samples preserved in WETs in the field had significantly higher PCR success rates and DNA Integrity Numbers compared to conventional traps. The WET addresses a critical challenge in the use of window traps, DNA degradation caused by water intrusion, and represents a significant advancement in fieldwork for genomic research in entomology. We provide the 3D printing template of the WET, enabling immediate, free use for all. Furthermore, it can be easily customized for different environments and purposes, and can be adapted to a variety of other traps, including pitfall traps, funnel traps and more.

A Taxonomic Study of Some Species of Hyperparasitoids Attacking the Primary Parasitoid Microterys nietneri of the Citricola Scale Insect, Coccus pseudomagnoliarum in Syria

Saleh, A.T. and A.M. Basheer. 2023. A Taxonomic Study of Some Species of Hyperparasitoids Attacking the Primary Parasitoid Microterys nietneri of the Citricola Scale Insect, Coccus pseudomagnoliarum in Syria. Arab Journal of Plant Protection, 41(3): 292-305. https://doi.org/10.22268/AJPP-41.3.292305 The citricola scale insect, Coccus pseudomagnoliarum, (Kuwana) (Hemiptera: Coccomorpha: Coccidae) is an economically important pest that occurs on citrus trees along the Syrian coast. The samples of host insects and infested plant parts were collected from April to June during 2016 and 2017 from two locations (Al-Sanobar, Dabba) in Lattakia Province and brought to the Biological Control Studies and Research Center (BCSRC), Faculty of Agriculture, Damascus University. Based on the information gathered, Microterys nietneri (Motschulsky) (Hymenoptera: Encyrtidae) may play an important role as a biological control agent of citricola scale insect, but high hyperparasitoids levels reduced M. nietneri efficiency as a citricola scale parasitoid. The objective of this study was to describe and identify naturally occurring hyperparasitoids. The following parasitoids were identified and reported for the first time in Syria: Cheiloneurus claviger (Thomson), Pachyneuron muscarum (Linnaeus), and Marietta leopardine (Motschulsky). Microscopic images were provided to illustrate the morphological and morphometric characteristics of these species. Keywords: Hyperparasitoids, Citricola scale, Microterys, Syria

The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria.

Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc). The rpf gene from Micrococcus luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in Escherichia coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla Actinomycetota and Bacillota . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

Biodiversity of herbivorous insects in rice organic and inorganic ecosystem

Since many years, presence and activities of herbivorous insect very crucial in the agricultural ecosystem. Purpose of the research is to study biodiversity of herbivorous insect in the rice organic and inorganic ecosystem. The research was conducted in Salassae village, Bulukumba Regency, South Sulawesi from June to August 2021. The herbivorous insect from organic and inorganic ecosystem was carried out and identified at the Pest Laboratory, Department of Plant Pest and Diseases, Faculty of Agriculture, Hasanuddin University Makassar. The places used in the research divided into organic and inorganic rice ecosystem, with measurement 25 m x 25 m, respectively. The insect samples collected used ubinan system methods with diagonal intersection applied for pitfall trap. The samples from organic and inorganic ecosystem analyzed by Shannon-Wiener Index Diversity. The result of Shannon-Wiener Index was showed the higher number of herbivorous insects in the inorganic rice ecosystem (H’ = 1.71) with population about 391 individual (51%). In contrast, result of organic ecosystem (H’ = 1.68) lower than inorganic. Conclusion of the research: 1) the higher number of herbivorous insects in the inorganic rice ecosystem (H’ = 1.71), the lowest result from organic ecosystem (H’ = 1.68); 2) the highest rice harvest higher the organic ecosystem (297 kg) and the lowest in the inorganic (279.2 kg).